Method for establishing learning and cognitive impairment model by periodically exposing to near-body-temperature cabin environment and application thereof

Abstract

This invention discloses a method for establishing a learning and cognitive impairment model induced by periodic exposure to a near-body temperature cabin environment and its application. The method involves placing mice in a stepped-heating environment for periodic heat exposure, and continuously monitoring the mice's rectal temperature, plasma and hippocampal lactate concentrations, behavioral changes, and the expression of mitochondrial damage molecules and inflammatory factors in the hippocampus. When all four criteria are met simultaneously, the learning and cognitive impairment model is confirmed to be successfully established. By placing mice in a stepped-heating environment for periodic heat exposure, the high frequency of heat exposure, comprehensive assessment, and high accuracy more realistically simulate the characteristics of gradually increasing and periodically changing temperature in a helicopter cabin environment. Compared with traditional heat exposure models, it is closer to the thermal environment conditions faced by pilots in non-sealed pressurized cabins and low-altitude environments during actual operations, which helps to further study the impact mechanism of near-body temperature cabin environment on pilots' learning and cognition.

Description

Technical Field

[0001] This invention relates to the field of animal model establishment technology, and in particular to a method for establishing a model of learning and cognitive impairment caused by periodic exposure to near-body temperature cabin environment and its application. Background Technology

[0002] With the continuous advancement of technology, helicopters have been widely used due to their unique flight capabilities and flexibility. However, the helicopter cabin environment presents many factors that are detrimental to flight operations. The unsealed pressurized cabin of a helicopter is small, the onboard equipment operates under heavy loads, and the heat sources within the cabin are complex and diverse (including solar radiation, electronic equipment, the cabin greenhouse effect, and the metabolic heat generated by the flight crew). Without effective cabin environmental protection measures or systems to mitigate the environmental heat load, flight operations in high temperatures have a serious impact on the physiological and psychological well-being of flight crews. This is especially true during the pre-takeoff, taxiing, takeoff waiting, and low-altitude flight phases, when flight crews are exposed to high temperatures for extended periods and frequently. Studies have shown that in summer, the ground temperature of a helicopter cabin can reach or even exceed 56°C; when the ambient temperature rises to 3°C... At 7°C, the error rate of pilots' operations increases significantly. When the ambient temperature rises to 38°C, problems such as operational incoordination, decreased sensitivity, and reduced target recognition ability will occur. High-temperature environments not only cause physiological dysfunction in pilots (such as reduced oxygen transport capacity of the circulatory system and imbalance of internal homeostasis), but also have a profound impact on the central nervous system. This manifests as exhaustion of attention resources, decreased performance in complex tasks due to dopamine secretion inhibition, and damage to hippocampal function, which in turn leads to memory loss and decision-making impairment. With the continuous rise in global average temperature and the increasing frequency and intensity of extreme weather events, the risk of pilots being exposed to near-body temperature high-temperature environments continues to rise. Moreover, pilots' exposure to the working environment is often periodic, and the central fatigue caused by prolonged and frequent exposure to high-temperature environments seriously affects flight safety and operational capabilities.

[0003] Currently, quantitative assessment and research on the damage mechanisms of helicopter pilots' learning and cognition after periodic exposure to near-body temperature cabin environments are difficult to conduct in real flight environments. This not only hinders a deeper understanding of the impact mechanisms of high-temperature environments on pilots but also impedes the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments. Although existing technologies have heat exposure models that use rodents to simulate cognitive impairment caused by high-temperature environments, they still have shortcomings: the environmental temperature settings of traditional heat exposure models differ significantly from the animal's core body temperature, making it difficult to reflect working conditions where there is no significant heat exchange between the animal and the cabin environment or the body's heat dissipation efficiency is significantly reduced; the frequency of heat exposure is too short, failing to objectively reflect the weight of the animal's heat adaptation process in periodic heat exposure; and there is insufficient research on the molecular mechanisms of damage assessment indicators after periodic heat exposure in key brain regions of learning and cognition (such as the hippocampus), especially lacking in-depth analysis of cellular level indicators such as mitochondrial functional impairment and inflammatory factor expression, as well as targeted protective measures. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies. Firstly, it provides a method for establishing a model of learning and cognitive impairment caused by periodic exposure to near-body temperature cabin environments. This method solves problems such as large differences between environmental temperature settings and animal core body temperature, short frequency of heat exposure, insufficient research on the molecular mechanisms of key brain regions involved in learning and cognition, and a lack of in-depth analysis of targeted protective measures. The successful establishment of this learning and cognitive impairment model provides theoretical support for in-depth research on the effects of periodic near-body temperature environmental exposure on learning and cognitive impairment and the prevention of heat damage. Secondly, it provides applications for the learning and cognitive impairment model caused by periodic exposure to near-body temperature cabin environments.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for establishing a model of learning and cognitive impairment caused by periodic exposure to near-body temperature cabin environments includes the following steps:

[0007] Mice were subjected to periodic heat exposure in a stepped heating environment, and their condition was continuously monitored to obtain a learning and cognitive impairment model.

[0008] The monitored indicators include the mouse's anal temperature, plasma and hippocampal lactate concentrations, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampus.

[0009] The criteria for judging whether the learning and cognitive impairment model has been successfully established include: ① elevated anal temperature in mice; ② elevated lactate concentration in plasma and hippocampus; ③ behavioral changes in anxiety behavior, spatial memory ability, and non-spatial memory ability; ④ increased expression levels of mitochondrial damage molecules and inflammatory factors in hippocampus.

[0010] All four criteria must be met simultaneously for the learning and cognitive impairment model to be considered successfully established.

[0011] This invention uses rodent mice as experimental animals to construct a learning and cognitive impairment model. Mice have similar genes, physiological functions, biological processes and metabolic characteristics to humans. When studying the effects of high temperature environment on learning and cognition, the neural responses and cognitive changes of mice can reflect the possible situations in humans to a certain extent. In addition, mice have the advantages of rapid reproduction, easy breeding, small size, easy operation and low cost.

