Construction method and application of severe heatstroke mouse model in composite environment

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a severe heatstroke mouse model in a composite environment. By constructing the severe heatstroke mouse model in the high-temperature, high-humidity and high-salt composite environment, the method has remarkable innovativeness and scientific significance. Through specific experimental data and control tests, the superiority of the method in the aspect of heatstroke model construction is verified, and a powerful tool is provided for pathological mechanism research of heatstroke and development of potential treatment schemes. According to the standardized modeling method provided by the invention, researchers can obtain consistent experimental results under different experimental conditions, and a unified platform is provided for related research.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a severe heatstroke mouse model in a composite environment and an application thereof. Background Art

[0002] With the progress of industrialization and urbanization, global warming has gradually intensified, extreme weather has occurred frequently, and the incidence of heat-related injuries has continued to rise, becoming an important factor affecting the quality of human life and life expectancy, bringing heavy burdens to individuals, families, and society. The complex climate environment based on high temperature, including the southern coastal areas, islands and reefs with high humidity and high salt, has been in a high temperature, high humidity, and high salt complex climate environment for a long time, and the human body will undergo a series of physiological function changes. At the same time, this high temperature, high humidity, and high salt environment has led to an increasing incidence and mortality rate of severe heat stroke, which seriously affects the quality of human life and increases the social and economic burden. Severe heat stroke (HS) is the most serious type of heat-related diseases, with a mortality rate of up to 63.2%. It is a serious disease characterized by an increase in core body temperature (mostly greater than 40.5℃) and central nervous system disorders (such as delirium, epilepsy, coma, etc.). In a high temperature, high humidity, and high salt environment, patients with severe heat stroke have multiple organ dysfunction, poor prognosis, and a high disability rate. Therefore, constructing an animal model of heatstroke with high temperature, high humidity and high salt is an important basis for in-depth research on severe heatstroke injury, and can provide new ideas for the development of clinical treatment.

[0003] However, there is currently limited research in China on the construction of animal models of severe heatstroke caused by high-temperature composite environment (high temperature, high humidity, and high salt) models, and very few studies have attempted to combine high temperature, high humidity, and high salt conditions in the same experiment. For example, some studies have constructed a heatstroke model in a composite environment by simultaneously increasing the ambient temperature and humidity and giving a high-salt diet. This method can simulate a complex natural environment more comprehensively, but due to the lack of research, the technical details are not yet mature, and the repeatability and stability of the model construction still need to be further verified. In addition, there is limited research on composite environment models in the existing technology, and there is no standardized model that can stably and reliably simulate severe heatstroke under high temperature, high humidity, and high salt conditions, which limits in-depth research in this field. Summary of the invention

[0004] In view of the defects and shortcomings of the prior art in constructing animal models of severe heatstroke, the present invention aims to solve the following technical problems: (1) Comprehensive simulation of complex environmental factors: In the prior art, most heatstroke models only consider a single factor (such as high temperature or high humidity) or high temperature and high humidity, and lack a comprehensive simulation of complex environmental factors such as high temperature, high humidity, and high salt. The present invention aims to develop a heatstroke mouse model that can comprehensively simulate these environmental factors, so as to more realistically reproduce the situation of severe heatstroke in the natural environment. (2) Model repeatability and stability: There are few studies on existing heatstroke models in complex environments, and the repeatability and stability of model construction are insufficient, making it difficult to provide a reliable experimental basis for the study of the pathological mechanism of heatstroke and drug screening. The present invention establishes a mouse model with high repeatability and stability by optimizing environmental parameters and operating procedures to ensure the reliability and repeatability of experimental results. (3) Lack of standardized modeling methods: There is currently no standardized construction method for mouse models of heatstroke in a complex environment of high temperature, high humidity, and high salt, which makes it difficult to compare and integrate data from different studies. The details are as follows:

[0005] The purpose of the first aspect of the present invention is to provide a method for constructing a severe heatstroke mouse model.

[0006] The purpose of the second aspect of the present invention is to provide an application of an animal model constructed by the method for constructing a severe heatstroke mouse model according to the first aspect of the present invention.

