A method for constructing a complex environmental heat stroke mouse model and application thereof

Abstract

The application belongs to the technical field of animal model construction, and particularly relates to a method for constructing a severe heat stroke mouse model in a composite environment and application thereof. The application has significant innovation and scientific significance by constructing a severe heat stroke mouse model in a high-temperature, high-humidity and high-salt composite environment. Through specific experimental data and control tests, the application verifies the superiority of the application in the construction of a heat stroke model, and provides a powerful tool for the study of the pathological mechanism of heat stroke and the development of potential treatment schemes. The standardized modeling method provided by the application enables researchers to obtain consistent experimental results under different experimental conditions, and provides a unified platform for related research.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing a mouse model of severe heatstroke in a complex environment and its application. Background Technology

[0002] With industrialization and urbanization, global warming is intensifying, extreme weather events are becoming more frequent, and the incidence of heat-related injuries is rising, becoming a significant factor affecting human quality of life and lifespan, placing a heavy burden on individuals, families, and society. Complex climatic environments based on high temperatures, including coastal and island areas with high humidity and salinity, cause a series of physiological changes in the human body when exposed to these conditions for extended periods. This high-temperature, high-humidity, and high-salt environment leads to an increasing incidence and mortality rate of severe heatstroke, seriously impacting human quality of life and increasing the socioeconomic burden. Severe heatstroke (HS) is the most serious type of heat-related illness, with a mortality rate still as high as 63.2%. It is a severe illness characterized by elevated core body temperature (often greater than 40.5°C) accompanied by central nervous system disorders (such as delirium, epilepsy, and coma). In high-temperature, high-humidity, and high-salt environments, patients with severe heatstroke experience multiple organ dysfunction, have a poor prognosis, and a high rate of disability. Therefore, constructing animal models of heatstroke under high temperature, high humidity, and high salt conditions is an important foundation for in-depth research on severe heatstroke injuries and can provide new ideas for clinical treatment.

[0003] However, current research in China on the construction of animal models of severe heatstroke caused by high-temperature combined environments (high temperature, high humidity, and high salinity) is relatively limited. Very few studies have attempted to combine high temperature, high humidity, and high salinity conditions in the same experiment. For example, some studies have constructed a heatstroke model under combined conditions by simultaneously increasing ambient temperature and humidity and administering a high-salt diet. This method can more comprehensively simulate complex natural environments, but due to limited research, the technical details are not yet mature, and the reproducibility and stability of the model construction still need further verification. Furthermore, the limited research on combined environment models in existing technologies means that there is no standardized model that can stably and reliably simulate severe heatstroke under high temperature, high humidity, and high salinity conditions, which limits in-depth research in this field. Summary of the Invention

[0004] In view of the deficiencies and shortcomings of existing technologies in constructing animal models of severe heatstroke, this invention aims to solve the following technical problems: (1) Comprehensive simulation of complex environmental factors: In existing technologies, most heatstroke models only consider a single factor (such as high temperature or high humidity) or high temperature and high humidity, lacking a comprehensive simulation of complex environmental factors such as high temperature, high humidity, and high salinity. This invention aims to develop a mouse model of heatstroke that can comprehensively simulate these environmental factors, thereby more realistically reproducing severe heatstroke in the natural environment. (2) Model repeatability and stability: There are few existing studies on 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. This 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: Currently, there is no standardized construction method for mouse models of heatstroke in complex environments of high temperature, high humidity, and high salinity, making it difficult to compare and integrate data from different studies. Specifically:

[0005] The first objective of this invention is to provide a method for constructing a mouse model of severe heatstroke.

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

[0007] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

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

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

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

[0011] In some embodiments of the present invention, the concentration of the salt spray is: NaCl concentration of 30 mg ± 5 mg / m³. 3 Preferably, the concentration of the salt spray is: NaCl concentration of 30 mg ± 3 mg / m³. 3 .

[0012] In some embodiments of the present invention, the monitored indicators include changes in rectal body temperature in 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) Impaired consciousness;

[0016] 3) Near death.