[0012] Based on the aforementioned technical means, this invention, through periodic heat exposure of mice in a stepped heating environment, can more realistically simulate the characteristics of a helicopter cockpit environment with gradually increasing and periodic temperature changes. Compared to traditional heat exposure models, this model more closely resembles the thermal environment faced by pilots in actual operations, facilitating in-depth research into the impact mechanism of near-body temperature cockpit environments on pilots' learning and cognition. Furthermore, the physiological indicators of mice are easily monitored. After periodic heat exposure, their learning and cognitive abilities can be assessed by monitoring multiple indicators such as rectal temperature, changes in serum lactate concentration, behavioral changes, and hippocampal tissue changes, from physiological and behavioral perspectives. To comprehensively assess the state changes of mice during heat exposure at multiple levels, including tissues, rectal temperature was used as a thermomonitoring point, which is closer to the core body temperature for rodents. Lactic acid is not only an intermediate product of cellular metabolism but also an important signaling molecule mediating cell communication. Pathological accumulation of lactic acid in the brain may lead to cognitive decline and the development of neurodegenerative diseases. Therefore, lactic acid can serve as an energy substrate for glycolysis in the central nervous system, meeting the central nervous system's urgent need for energy uptake. However, after environmental exposure and central nervous system damage, the continued accumulation of lactic acid, as a signaling molecule, can affect cellular energy... Damage to mitochondria, the metabolic organelles, leads to structural and functional abnormalities in the nervous system, resulting in changes in plasma and tissue lactate concentrations, which can serve as important candidate parameters for metabolic function. This invention reflects the degree of physiological stress response in mice by monitoring rectal temperature and serum lactate concentration; it directly reflects whether the learning and cognitive functions of mice are affected through behavioral changes; and it reveals the possible mechanisms of learning and cognitive impairment from a neurobiological perspective through changes in hippocampal tissue, providing rich data support for a comprehensive assessment of learning and cognitive impairment. Furthermore, this invention establishes a rigorous and comprehensive set of criteria for the successful establishment of a learning and cognitive impairment model, ensuring high consistency and accuracy of the established model and avoiding unreliability issues caused by ambiguous criteria, thus providing reliable experimental subjects for subsequent research. The successful establishment of the learning and cognitive impairment model helps to understand the impact mechanism of high-temperature environments on flight personnel's learning and cognition, providing a scientific basis for the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments. Through research on the learning and cognitive impairment model, effective protective measures can be explored in a targeted manner to reduce the risk of learning and cognitive impairment in flight personnel in high-temperature cockpit environments, thereby improving flight safety and operational capabilities.

[0013] Furthermore, the mitochondrial damage molecules in the mouse hippocampus include ATP5H; the inflammatory factors include TNF-α and IL-6.

[0014] Based on the aforementioned technical methods, ATP5H is an important component of mitochondrial ATP synthase, and its changes can reflect the degree of mitochondrial functional impairment. TNF-α and IL-6 are classic inflammatory factors, and changes in their expression levels reflect the inflammatory response in hippocampal tissue under high-temperature conditions. Excessive inflammatory responses may lead to nerve cell damage and death. By using ATP5H as the molecule causing mitochondrial damage in mouse hippocampal tissue, and TNF-α and IL-6 as inflammatory factors, this invention provides clear biomarkers for assessing learning and cognitive impairment caused by high-temperature environments. By detecting the expression levels of these molecules, this invention can accurately determine the degree of mitochondrial damage and inflammatory response in mouse hippocampal tissue under high-temperature conditions, thereby quantifying learning and cognitive impairment and more objectively and accurately determining whether a learning and cognitive impairment model has been successfully established.

[0015] Furthermore, a learning and cognitive impairment model is considered to have been successfully established when mice simultaneously meet the following four criteria.

[0016] ① The rectal temperature of mice increased to 38-42℃; ② The plasma lactate concentration increased by 5.56%-65.57%, and the hippocampal lactate concentration increased by 10.7%-55.2%; ③ Behavioralally, the spontaneous exploration ability of mice weakened, anxiety was observed, and spatial and non-spatial memory abilities were impaired; ④ The expression levels of mitochondrial damage molecules in the hippocampus increased by 13.86%-56.38%; the expression levels of TNF-α increased by 22.43%-212.80%, and the expression levels of IL-6 increased by 13.58%-234.80%.

[0017] Based on the above technical means, when the monitoring indicators of mice meet the above four criteria at the same time, the heat exposure ends and it is judged that the learning and cognitive impairment model has been successfully established. The above judgment method not only enhances the adaptability of experimental mice to periodic heat exposure and improves the experimental compliance of experimental mice, but also facilitates the accurate observation and collection of behavioral indicators. The method is simple and the results are reliable.

[0018] Furthermore, the stepped heating conditions are as follows: the temperature is gradually increased from an initial temperature of 25°C to 39°C at a rate of 0.67°C / min, and the relative humidity is 60%. The periodic heat exposure lasts for 14 consecutive days, with 4 hours of heat exposure per day.

[0019] Based on the aforementioned technical methods, the initial temperature was increased from room temperature (25℃) to 39℃ in a stepwise manner at a rate of 0.67℃ / min, with a relative humidity of 60%, and the heat exposure lasted for 4 hours. This approach closely approximates the high-temperature warning temperature for the human body, simulating the temperature that a helicopter cabin might reach in near-ground high-temperature weather with poor heat dissipation. This method more closely reflects the actual thermal environment and more realistically reflects the heat stress state of flight personnel in the actual cabin environment. Furthermore, the stepwise temperature increase condition with 4 hours of heat exposure per day for 14 consecutive days better matches the periodic high-temperature exposure faced by flight personnel in actual flight operations. This allows for a more accurate simulation of flight personnel's work patterns, enabling precise research into the damage to their learning and cognition after long-term exposure to high-temperature environments. This provides a scientific basis for the protection of flight personnel in high-temperature cabin environments, improving flight safety and operational capabilities.