[0007] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a method for constructing a severe heatstroke mouse model, comprising the following steps:

[0009] The mice were placed in a high temperature and high humidity environment and treated with salt spray. The condition of the mice was continuously monitored, the modeling was terminated, and the test was repeated to obtain a severe heatstroke mouse model.

[0010] In some embodiments of the present invention, the conditions of the high temperature and high humidity environment are: temperature 37°C ± 2°C, humidity 75% ± 5%; preferably, the conditions of the high temperature and high humidity environment are: temperature 37°C ± 1°C, humidity 75% ± 3%

[0011] In some embodiments of the present invention, the concentration of the salt spray is: NaCl concentration is 30 mg ± 5 mg / m 3 ; Preferably, the concentration of the salt mist is: NaCl concentration is 30mg±3mg / m 3 .

[0012] In some embodiments of the present invention, the monitored indicators include changes in the rectal temperature of mice.

[0013] In some embodiments of the present invention, the modeling is terminated when the mouse exhibits any of the following conditions 1) to 3);

[0014] 1) Rectal temperature ≥ 42.7℃

[0015] 2) Impairment of consciousness;

[0016] 3) Near death.

[0017] In some embodiments of the present invention, the duration of the consciousness disorder is greater than 5 seconds.

[0018] In some embodiments of the invention, the symptoms of impaired consciousness include the inability of mild, painless stimulation to drive the animal to crawl or change position; or the absence of voluntary movement.

[0019] In some embodiments of the present invention, the symptoms of dying include obvious convulsions and significant changes in respiratory rhythm.

[0020] In some embodiments of the present invention, after the modeling is terminated, the mice are placed in a ventilated environment, and the rectal temperature is remeasured after intraperitoneal injection of physiological saline.

[0021] In some embodiments of the present invention, the temperature of the ventilation environment is 22.0°C±5°C; preferably, the temperature of the ventilation environment is 22.0°C±2°C.

[0022] In some embodiments of the present invention,

[0023] In some embodiments of the present invention, the dosage of the intraperitoneal injection of normal saline is 30 mL±5 mL / kg.

[0024] In some embodiments of the present invention, when the rectal temperature is ≥42.7°C after re-measurement and the mouse is accompanied by impaired consciousness, it is determined that the heat stroke model is successfully established.

[0025] In some embodiments of the present invention, if the rectal temperature is <42.7°C when retested, it is recorded as modeling failure; if the mouse dies in the simulation chamber or dies within 30 minutes after leaving the chamber, it is recorded as modeling death.

[0026] In some embodiments of the present invention, mice are pretreated before modeling, and the pretreatment includes the following steps: the mice are adaptively raised in the laboratory for 1 to 2 weeks, the raising environment temperature is 22-25°C, the humidity is 40-60%, the light cycle is 12 hours light / 12 hours dark, and regular feed and tap water are provided for free intake.

[0027] The second aspect of the present invention provides a method for constructing a severe heatstroke mouse model according to the first aspect of the present invention, and uses the animal model constructed in the study of the pathological mechanism of heatstroke and the screening of potential therapeutic drugs for heatstroke.

[0028] The beneficial effects of the present invention are:

[0029] 1. More realistic heat stroke environment simulation

[0030] The present invention successfully constructed a mouse heatstroke model that is closer to the natural extreme environment by comprehensively considering the three environmental factors of high temperature, high humidity, and high salt. In reality, high temperature environments are often accompanied by high humidity and high salt (such as coastal operations, high-salt mining areas, etc.). The combined effect of these factors will aggravate the occurrence and development of heatstroke. The present invention innovatively constructs a composite environment of high temperature (37°C), high humidity (75%), and high-salt aerosol (3% NaCl), making the experimental conditions closer to the actual human heatstroke situation, thereby making the mouse model more clinically relevant. When the mice in the experimental group were exposed to a composite environment of high temperature, high humidity, and high-salt aerosols, compared with the control group (only high temperature and high humidity environment), the inflammatory indicators and chemokines of the mice showed an upward trend, and the degree of organ damage was aggravated, especially the inflammation and necrosis of the liver and kidney tissues were more obvious.