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

[0018] In some embodiments of the present invention, the symptoms of the disorder of consciousness include the inability to drive an animal to crawl or change position due to mild, painless stimuli; or the lack of voluntary activity.

[0019] In some embodiments of the present invention, the symptoms of impending death include the appearance of obvious convulsions and a significant change in respiratory rhythm.

[0020] In some embodiments of the present invention, after terminating the modeling, the mice are placed in a ventilated environment, and their 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℃±5℃; preferably, the temperature of the ventilation environment is 22.0℃±2℃.

[0022] In some embodiments of the present invention

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

[0024] In some embodiments of the present invention, when the rectal temperature is ≥42.7°C after retesting and the mouse has impaired consciousness, the heatstroke model is considered to have been successfully established.

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

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

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

[0028] The beneficial effects of this invention are:

[0029] 1. A more realistic simulation of heatstroke environment

[0030] This invention successfully constructed a mouse heatstroke model that more closely resembles natural extreme environments by comprehensively considering three environmental factors: high temperature, high humidity, and high salinity. Real-world high-temperature environments are often accompanied by high humidity and high salinity (such as coastal operations and high-salt mining areas), and these factors combined exacerbate the occurrence and development of heatstroke. This invention innovatively constructs a composite environment of high temperature (37°C), high humidity (75%), and high-salt aerosols (3% NaCl), making the experimental conditions closer to the actual human heatstroke situation, thus making the mouse model more clinically relevant. When the experimental group mice were exposed to the composite environment of high temperature, high humidity, and high-salt aerosols, compared with the control group (high temperature and high humidity environment only), the mice showed an increased trend in inflammatory markers and chemokines, and the degree of organ damage was aggravated, especially the inflammation and necrosis of the liver and kidney tissues.

[0031] 2. Exacerbation of organ damage

[0032] Experimental data showed that in mice exposed to high temperature, high humidity, and high salinity, the damage to the liver and kidneys was significantly aggravated. Histopathological analysis revealed significant necrosis and edema in the hepatocytes of the experimental group mice, as well as significant necrosis and inflammatory responses in the renal tubules. In contrast, while the control group mice also showed organ damage, the degree was significantly milder. Specifically, the experimental group mice showed elevated IL-6 levels and a trend of increased chemokines CXCL-1 and CXCL-6, while the control group only showed a slight increase. These data indicate that exposure to high-salt aerosols leads to more severe organ damage, especially exacerbating liver and kidney dysfunction.

[0033] 3. Establishment of a multi-factor superposition model for heatstroke

[0034] The technical solution of this invention combines high temperature, high humidity, and high salinity to provide a more complex and realistic mouse model of severe heatstroke. This model not only makes heatstroke occur faster and the symptoms more severe, but also more comprehensively simulates the combined effects of factors encountered in real life, such as those encountered by workers in coastal areas or high-salt environments, on heatstroke.

[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 treatment interventions (such as fluid replacement, cooling drugs, and anti-inflammatory drugs) under combined conditions can be tested, providing more instructive experimental data for clinical heatstroke treatment.

[0037] 5. The heatstroke model of this invention provides an innovative tool for studying the pathological mechanism of heatstroke, helping to reveal the pathogenesis and aggravating factors of heatstroke, especially the mechanism of heatstroke under combined high temperature, high humidity, and high salinity environments. This model not only has important value in the field of basic scientific research, but also provides a new means for the prevention and treatment of heatstroke and drug screening, and is expected to reduce the health risks caused by heatstroke, especially in industries prone to heatstroke such as coastal operations, mining areas, and high-temperature factories, with significant social and economic benefits. Attached Figure Description

[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 This study investigated the effects of high temperature, high humidity, and high salt (at different salt concentrations) on the lungs, kidneys, and liver of mice. Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] The design concept of this invention is as follows:

[0043] 1) Mouse selection and preparation

[0044] According to current reports, animal models of spontaneous hypothermia (HS) mainly include sheep, rabbits, pigs, baboons, rats, and mice. Larger animals often require restraint and anesthesia due to their poor cooperation and manipulability. However, anesthetic drugs can affect the animal's physiological state, and restraint can affect its spontaneous thermoregulation; both factors can impact experimental studies of spontaneous HS. Therefore, small, easily manipulated, conscious, and freely moving mice have become the most commonly used model animals in HS-related research in recent years. Thus, mice were selected as the research model in this embodiment.