[0020] Furthermore, the daily heat exposure time is any consecutive 4 hours between 8:00 and 14:00.

[0021] Based on the aforementioned technical methods, heat exposure was conducted between 8:00 and 14:00. This allows different mice to receive periodic heat exposure within the same circadian rhythm cycle, reducing systematic errors among the tested indicators of different mice. At the same time, this period coincides with the regular flight operation time of pilots and is the main period of cabin temperature rise during the high-temperature season, which is more in line with the actual flight environment and ensures the authenticity and reliability of the model.

[0022] Application of a learning and cognitive impairment model obtained according to the above-described method in the preparation of drugs for the prevention or treatment of thermal injury.

[0023] A method for screening drugs for the prevention or treatment of heat injury includes the step of using the drug to be screened in a learning-cognitive injury model obtained according to the above-described method.

[0024] An experimental system for the above-described establishment method includes:

[0025] An exposure chamber is used to control temperature and humidity to simulate a near-body temperature cabin environment;

[0026] A temperature measuring device used to monitor the rectal temperature of mice;

[0027] Behavioral testing equipment used to test anxiety behavior, spatial memory ability, and non-spatial memory ability in mice;

[0028] A molecular detection device was developed to analyze serum lactate concentration, mitochondrial damage molecules, and inflammatory factors in mouse tissue.

[0029] Based on the above technical means, the exposure chamber simulates the environment of a helicopter cockpit and precisely controls the temperature and humidity inside the exposure chamber to restore the complex thermal environment of pilots during flight operations. This provides mice with a stable thermal exposure environment that meets the experimental requirements, ensuring the reliability and repeatability of the experimental results. It can more realistically reflect the thermal stress state of flight personnel in the actual cockpit environment, making the experimental results more valuable for practical reference.

[0030] By monitoring the rectal temperature of mice in real time using a temperature measuring device, changes in rectal temperature can directly reflect the mice's thermoregulation and the degree of heat stress, providing important data for assessing the physiological stress response of mice in high-temperature environments.

[0031] The behavioral testing equipment accurately tests the anxiety behavior, spatial memory ability and non-spatial memory ability of mice, which directly reflects the changes in the learning and cognitive functions of mice and helps to further study the mechanism of the influence of high temperature environment on learning and cognition.

[0032] By analyzing the concentration of lactate in the plasma and hippocampus of mice, as well as the expression levels of mitochondrial damage molecules (ATP5H) and inflammatory factors (TNF-α and IL-6) in the hippocampus, molecular detection devices were used to reveal the mechanism of learning and cognitive impairment caused by high temperature environment at the molecular level. Combined with the rectal temperature of mice, the physiological state of mice under high temperature environment was more comprehensively understood. This provides key data for a deeper understanding of the damage of high temperature environment to the nervous system and helps to discover potential therapeutic targets and protective measures.

[0033] In summary, by integrating the exposure chamber, temperature measurement device, behavioral testing equipment, and molecular detection device into one system, the experimental process is automated and integrated, which greatly saves experimental time and labor costs and improves research efficiency.

[0034] Furthermore, the exposure chamber is connected to a temperature regulation feedback device and a humidity regulation feedback device for regulating the temperature and humidity inside the exposure chamber.

[0035] Based on the aforementioned technical means, this invention can monitor the temperature and humidity inside the exposure chamber in real time through temperature and humidity regulation feedback devices. It can also precisely adjust the temperature and humidity according to preset stepped heating conditions (from an initial temperature of 25°C to 39°C in steps, with a heating rate of 0.67°C / min, relative humidity of 60%, 4 hours of heat exposure per day for 14 consecutive days). If the temperature or humidity inside the exposure chamber deviates from the set value, the feedback device will immediately activate the adjustment mechanism to ensure that the temperature and humidity remain within the target range. This more accurately simulates the near-body temperature cabin environment, making the experimental results closer to reality and ensuring the reliability and accuracy of the experiment.

[0036] Furthermore, the behavioral testing equipment includes an open field test chamber, a Y-maze device, and a novel object recognition test device. The open field test chamber is used to test the anxiety behavior of mice, the Y-maze device is used to test the spatial memory ability of mice, and the novel object recognition test device is used to test the non-spatial memory ability of mice.

[0037] Based on the aforementioned technologies, the open field test chamber effectively assesses mice's anxiety behavior by recording their activities in an open environment, such as the time spent in the central area, the distance traveled, and the speed. Anxious mice typically tend to stay at the edges of the open field and are less likely to enter the central area. The Y-maze device utilizes mice's natural tendency to explore new environments, accurately testing their spatial memory ability by recording the number of times and the time taken to enter each arm of the maze. The new object recognition test device assesses mice's non-spatial memory ability by measuring the difference in exploration time between new and old objects. These three devices evaluate mouse behavior from different perspectives, comprehensively covering multiple aspects such as anxiety behavior, spatial memory ability, and non-spatial memory ability, providing rich behavioral data for in-depth research on learning and cognitive impairment. Furthermore, they can acquire mouse behavioral data in a relatively short time, improving experimental efficiency, reliability, and effectiveness.

[0038] The beneficial effects achieved by this invention are as follows:

[0039] 1. This invention exposes mice to heat in a stepped heating environment with high frequency, which can more realistically simulate the gradual increase and periodic changes in temperature in a helicopter cabin environment. Compared with traditional heat exposure models, it is closer to the thermal environment faced by pilots in actual operations, and helps to study the influence mechanism of near-body temperature cabin environment on pilots' learning and cognition.