[0031] 2. Aggravating organ damage

[0032] Experimental data show that the degree of liver and kidney damage is significantly aggravated in mice exposed to high temperature, high humidity and high salt environment. Histopathological analysis found that the liver cells of the experimental group of mice showed obvious necrosis and edema, and the renal tubules also had significant necrosis and inflammatory response. In contrast, although the mice in the control group also suffered organ damage, the degree was significantly milder. Specifically, the IL-6 of the experimental group of mice increased, and the chemokines CXCL-1 and CXCL-6 tended to increase, while the control group only had a slight increase. These data show that after exposure to high-salt aerosols, organ damage is more serious, especially the liver and kidney function damage is aggravated.

[0033] 3. Establishment of a heat stroke model with multiple factors superimposed

[0034] The technical solution of the present invention combines high temperature, high humidity and high salt to provide a more complex and realistic severe heatstroke mouse model. This model not only makes heatstroke occur faster and the symptoms more severe, but also more comprehensively simulates the impact of complex factors on heatstroke encountered in real life, such as coastal operations and high-salt environment workers.

[0035] 4. Application value of drug screening and treatment research

[0036] This severe heatstroke model can provide an ideal experimental platform for subsequent drug screening and treatment research. Through this model, the effectiveness of different therapeutic interventions (such as fluid replacement, cooling drugs, anti-inflammatory drugs, etc.) under complex conditions can be tested, providing more instructive experimental data for clinical heatstroke treatment.

[0037] 5. The heat stroke model of the present invention provides an innovative tool for the study of the pathological mechanism of heat stroke, which helps to reveal the pathogenesis and aggravating factors of heat stroke, especially the heat stroke mechanism under the complex environment of high temperature, high humidity and high salt. This model is not only of great value in the field of basic scientific research, but also provides a new means for preventing and treating heat stroke and screening drugs, and is expected to reduce the health risks caused by heat stroke, especially in industries prone to heat stroke such as coastal operations, mining areas, and high-temperature factories, which has significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0039] Figure 1 The effects of different high salt concentrations on inflammatory factors and chemokines in mice.

[0040] Figure 2 The damage to the lungs, kidneys and liver of mice caused by high temperature, high humidity and high salt (different salt concentrations). DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0042] The design ideas of the present invention are as follows:

[0043] 1) Mouse selection and preparation

[0044] According to current relevant reports, animal models for HS mainly include sheep, rabbits, pigs, baboons, rats and mice, among which large animals often need to be restrained, anesthetized and other treatments due to poor cooperation and operability. However, anesthetic drugs will affect the physiological state of animals, and restraint will also affect the spontaneous temperature regulation of animals. The above factors will affect the experimental research of spontaneous HS. Therefore, mice with small size, easy operation, sober and free movement have become the most commonly used model animals in HS-related research in recent years, so mice are selected as research models in this embodiment.

[0045] 2) Simulation of high temperature, high humidity and high salt environment

[0046] Salt fog in the atmosphere mainly comes from the ocean, accounting for about 90%, and the rest is salty dust, burning plants, etc. Since salt fog easily migrates with external factors such as wind, the salt fog content in the environment is variable and has a wide distribution range. Even in the same region, the salt fog content in the atmosphere varies greatly. The salinity of seawater in various sea areas in my country varies with summer and winter, with an average fluctuation of about 3%. Therefore, the present invention selects a salt concentration of 3% in the salt fog setting, that is, 30mg / m3.

[0047] The experimental environment is controlled by a specially designed environmental simulation chamber. In this chamber, the temperature is controlled at 37°C and the humidity is controlled at 75%. The high-salt environment is achieved by releasing aerosol containing salt in the chamber. The specific method is to atomize saturated salt water (3wt% NaCl) into the air through an ultrasonic atomizer so that the salt concentration in the air in the chamber reaches the level of inhalable salt particles. The goal of this environment is to simulate a high-salt environment (such as seaside operations, salt lakes or mining areas, etc.).