[0045] 2) Simulation of high temperature, high humidity, and high salinity environments

[0046] Atmospheric salt fog mainly originates from the ocean, accounting for about 90%, with the remainder coming from sources such as salty dust and plant combustion. Because salt fog easily migrates with external factors such as wind, its concentration in the environment is highly variable and widely distributed. Even within the same region, the concentration of salt fog in the atmosphere varies considerably. The salinity of seawater in various sea areas of my country varies with summer and winter, fluctuating by an average of about 3%. Therefore, this invention uses a salt concentration of 3%, or 30 mg / m³, for the salt fog setting.

[0047] The experimental environment was controlled using a specially designed environmental simulation chamber. Inside this chamber, the temperature was maintained at 37°C and the humidity at 75%. The high-salt environment was achieved by releasing salt-containing aerosols within the chamber. Specifically, saturated salt water (3 wt% NaCl) was atomized into the air using an ultrasonic nebulizer, raising the salt concentration in the chamber air to a level suitable for inhaling salt particles. The goal of this environment was to simulate high-salt environments (such as coastal operations, salt lakes, or mining areas).

[0048] 3) Construction of a heatstroke model

[0049] Both experimental and control mice were placed in the aforementioned high-temperature, high-humidity, and high-salt composite environment. During the experiment, the mice were fasted and deprived of water, and kept in standard cages (3-5 mice / cage). The simulated climate chamber was set at 37℃, 75% humidity, and 30 mg / m³ (3wt% NaCl) and stabilized for at least 1 hour before the mouse cages were directly placed into the chamber for heat exposure. Throughout the process, the experimental group mice were continuously exposed to this composite environment, while the control group was only exposed to the high-temperature and high-humidity environment, without any salt aerosol atomization treatment. The blank control group mice were kept at room temperature (22.0±1.6)℃.

[0050] The specific experimental data for this invention are as follows.

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

[0052] 1. Experimental Materials

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

[0054] Salt spray generator and measuring device: Model CEWS-SSMD; High-precision particulate matter controller: Model CEWS-APMC. Manufacturer: Chongqing Guoke Chengyuan Environmental Technology Co., Ltd.

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

[0056] Temperature monitoring equipment: small animal rectal thermometer, with a measurement accuracy of ±0.1℃.

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

[0058] 2. Experimental Procedure

[0059] 1) Mouse preparation: Purchase adult male C57BL / 6 mice weighing 20-25g from the supplier. Adapt the mice to living conditions in the laboratory for one week at a temperature of 22-25℃ and a humidity of 40-60%, with a 12-hour light / 12-hour dark cycle. Provide them with regular feed and free access to tap water.

[0060] 2) Environment settings:

[0061] The experimental environment was controlled using a specially designed environmental simulation chamber. Inside this chamber, the temperature was maintained at 37°C and the humidity at 75%. The high-salt environment was achieved by releasing salt-containing aerosols within the chamber. Specifically, saturated saline solution (3% NaCl) was atomized into the air using an ultrasonic nebulizer, ensuring that the salt concentration in the chamber air reached a level where inhalable salt particles could be produced. The nebulizer was set to continuous atomization to ensure that the salt concentration within the chamber remained stable.

[0062] 3) Experimental treatment:

[0063] The mice were divided into three groups of 6-7 mice each.

[0064] Experimental group: placed in a combined environment of high temperature (37℃), high humidity (75%), and high salinity aerosols. The high salinity aerosol was used as a control group and a concentration of 0 mg / m³. 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 in a room temperature environment of (22.0±1.6)℃.