[0040] 2. This invention comprehensively assesses the state changes of mice during heat exposure from multiple levels, including physiological, behavioral, and tissue aspects, by continuously monitoring multiple indicators such as rectal temperature, plasma and hippocampal lactate concentration, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in hippocampal tissue. Among these, rectal temperature and lactate concentration can reflect the degree of physiological stress response in mice; behavioral changes can directly reflect whether the learning and cognitive functions of mice are affected; and the expression levels of mitochondrial damage molecules and inflammatory factors reveal the possible mechanisms of learning and cognitive impairment from a neurobiological perspective, providing rich data support for the comprehensive assessment of learning and cognitive impairment.

[0041] 3. The present invention establishes a rigorous and comprehensive set of criteria for judging the success of the learning and cognitive impairment model. These criteria require that the mice's rectal temperature and lactate concentration rise to a preset range, exhibit changes in anxiety behavior, spatial memory ability, and non-spatial memory ability, and change in the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampus. All four criteria must be present simultaneously, ensuring that the established learning and cognitive impairment model has a high degree of consistency and accuracy. This avoids the problem of unreliability of the model due to ambiguity in the criteria and provides reliable experimental subjects for subsequent research.

[0042] 4. The successful establishment of the learning and cognitive impairment model in this invention helps to gain a deeper understanding of the impact mechanism of high temperature environment on flight personnel's learning and cognition, and provides a scientific basis for the construction and improvement of flight safety protection measures and early warning system in high temperature environment. Through the research on the learning and cognitive impairment model, effective protective measures can be explored in a targeted manner to reduce the risk of learning and cognitive impairment of flight personnel in high temperature cockpit environment and improve flight safety and operational capabilities. Attached Figure Description

[0043] Figure 1 This is a comparison of rectal temperatures on days 2-5 of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group subjected to periodic exposure to a near-body temperature cabin environment in this invention.

[0044] Figure 2 This is a comparison of rectal temperatures on days 6-9 of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group subjected to periodic exposure to a near-body temperature cabin environment in this invention.

[0045] Figure 3 This is a comparison of rectal temperatures on days 10-14 of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to a near-body temperature cabin environment in this invention.

[0046] Figure 4 This is a comparison of rectal temperature changes over exposure time in mice subjected to periodic exposure to a near-body temperature cabin environment in this invention, specifically in the normal group, the heat exposure group, and the heat exposure + lactate inhibition group.

[0047] Figure 5 The graph shows the body weight and food intake of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to a near-body temperature cabin environment in this invention.

[0048] Figure 6 The graph shows the plasma lactate and hippocampal lactate concentrations of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group subjected to periodic exposure to near-body temperature cabin environment in this invention.

[0049] Figure 7This diagram illustrates the anxiety behavior of mice in the open field experiment used in this invention to assess the anxiety behavior of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group under periodic exposure to a near-body temperature cabin environment.

[0050] Figure 8 This invention provides Y-maze test diagrams to evaluate the spatial memory and exploratory behavior of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to a near-body temperature cabin environment in this invention.

[0051] Figure 9 This diagram illustrates the non-spatial learning and memory abilities of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to a near-body temperature cabin environment in this invention, used to evaluate the new object recognition experiment of mice in the following groups:

[0052] Figure 10 The image shows the detection of ATP5H and TNF-α and IL-6 protein molecules in the hippocampal tissues of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group subjected to periodic exposure to near-body temperature cabin environment in this invention.

[0053] Figure 11 These are hematoxylin-eosin staining images of hippocampal tissues from mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to near-body temperature cabin environment in this invention.

[0054] Figure 12 These are Nissl staining images of hippocampal tissue from mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to near-body temperature cabin environment in this invention.

[0055] Figure 13 Golgi staining images of hippocampal tissues of mice in the normal group, heat-exposed group, and heat-exposed + lactate-inhibited group subjected to periodic exposure to near-body temperature cabin environment in this invention. Detailed Implementation

[0056] To better understand the technical solution, achieve the objectives, and beneficial effects of this invention, the following embodiments further illustrate the invention in detail. However, these embodiments should not be construed as limiting the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are also considered to fall within the scope of protection of this invention. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the conditions recommended by the reagent manufacturer. Unless otherwise specified, the reagents and equipment used in this invention are conventional reagents and equipment in this technical field.

[0057] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0058] In this application Figures 5-13 The values ​​involved are *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns: P>0.05.

[0059] Example 1: Establishment of a model of learning and cognitive impairment caused by periodic exposure to near-body temperature cabin environment

[0060] 1. Animal selection and preparation

[0061] 1) Select healthy adult male C57 / BL6 mice that are 6-8 weeks old and weigh 20-24g as the research model;

[0062] 2) Allow the healthy C57 / BL6 male mice to drink water, eat, and sleep normally, and set them aside for later use.

[0063] 2. Model Establishment

[0064] The healthy male C57 / BL6 mice were randomly divided into a normal group and a heat exposure group, with 18 mice in each group. Each group was placed in an exposure chamber, and the temperature and humidity in the exposure chamber were adjusted using temperature and humidity feedback devices, respectively. The mice were then treated as follows:

[0065] Normal group: As a blank control, each mouse was exposed to heat for 4 hours at random times between 8:00 and 14:00 every day for 14 consecutive days in an environment with a constant temperature of 25℃ and a relative humidity of 60%.

[0066] Heat exposure group: Each mouse was subjected to a stepwise temperature increase from an initial temperature of 25℃ to 39℃ at a rate of 0.67℃ / min, and a relative humidity of 60% for 4 consecutive hours between 8:00 and 14:00 each day for 14 consecutive days.