[0048] 3) Construction of heat stroke model

[0049] The mice in the experimental and control groups were placed in the above-mentioned high temperature, high humidity, and high salt complex environment. During the experiment, the mice were fasted and deprived of water and placed in conventional cages (3-5 / cage). The simulated climate chamber temperature was 37°C, humidity was 75%, and salt concentration was 30 mg / m3 (3wt% NaCl) and then stably operated for more than 1 hour. The mouse cages were directly placed in the cabin for heat exposure. Throughout the process, the mice in the experimental group were continuously exposed to this complex environment, while the control group was only exposed to a high temperature and high humidity environment without salt aerosol atomization treatment. The mice in the blank control group were always in a room temperature environment of (22.0±1.6)°C.

[0050] The specific experimental data of the present invention are as follows.

[0051] Example Construction of a severe heatstroke mouse model under high temperature, high humidity, and high salt conditions

[0052] 1. Experimental Materials

[0053] Mice: Adult C57BL / 6 mice, weighing 20-25 g, purchased from Guangzhou Zhiyuan Biological Co., Ltd., in good health. High temperature and high humidity environment equipment: Environmental simulation chamber equipped with precise temperature and humidity control system.

[0054] Salt spray generation and measurement device: Model CEWS-SSMD; High-precision particle controller: Model CEWS-APMC. Manufacturer: Chongqing Guoke Chengyuan Environmental Technology Co., Ltd.

[0055] Salt aerosol equipment: Ultrasonic nebulizer, used to nebulize 3% NaCl solution into aerosol.

[0056] Body temperature monitoring equipment: small animal rectal thermometer, measuring accuracy is ±0.1℃.

[0057] Other equipment: Histopathology equipment (for pathological sections and microscopic analysis), microplate reader (for testing serum ELISA).

[0058] 2. Experimental steps

[0059] 1) Mouse preparation: Adult C57BL / 6 male mice weighing 20-25 g were purchased from a supplier. The mice were adapted to feeding in the laboratory for 1 week, with a temperature of 22-25°C, a humidity of 40-60%, a light cycle of 12 hours light / 12 hours dark, and regular feed and tap water were provided ad libitum.

[0060] 2) Environment settings:

[0061] The experimental environment is controlled by a specially designed environmental simulation cabin. In this cabin, the temperature is controlled at 37°C and the humidity is controlled at 75%. The high-salinity environment is achieved by releasing salt aerosol in the cabin. The specific method is to atomize saturated salt water (3% NaCl) into the air through an ultrasonic atomizer so that the salt concentration in the cabin air reaches the level of inhalable salt particles. The atomizer is set to continuous atomization to ensure that the salt concentration in the cabin remains stable.

[0062] 3) Experimental treatment:

[0063] The mice were divided into three groups, with 6-7 mice in each group.

[0064] Experimental group: placed in a high temperature (37°C), high humidity (75%) and high salt aerosol compound environment. Among them, the high salt aerosol control group and 0mg / m 3 , 0mg / m 3 , 5mg / m 3 , 10mg / m 3 , 30mg / m 3 .

[0065] Control group: placed in the same high temperature (37°C) and high humidity (75%) environment, but without salt aerosol exposure.

[0066] The blank control group was placed at room temperature of (22.0±1.6)℃.

[0067] During the treatment period, the core body temperature of the mice was monitored every 15 minutes using a rectal thermometer, and the activity patterns and consciousness state of the mice were observed.

[0068] 4) Determination of heat stroke:

[0069] After placing the mice in the experimental chamber, closely observe their activity patterns and consciousness. When the mice show any of the following conditions, it is considered the end point of modeling and they need to be immediately transferred out of the chamber for further treatment: (1) Tr ≥ 42.7℃; (2) impaired consciousness, that is, no autonomous activity (mild painless stimulation cannot drive the animal to crawl or change position) for > 5s; (3) dying, that is, obvious convulsions and obvious changes in respiratory rhythm. After leaving the chamber, the Tr of the mice was re-measured and they were quickly transferred to a ventilated environment of (22.0±1.6)℃, and normal saline (30ml / kg) was injected intraperitoneally. If Tr < 42.7℃ during the re-measurement, it was recorded as modeling failure; if the mice died in the simulation chamber or died within 30 minutes after leaving the chamber, it was recorded as modeling death; if Tr ≥ 42.7℃, accompanied by impaired consciousness, it was judged as a successful heatstroke modeling.