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

[0068] 4) Determination of heatstroke:

[0069] After placing the mice in the experimental chamber, their activity patterns and consciousness status were closely observed. The modeling endpoint was considered to be reached when any of the following conditions were observed in the mice, and they should be immediately transferred out of the chamber for further treatment: (1) Tr ≥ 42.7℃; (2) Impaired consciousness, i.e., no spontaneous activity (mild painless stimulation cannot drive the animal to crawl or change position) for more than 5 seconds; (3) Near death, i.e., obvious convulsions and obvious changes in respiratory rhythm. After the mice were removed from the chamber, Tr was retested and they were quickly transferred to a ventilated environment at (22.0±1.6)℃ and injected intraperitoneally with physiological saline (30ml / kg). If Tr < 42.7℃ during the retest, it was recorded as a failure of modeling; if the mice died in the simulated chamber or within 30 minutes after being removed from the chamber, it was recorded as modeling death; if Tr ≥ 42.7℃, accompanied by impaired consciousness, it was determined that the heatstroke modeling was successful.

[0070] Temperature monitoring uses a rectal thermometer. A flexible 1.3mm diameter temperature probe is inserted 2-3cm into the mouse rectum, and the thermometer is attached to the outside of the anus with medical tape. A dedicated app (Feiminger, SV224) is downloaded to the mobile phone. The measurement and transmission rate is 10 seconds / recording interval of 5 minutes. The temperature data is wirelessly sent to the mobile smart terminal to monitor temperature changes in real time.

[0071] 5) Tissue Sampling and Pathological Examination: After the experiment, the hearts of mice in both the experimental and control groups were perfused and fixed, and major organs such as the brain, heart, liver, and kidneys were collected for pathological sections. After staining, the pathological changes in the organs, such as cell degeneration, inflammatory reactions, and necrosis, were observed under a microscope.

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

[0073] The ELISA test 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, compared with the control group, the experimental group mice had higher proportions of inflammatory factors and chemokines in a high-temperature, high-humidity, and high-salt environment. The experimental group mice showed elevated IL-6 levels and a trend of increased chemokines CXCL-1 and CXCL-6, while the control group only showed a slight increase. These data indicate that exposure to high-salt aerosols leads to more severe organ damage, especially exacerbating liver and kidney dysfunction. The increase in inflammatory factors and chemokines intensified with increasing salt concentration, reaching a higher level at 30 mg / m³. 3 The highest time.

[0076] Histopathological results: The results are as follows Figure 2 As shown, in mice exposed to high temperature, high humidity, and high salinity, the damage to the liver and kidneys was significantly aggravated. Histopathological analysis revealed significant necrosis and edema in the hepatocytes of the experimental group mice, as well as significant necrosis and inflammatory responses in the renal tubules. In contrast, while the control group mice also showed organ damage, the degree was significantly milder. The severity of organ damage increased with increasing salt concentration, reaching its peak at 30 mg / m³. 3 The highest time.

Claims

1. A method for constructing a mouse model of heatstroke, characterized in that, Includes the following steps: Mice were placed in a high-temperature and high-humidity environment and given salt spray treatment. The mice were continuously monitored, the modeling was terminated, and the test was repeated to obtain a mouse model of severe heatstroke. The conditions for the high temperature and high humidity environment are: temperature 37℃±2℃, humidity 75%±5%; The concentration of the salt spray is: NaCl concentration of 30 mg ± 5 mg / m³. 3 ; The monitored indicators include changes in rectal body temperature in mice; The modeling process should be terminated when mice exhibit any of the following conditions 1) to 3); 1) Rectal temperature ≥ 42.7℃ 2) Impaired consciousness; 3) Near death; After terminating the modeling process, the mice were placed in a well-ventilated environment, and their rectal temperature was remeasured after intraperitoneal injection of physiological saline. If the rectal temperature is ≥42.7℃ after retesting, and the mouse exhibits impaired consciousness, then the heatstroke model is considered successfully established.

2. The construction method according to claim 1, characterized in that: The temperature of the ventilated environment is 22.0℃±5℃.

3. The construction method according to claim 1, characterized in that: The dosage of the intraperitoneal injection of physiological saline is 30 mL ± 5 mL / kg.

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