[0067] 3. Indicator Monitoring

[0068] 1) Rectal temperature monitoring in mice

[0069] 1.1 The temperature measuring device used is a high-precision electronic thermometer (±0.1℃ error range), with a probe diameter ≤2mm and a length of 15-20mm (ISO 10993 certified), and is equipped with a medical sterile water-based lubricant;

[0070] 1.2 Before monitoring, the ambient temperature was kept stable at 22±1℃, the relative humidity was maintained at 45-55%, and the pre-acclimatization time was ≥30 minutes (the metabolic stabilization period of mice);

[0071] 1.3 The mice were fixed in a 45° supine position using a special fixator. The probe was inserted into the anus to a depth of 2-2.5 cm (standard for adult ICR mice). The measurement time was ≥10 seconds while maintaining the probe stable. The anal temperature changes of each group of mice were monitored and recorded.

[0072] 2) Monitoring of lactate concentration in mice

[0073] Experiments were conducted on a normal group and a heat-exposed group. During the modeling of continuous periodic heat exposure, plasma and hippocampal lactate levels in mice of each group were monitored on days 7 and 14 using a molecular detection device, and changes in plasma and hippocampal lactate levels in each group were recorded.

[0074] 3) Observation of behavioral changes in mice

[0075] 3.1 Assessing anxiety behavior in mice using the open field test.

[0076] Each group of mice was placed in the center of an open field with their backs to the experimenter. The experimenter quickly left, allowing the mice to move freely in the open field test chamber. The video capture software or video recording system was quickly activated to record the activity of each group of mice in the open field for 5-10 minutes (the software can also be activated in advance to capture data, and the preparatory time can be deducted during the software analysis). After the monitoring time was over, the mice were removed and returned to their cages. The open field test chamber was thoroughly cleaned with 75% alcohol or laboratory disinfectant to prevent residual information from the mice (such as feces and odor) from affecting the results of the next test. After drying, the mice were replaced and the above experiment was repeated, recording the anxiety level of each group of mice.

[0077] 3.2 The spatial memory and exploratory behavior of mice were assessed using the Y maze test.

[0078] Each group of mice was placed in the laboratory to acclimatize to the environment for at least one day to reduce environmental stress. Then, on days 7 and 14 of continuous periodic heat exposure, the mice were placed in the central area of ​​the Y maze device (where the three arms meet), facing one of the arms, and allowed to freely explore the maze for 5-10 minutes. The number of times each group of mice entered each arm and the sequence of entering different arms were recorded.

[0079] 3.3 Novel Object Recognition Test (NORT) to assess mice's non-spatial learning and memory abilities

[0080] Before the experiment, mice in each group were petted daily to eliminate unfamiliarity and avoid stimulating them during the procedure. The experiment was then conducted in three phases on days 7 and 14 of continuous periodic heat exposure:

[0081] The first stage is the adaptation period: each mouse is placed in the new object recognition experimental device (without objects) and allowed to move freely for 10 minutes;

[0082] The second stage is the familiarization period: Two identical objects (A and B, ensuring that the objects are odorless and fixed and cannot be moved) are placed in the new object recognition experimental device. The objects are about 10cm away from the two side walls (to give the experimental animals space to explore). The mouse is placed in the device from an equal distance with its back to the object. The exploration time of the mouse on each object is recorded by the camera equipment and software (the exploration of the object is defined as the mouse's mouth or nose touching the object or getting close to the object within about 2-3cm). The number of times, time and distance of the mouse exploring each object are measured and recorded within 5 minutes.

[0083] The third stage is the testing period: 1 hour after the completion of the second stage is selected as the time interval for testing memory. One of the two identical objects is replaced with a different object and placed in the device (AC or BC). Similarly, the mouse is placed in the device with its back to the object from an equal distance from the object for 5 minutes and the mouse's memory ability is recorded.

[0084] To quantify memory ability, the discrimination index is calculated as follows: (exploration time of old objects - exploration time of new objects / [exploration time of old objects]) × 100%; the recognition index is calculated as follows: (exploration time of new objects / [exploration time of new objects + exploration time of old objects]) × 100%.

[0085] Three objects were used in the experiment: A and B were exactly the same, and C was significantly different from A and B. The diameter of the objects in the mouse experiment was generally 3 cm.

[0086] 4) Analysis of cognitive impairment mechanisms in mice

[0087] 4.1 Detection of ATP5H, TNF-α, and IL-6 proteins in mouse hippocampus

[0088] During the modeling of continuous periodic heat exposure, mice in each group were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) on ​​days 7 and 14. After craniotomy, hippocampal tissue was dissected, total hippocampal protein was extracted and its concentration was quantitatively determined. The expression levels of ATP5H, TNF-α and IL-6 proteins were detected by Western blotting.

[0089] 4.2 Staining analysis of mouse hippocampal tissue

[0090] Hematoxylin-eosin (HE) staining: In the assessment of tissue damage after heat exposure, this embodiment uses HE staining to stain the hippocampal tissues of each group to compare the basic morphological characteristics of the hippocampal tissues of each group. Through the changes in the cell nucleus and cytoplasm, it provides an intuitive basis for assessing the degree of cell damage caused by heat exposure. Specifically, HE staining makes the cell nucleus appear blue or purple, while eosin staining makes the cytoplasm and extracellular matrix appear red.

[0091] Nissl staining: Nissl can clearly show Nissl bodies in hippocampal neurons, which are widely distributed in various neurons. In this embodiment, Nissl staining was used to stain each group of hippocampal tissues to observe the shape, size and number of Nissl bodies. These characteristics are closely related to the functional state of neurons.

[0092] Golgi staining: Golgi staining is used to assess hippocampal tissue damage in mice after heat exposure. In this embodiment, Golgi staining was used to stain the hippocampal tissue of each group, which can clearly show the dendrites of hippocampal neurons, including dendritic spines, to reveal the subtle morphological changes of hippocampal neurons caused by heat exposure.