[0070] Among them, temperature monitoring uses a rectal thermometer. After inserting a 1.3mm diameter flexible temperature probe into the mouse rectum 2-3cm, the thermometer is pasted to the outside of the anus with medical tape. The mobile phone downloads the corresponding exclusive App (Fimminger, SV224), measures the transmission rate of 10 seconds / recording interval of 5 minutes, and sends the temperature data wirelessly to the mobile phone smart terminal to monitor the temperature changes in real time and dynamically.

[0071] 5) Tissue sampling and pathological examination: After the experiment, the mice in the experimental group and the control group were fixed by cardiac perfusion, and the main organs such as brain, heart, liver, and kidney were taken for pathological sections. After the tissue sections were stained, the pathological changes of the organs, such as cell degeneration, inflammatory reaction, and necrosis, were observed under a microscope.

[0072] 6) Blood ELISA and biochemical tests: Blood was collected from mice after heat stroke to detect serum levels of IL-6, IL-4, CXCL-1, CXCL-2, ALT (alanine aminotransferase), LDH (lactate dehydrogenase), creatinine and other indicators to assess the degree of liver and kidney damage.

[0073] The ELISA detection kits are as follows: IL-6: Bioswamp, SHM20002; IL-4: Bioswamp, MU30385; CXCL-1: Bioswamp, MU30699; CXCL-2: Bioswamp, MU30191; ALT: Bioswamp, BTK060; LDH: Bioswamp, MU30023.

[0074] 3. Experimental results

[0075] like Figure 1As shown in the data, compared with the control group, the experimental group mice had higher proportions of inflammatory factors and chemokines in the high temperature, high humidity, and high salt environment. The IL-6 of the experimental group mice increased, and the chemokines CXCL-1 and CXCL-6 tended to increase, while the control group only slightly increased. These data indicate that after exposure to high-salt aerosols, organ damage is more severe, especially liver and kidney damage. Among them, the increase in inflammatory factors and chemokines intensified with the increase in salt concentration, and at 30mg / m 3 When the highest.

[0076] Histopathological results: The results are as follows Figure 2 As shown in the study, the degree of liver and kidney damage in mice exposed to high temperature, high humidity and high salt environment was significantly aggravated. Histopathological analysis showed that the liver cells of the experimental group of mice showed obvious necrosis and edema, and the renal tubules also showed significant necrosis and inflammatory response. In contrast, although the mice in the control group also showed organ damage, the degree was significantly milder. The degree of organ damage was aggravated with the increase of salt concentration. At 30mg / m 3 When the highest.

Claims

1. A method for constructing a heat stroke mouse model, characterized in that: The following steps are involved: The mice were placed in a high temperature and high humidity environment and treated with salt spray. The condition of the mice was continuously monitored, the modeling was terminated, and the test was repeated to obtain a severe heatstroke mouse model.

2. The construction method according to claim 1, characterized in that: The conditions of the high temperature and high humidity environment are: temperature 37°C±2°C, humidity 75%±5%.

3. The construction method according to claim 1, characterized in that: The concentration of the salt spray is: NaCl concentration is 30mg±5mg / m 3 .

4. The construction method according to claim 1, characterized in that: The monitored indicators include changes in the rectal body temperature of mice.

5. The construction method according to claim 1, characterized in that: The modeling was terminated when the mice showed any of the following conditions 1) to 3); 1) Rectal temperature ≥ 42.7°C 2) Impairment of consciousness; 3) Near death.

6. The construction method according to claim 1, characterized in that: After the modeling was terminated, the mice were placed in a ventilated environment, and the rectal temperature was remeasured after intraperitoneal injection of normal saline.

7. The construction method according to claim 6, characterized in that: When the rectal temperature was ≥42.7°C after remeasurement and the mice were accompanied by impaired consciousness, the heat stroke model was determined to be successfully established.

8. The construction method according to claim 6, characterized in that: The temperature of the ventilation environment is 22.0°C±5°C.

9. The construction method according to claim 6, characterized in that: The dosage of the intraperitoneal injection of normal saline is 30 mL±5 mL / kg.

10. Use of the animal model constructed by the construction method according to any one of claims 1 to 9 in the study of the pathological mechanism of heat stroke and the screening of potential therapeutic drugs for heat stroke.

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