[0093] 4. Criteria for judging successful model construction

[0094] ① The rectal temperature of mice increased to 38-42℃; ② The plasma lactate concentration increased by 5.56%-65.57%, and the hippocampal lactate concentration increased by 10.7%-55.2%; ③ The mice exhibited a state of anxiety, with reduced spontaneous exploration ability, specifically manifested as a decrease in the distance traveled and the time spent in the central region during open field behavioral assessment; spatial and non-spatial memory abilities were impaired, specifically manifested as a decrease in the discrimination index and recognition index; ④ The expression level of mitochondrial damage molecule ATP5H protein increased by 13.86%-56.38% in hippocampal tissue; the protein expression level of inflammatory factor TNF-α increased by 22.43%-212.80%, and the protein expression level of IL-6 increased by 13.58%-234.80%;

[0095] All four criteria must be met simultaneously for the learning and cognitive impairment model to be considered successfully established.

[0096] 5. Experimental Results

[0097] 1) Monitoring rectal temperature in mice, such as Figures 1-4 As shown:

[0098] Normal group: As shown in Table 1, the average rectal temperature of mice in the normal group after 4 hours of heat exposure for 14 consecutive days was 37.39℃.

[0099] Heat exposure group: As shown in Table 2, the average rectal temperature of mice in the heat exposure group after 4 hours of heat exposure for 14 consecutive days was 39.28℃, which met the judgment criterion ①.

[0100] Table 1. Results of rectal temperature monitoring in normal mice.

[0101] Exposure time 0h 1h 2h 3h 4h Mean / ℃ 37.73 37.43 37.20 37.28 37.39 Standard deviation 0.65 0.52 0.41 0.38 0.45

[0102] Table 2. Results of rectal temperature monitoring in the heat-exposed mice group.

[0103] Exposure time 0h 1h 2h 3h 4h Mean / ℃ 37.85 39.64 39.15 39.25 39.28 Standard deviation 0.68 0.64 0.90 0.60 0.52

[0104] 2) Monitoring lactate concentration in mice, such as Figure 6 As shown:

[0105] Normal group: As shown in Table 3, the concentrations of lactate in the plasma and hippocampus of mice in the normal group were at normal levels;

[0106] Heat exposure group: As shown in Table 4, on the 7th day of heat exposure, the plasma lactate concentration of mice in the heat exposure group increased by 65.57% compared with the normal group, and the hippocampal lactate concentration increased by 42.74% compared with the normal group; on the 14th day of heat exposure, the plasma lactate concentration of mice in the heat exposure group increased by 55.02% compared with the normal group, and the hippocampal lactate concentration increased by 12.73% compared with the normal group. The increase in plasma and hippocampal lactate concentrations monitored on the 7th and 14th days of heat exposure in the heat exposure group met the judgment criteria ②.

[0107] Table 3. Results of lactate concentration monitoring in normal mice.

[0108] organize Day 7 Day 14 Mean plasma concentration / mmol / L 6.10 5.18 Mean hippocampal concentration (mmol / g) 1.24 1.10

[0109] Table 4. Results of lactate concentration monitoring in the heat-exposed mice group.

[0110] organize Day 7 Day 14 Mean plasma concentration / mmol / L 10.10 8.03 Mean hippocampal concentration (mmol / g) 1.77 1.24

[0111] 3) Observation of behavioral changes in mice, such as... Figures 7-9 As shown:

[0112] Normal group: No abnormalities were found in the open field, Y-maze, and new object recognition experiments;

[0113] Heat exposure group: Abnormalities in open field, Y maze and new object recognition tests, specifically manifested as reduced movement distance and dwell time in the central area during open field behavioral assessment, weakened spontaneous exploration ability of mice, and anxiety state; during Y maze and new object recognition test assessment, the discrimination index and recognition index decreased, and spatial memory and non-spatial memory abilities were reduced, meeting the judgment criteria ③.

[0114] 4) Analysis of cognitive impairment mechanisms in mice, such as... Figure 10-13 As shown:

[0115] Normal group: As shown in Table 5, ATP5H, TNF-α and IL-6 proteins were normally expressed in the hippocampus of mice, and no abnormalities were observed in the staining of the hippocampus.

[0116] Heat exposure group: As shown in Table 6, on day 7, the protein expression levels of ATP5H, TNF-α, and IL-6 in the hippocampus of mice increased by 15.22%, 54.32%, and 49.72% respectively compared to the normal group; on day 14, the protein expression levels of ATP5H, TNF-α, and IL-6 in the hippocampus of mice increased by 27.47%, 63.42%, and 55.22% respectively compared to the normal group.

[0117] Furthermore, staining of the hippocampus tissue showed significant damage to neuronal structure and an abnormal number of Nissl bodies, with a decrease in the number of neuronal dendritic branches and dendritic spine density compared to the control group;

[0118] In summary, the changes in vital signs of mice in the heat exposure group showed good stability. Using this method to conduct periodic exposure experiments in near-body temperature cabin environments and construct a learning and cognitive impairment model can provide a scientific basis for the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments.

[0119] Table 5. Expression results of ATP5H, TNF-α, and IL-6 proteins in normal mice.

[0120] index Day 7 Day 14 ATP5H 1 1 TNF-α 1 1 IL-6 1 1

[0121] Table 6. Expression results of ATP5H, TNF-α, and IL-6 proteins in mice in the heat exposure group.

[0122] index Day 7 Day 14 ATP5H 1.15 1.27 TNF-α 1.54 1.63 IL-6 1.50 1.55

[0123] Example 2: Analysis of the effect of lactate level intervention on the improvement of a heat exposure model

[0124] 1. Animal selection and preparation

[0125] Same as Example 1.

[0126] 2. Model Establishment

[0127] The healthy male C57 / BL6 mice were randomly divided into a normal group and a heat exposure + lactate inhibition group, with 18 mice in each group. Each group was placed in an exposure chamber, and the temperature and humidity in the exposure chamber were adjusted using temperature and humidity feedback devices, respectively. The mice were then treated as follows:

[0128] Pretreatment: Mice in the heat exposure + lactate inhibition group were injected intraperitoneally with a dose of 300 mg / kg sodium oxalate saline solution 1 hour before heat exposure; at the same time, mice in the normal group were injected intraperitoneally with an equal volume of saline.

[0129] Normal group: As a blank control, mice after each treatment were kept in an environment with a constant temperature of 25°C and a relative humidity of 60% and were exposed to heat for 4 hours at random times between 8:00 and 14:00 every day for 14 consecutive days.

[0130] Heat exposure + lactate inhibition group: The treated mice were subjected to a stepwise increase in temperature from an initial temperature of 25°C to 39°C at a rate of 0.67°C / min, and a relative humidity of 60%. They were exposed to heat for 4 hours at random times between 8:00 and 14:00 every day for 14 consecutive days.

[0131] 3. Indicator Monitoring

[0132] Same as Example 1.

[0133] 4. Experimental Results

[0134] 1) Monitoring rectal temperature in mice, such as Figures 1-4 As shown:

[0135] Normal group: As shown in Table 7, the average rectal temperature of mice in the normal group after 4 hours of heat exposure for 14 consecutive days was 37.39℃.

[0136] Heat exposure + lactate inhibition group: As shown in Table 8, the average rectal temperature of mice in the heat exposure group after 4 hours of heat exposure for 14 consecutive days was 38.63℃.

[0137] Table 7. Results of rectal temperature monitoring in normal mice.

[0138] Exposure time 0h 1h 2h 3h 4h Mean / ℃ 37.73 37.43 37.20 37.28 37.39 Standard deviation 0.65 0.52 0.41 0.38 0.45

[0139] Table 8. Results of rectal temperature monitoring in mice in the heat exposure + lactate inhibition group.

[0140] Exposure time 0h 1h 2h 3h 4h Mean / ℃ 37.90 38.75 38.62 38.78 38.63 Standard deviation 0.71 0.97 0.75 0.80 0.86

[0141] 2) Monitoring lactate concentration in mice, such as Figure 6 As shown:

[0142] Normal group: As shown in Table 9, the lactate concentrations in the plasma and hippocampus of mice in the normal group were at normal levels;

[0143] Heat exposure + lactate inhibition group: As shown in Table 10, the plasma lactate concentration and hippocampal lactate concentration of mice in the heat exposure + lactate inhibition group were significantly lower than those in the heat exposure group in Example 1 on days 7 and 14, and the differences were significantly smaller compared with the normal group.

[0144] Table 9. Results of lactate concentration monitoring in normal mice.

[0145] organize Day 7 Day 14 Mean plasma concentration / mmol / L 6.10 5.18 Mean hippocampal concentration (mmol / g) 1.24 1.10

[0146] Table 10. Results of lactate concentration monitoring in mice in the heat exposure + lactate inhibition group.

[0147] organize Day 7 Day 14 Mean plasma concentration / mmol / L 8.67 7.47 Mean hippocampal concentration (mmol / g) 1.42 0.91

[0148] 3) Observation of behavioral changes in mice, such as... Figure 7-9 As shown:

[0149] Normal group: No abnormalities were found in the open field, Y-maze, and new object recognition experiments;

[0150] The heat exposure + lactate inhibition group showed significant improvement in open field, Y maze, and new object recognition tests, with improved spontaneous exploration ability and increased distance and dwell time in the central area compared to the heat exposure group during open field behavioral tests. Spatial and non-spatial memory abilities were also improved, with increased discrimination and recognition indices compared to the heat exposure group.

[0151] 4) Analysis of cognitive impairment mechanisms in mice

[0152] Normal group: As shown in Table 11, ATP5H, TNF-α and IL-6 proteins were normally expressed in the hippocampus of mice, and no abnormalities were observed in the staining of the hippocampus.

[0153] Heat exposure + lactate inhibition group: As shown in Table 12, on days 7 and 14, the normal expression of ATP5H, TNF-α, and IL-6 proteins in the hippocampus of mice was significantly inhibited. Specifically, on day 7, the protein expression level of ATP5H in the hippocampus of mice was reduced by 12.20% compared to the heat exposure group in Example 1, the protein expression level of TNF-α was reduced by 32.20% compared to the heat exposure group in Example 1, and the protein expression level of IL-6 was reduced by 38.32% compared to the heat exposure group in Example 1; on day 14, the normal expression level of ATP5H, TNF-α, and IL-6 proteins in the hippocampus of mice was reduced by 32.20% compared to the heat exposure group in Example 1; on day 14, the normal expression level of ATP5H, TNF-α, and IL-6 proteins in the hippocampus of mice was reduced by 32.20% compared to the heat exposure group in Example 1; on day 14, the normal expression level of TNF-α, TNF-α, and IL-6 proteins in the hippocampus of mice was reduced by 38.32% compared to the heat exposure group in Example 1; on day 14, the normal expression level of TNF-α, TNF-α, and IL-6 proteins in the hippocampus of mice was reduced by 32.20 ... At 14 days, the protein expression levels of ATP5H in the hippocampus of mice were reduced by 21.42% compared to the heat exposure group in Example 1, the protein expression levels of TNF-α were reduced by 33.52% compared to the heat exposure group in Example 1, and the protein expression levels of IL-6 were reduced by 29.42% compared to the heat exposure group in Example 1. Furthermore, staining of the hippocampus showed that neuronal structural damage and abnormal Nissl bodies were reduced compared to the heat exposure group in Example 1, while the number of dendritic branches and the density of dendritic spines were increased in the hippocampal neurons compared to the heat exposure group in Example 1.

[0154] In summary, sodium oxalate saline solution, as a lactate synthesis inhibitor, inhibits the conversion of pyruvate to lactate through lactate dehydrogenase. In the heat-exposed group, after the lactate synthesis inhibition measure, the concentration of lactate in the plasma and hippocampus of mice in this example was significantly reduced compared to the heat-exposed group in Example 1, and learning and cognitive impairment was significantly improved.

[0155] And by Figure 5 It was found that before the experiment, there were no significant differences in body weight and daily food intake among the normal group, the heat exposure group, and the heat exposure + lactate inhibition group. After 14 days of heat exposure, the normal group mice gained about 1.5g in weight, while the heat exposure group mice gained only about 0.5g, indicating that heat exposure significantly inhibited the weight gain of mice. In contrast, the heat exposure + lactate inhibition group mice gained about 1.2g in weight, which was significantly higher than the heat exposure group but lower than the normal group. The results show that lactate intervention can alleviate the effect of periodic near-body temperature heat exposure on the body weight of mice, further confirming the reliability of constructing a learning and cognitive impairment model, and providing a scientific basis for the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments.

[0156] Table 11. Expression results of ATP5H, TNF-α, and IL-6 proteins in normal mice.

[0157]

[0158]

[0159] Table 12. Expression results of ATP5H, TNF-α, and IL-6 proteins in mice in the heat exposure + lactate inhibition group.

[0160] Day 7 Day 14 ATP5H 1.01 1.00 TNF-α 1.04 1.08 IL-6 0.93 1.09

[0161] Example 3: Stability Experiment of Thermal Exposure Model

[0162] Eighteen healthy mice, identical to those in Example 1, were used to obtain a learning and cognitive impairment model again under the same conditions as the heat exposure group in Example 1. The indicators in Example 1 were used to determine whether the model was successfully obtained, in order to verify the stability of the method used in this invention.

[0163] The results showed that the rectal temperature of mice subjected to periodic near-body temperature heat exposure exhibited the same pattern of change during the 4-hour heat exposure period each day over 14 consecutive days; plasma and hippocampal lactate concentrations increased significantly; open field, Y-maze, and new object recognition test indicators were abnormal; and the normal expression of ATP5H, TNF-α, and IL-6 proteins in hippocampal tissue was increased, indicating that the method described in this invention can stably establish a learning and cognitive impairment model caused by periodic exposure to near-body temperature cabin environment.

[0164] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for establishing a model of learning and cognitive impairment caused by periodic exposure to near-body temperature cabin environments, characterized in that, Includes the following steps: Mice were subjected to periodic heat exposure in a stepped heating environment, and their condition was continuously monitored to obtain a learning and cognitive impairment model. The monitored indicators include the mouse's anal temperature, plasma and hippocampal lactate concentrations, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampus. The criteria for judging whether the learning and cognitive impairment model has been successfully established include: ① elevated anal temperature in mice; ② elevated lactate concentration in plasma and hippocampus; ③ behavioral changes in anxiety behavior, spatial memory ability, and non-spatial memory ability; ④ increased expression levels of mitochondrial damage molecules and inflammatory factors in hippocampus. All four criteria must be met simultaneously for the learning and cognitive impairment model to be successfully established. The stepped heating conditions involved gradually increasing the temperature from an initial temperature of 25℃ to 39℃ at a rate of 0.67℃ / min, with 4 hours of heat exposure per day.

2. The method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cabin environment according to claim 1, characterized in that, The mitochondrial damage molecules in the hippocampus of the mice included ATP5H; the inflammatory factors included TNF-α and IL-6.

3. The method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cabin environment according to claim 2, characterized in that, A mouse is considered to have successfully established a learning and cognitive impairment model when it meets all four of the following criteria. ① The rectal temperature of mice increased to 38-42 ℃; ② The plasma lactate concentration increased by 5.56% - 65.57%, and the hippocampal lactate concentration increased by 10.7% - 55.2%; ③ Behavioralally, mice showed reduced spontaneous exploration ability, anxiety, and decreased spatial and non-spatial memory; ④ The expression levels of mitochondrial damage molecules in the hippocampus increased by 13.86% - 56.38%; the expression levels of TNF-α increased by 22.43% - 212.80%, and the expression levels of IL-6 increased by 13.58% - 234.80%.

4. The method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cabin environment according to claim 1, characterized in that, The relative humidity of the stepped heating environment is 60%; the periodic heat exposure lasts for 14 consecutive days.

5. The method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cabin environment according to claim 4, characterized in that, Daily heat exposure time is any consecutive 4 hours between 8:00 and 14:

00.

6. The application of a learning and cognitive impairment model obtained by the method according to any one of claims 1-5 in the preparation of a medicament for the prevention or treatment of thermal injury.

7. A method for screening drugs for the prevention or treatment of heat injury, characterized in that, This includes the step of using the drug to be screened in the learning and cognitive impairment model obtained by the method described in any one of claims 1-5.

8. An experimental system for the establishment method according to any one of claims 1-5, characterized in that, include: An exposure chamber is used to control temperature and humidity to simulate a near-body temperature cabin environment; A temperature measuring device used to monitor the rectal temperature of mice; Behavioral testing equipment used to test anxiety behavior, spatial memory ability, and non-spatial memory ability in mice; A molecular detection device was developed to analyze serum lactate concentration, mitochondrial damage molecules, and inflammatory factors in mouse tissue.

9. The experimental system according to claim 8, characterized in that, The exposure chamber is connected to a temperature regulation feedback device and a humidity regulation feedback device, which are used to regulate the temperature and humidity inside the exposure chamber.

10. The experimental system according to claim 8, characterized in that, The behavioral testing equipment includes an open field test chamber, a Y-maze device, and a novel object recognition test device. The open field test chamber is used to test the anxiety behavior of mice, the Y-maze device is used to test the spatial memory ability of mice, and the novel object recognition test device is used to test the non-spatial memory ability of mice.

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