Establishment method of learning cognitive impairment model caused by periodic exposure of near-body-temperature cabin environment and application thereof

Abstract

The invention discloses an establishment method and application of a learning cognitive impairment model caused by periodic exposure of a near-body-temperature cabin environment, and the establishment method comprises the following steps: placing a mouse in a stepped heating environment for periodic thermal exposure; continuously monitoring the anus temperature, plasma and hippocampal tissue lactic acid concentration and behavioral change of the mouse and the expression of mitochondrial injury molecules and inflammatory factors in hippocampal tissue, and when the four standards exist at the same time, confirming that the learning cognitive impairment model is successfully established to obtain the learning cognitive impairment model; according to the model, the mouse is subjected to periodic thermal exposure in the stepped heating environment, the thermal exposure frequency is high, evaluation is comprehensive, the accuracy is high, and the characteristic that the temperature in the helicopter cabin environment rises gradually and changes periodically is simulated more truly; the method is closer to thermal environment conditions faced by flight personnel in a non-closed pressurized cabin and a low-altitude environment in actual operation, and is helpful for deeply researching an influence mechanism of a near-body-temperature cabin environment on learning and cognition of the flight personnel.

Description

Technical Field

[0001] The present invention relates to the technical field of establishing animal models, and particularly relates to a method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cockpit environment and its application. Background Art

[0002] With the continuous progress of technology, helicopters have been widely used in both military and civilian fields due to their unique flight capabilities and flexibility. However, there are many factors in the helicopter cockpit environment that are unfavorable for the operation of flight crew; the non-hermetic pressurized cockpit of a helicopter is small in space, the operating load of on-board equipment is large, and the heat sources in the cockpit are complex and diverse (including solar radiation, electronic equipment, cockpit greenhouse effect, and the metabolic heat production of flight crew themselves). In the absence of effective cockpit environment protection measures or a protection system to reduce the environmental heat load, flight operations in high-temperature weather have a serious impact on the physiology and psychology of flight crew. Especially during takeoff, ground taxiing, waiting for takeoff, and low-altitude flight, flight crew are exposed to high-temperature environments for a long time and frequently. Research shows that the ground temperature in a helicopter cockpit can reach or even exceed 56°C in summer at high temperatures; when the environmental temperature rises to 37°C, the operation error rate of flight crew increases significantly; when the environmental temperature rises to 38°C, problems such as incoordination in operation, reduced sensitivity, and decreased ability to identify targets will occur. The high-temperature environment not only causes disorders in the physiological functions of flight crew (such as reduced oxygen transport capacity of the circulatory system and imbalance of the internal environment homeostasis), but also has a profound impact on the central nervous system, manifested as depletion of attention resources, decreased execution ability of complex tasks caused by inhibition of dopamine secretion, and impairment of hippocampal function, which in turn leads to memory loss and decision-making ability disorders; with the continuous rise of the global average temperature and the increasing frequency and intensity of extreme weather events, the risk of flight crew being exposed to near-body temperature high-temperature environments continues to increase, and the exposure of flight crew to the operating environment is often periodic. The central fatigue caused by long-term and frequent exposure to high-temperature environments seriously affects flight safety and operating ability.

[0003] At present, it is difficult to conduct research on the quantitative evaluation and damage mechanism of helicopter pilots' learning and cognition after periodic exposure to a near-body-temperature cockpit environment in a real flight environment. This not only hinders in-depth understanding of the impact mechanism of high-temperature environment on pilots, but also impedes the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments. Although there are existing thermal exposure models in the prior art that use rodents to simulate cognitive damage caused by high-temperature environments, they still have deficiencies: there is a large difference between the environmental temperature settings of traditional thermal exposure models and the core body temperature of animals, making it difficult to reflect the operating conditions where there is no obvious heat exchange between the animal and the cockpit environment or the body's heat dissipation efficiency is significantly reduced; the frequency of thermal exposure is relatively short, and it is impossible to objectively reflect the weight of the animal's heat acclimation process in periodic thermal exposure; the damage evaluation indicators after periodic thermal exposure lack in-depth research on the molecular mechanisms of key brain regions for learning and cognition (such as the hippocampus), especially the lack of in-depth analysis of cellular level indicators such as mitochondrial function damage and inflammatory factor expression, as well as targeted protection measures. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the first object of the present invention is to provide a method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body-temperature cockpit environment, which solves the problems of large differences between environmental temperature settings and animal core body temperature, relatively short thermal exposure frequency, insufficient research on the molecular mechanisms of key brain regions for learning and cognition, and lack of in-depth analysis of targeted protection measures. A learning and cognitive impairment model has been successfully established, providing theoretical support for in-depth research on the damage of learning and cognition caused by periodic near-body-temperature environment exposure and the prevention of heat damage; the second object is to provide the application of a learning and cognitive impairment model caused by periodic exposure to a near-body-temperature cockpit environment.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body-temperature cockpit environment, comprising the following steps:

[0007] Place the mice in a stepped heating environment for periodic thermal exposure, and continuously monitor the state of the mice to obtain a learning and cognitive impairment model;

[0008] The monitored indicators include the rectal temperature of the mice, the lactate concentration in plasma and hippocampal tissue, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in hippocampal tissue;

[0009] The judgment criteria for whether the learning and cognitive impairment model is successfully established include: ① the rectal temperature of the mice increases; ② the lactate concentration in plasma and hippocampal tissue increases; ③ anxiety behavior, changes in spatial memory ability and non-spatial memory ability occur behaviorally; ④ the expression levels of mitochondrial damage molecules and inflammatory factors in hippocampal tissue increase;

[0010] The above four criteria must exist simultaneously to confirm the successful establishment of the learning and cognitive impairment model.

[0011] The present invention uses rodent mice as experimental animals to construct a learning and cognitive impairment model. The genes, physiological functions, biological processes, and metabolic characteristics of mice are similar to those of humans. When studying the effects of high-temperature environments on learning and cognition, the neural responses and cognitive changes in mice can reflect to a certain extent the situations that may occur in humans. Moreover, mice have the advantages of fast reproduction, easy feeding, small size, easy operation, and low cost.

[0012] According to the above technical means, during the experiment of the present invention, by placing mice in a stepped temperature-rising environment for periodic heat exposure, it is possible to more realistically simulate the characteristics of gradually increasing and periodically changing temperature in the helicopter cockpit environment. Compared with the traditional heat exposure model, it is closer to the thermal environment conditions faced by flight personnel during actual operations, which helps to deeply study the influence mechanism of near-body temperature cockpit environment on the learning and cognition of flight personnel. Moreover, the physiological indicators of mice are easy to monitor. After periodic heat exposure, the learning and cognitive ability of mice is comprehensively evaluated from multiple levels such as physiology, behavior, and tissue by monitoring multiple indicators such as the rectal temperature of mice, changes in serum lactate concentration, behavior changes, and hippocampal tissue changes. Among them, using the rectal temperature of mice as a body temperature monitoring point is closer to the core body temperature for rodents. And lactate is not only an intermediate product of cell metabolism but also an important signaling molecule mediating cell communication. The pathological accumulation of lactate in the brain may lead to a decline in cognitive function and the development of neurodegenerative diseases. Therefore, lactate can be used as an energy metabolism substrate for glycolysis in the central nervous system to meet the urgent need of the central nervous system for energy uptake. However, when the central nervous system is damaged after environmental exposure, the continuous accumulation of lactate as a signaling molecule can damage the cell energy metabolism organelle - mitochondria, resulting in abnormal structure and function of the nervous system. Thus, the changes in plasma and tissue levels of lactate concentration can be used as important candidate parameters for the body's metabolic function. The present invention reflects the degree of physiological stress response of mice by monitoring the rectal temperature and serum lactate concentration of mice; directly reflects whether the learning and cognitive function of mice is affected through behavior changes; reveals the possible mechanism of learning and cognitive impairment from the perspective of neurobiology through hippocampal tissue changes, providing rich data support for comprehensively evaluating learning and cognitive impairment; and the present invention establishes a successful judgment standard for the learning and cognitive impairment model through strict and comprehensive means, ensuring that the established learning and cognitive impairment model has high consistency and accuracy, avoiding the problem of unreliable models caused by vague standards, and providing a reliable experimental object for subsequent research; the successful establishment of the learning and cognitive impairment model helps to deeply understand the influence mechanism of high-temperature environment on the learning and cognition of flight personnel, providing a scientific basis for the construction and improvement of flight safety protection measures and early warning systems in high-temperature environments; through the research on the learning and cognitive impairment model, effective protection measures can be explored targeted to reduce the risk of learning and cognitive impairment of flight personnel in high-temperature cockpit environments and improve flight safety and operation ability.

[0013] Further, the mitochondrial damage molecules in the hippocampal tissue of the mice include ATP5H; the inflammatory factors include TNF-α and IL-6.

[0014] According to the above technical means, ATP5H is an important component of mitochondrial ATP synthase, and its changes can reflect the degree of mitochondrial function damage. TNF-α and IL-6 are classical inflammatory factors, and the changes in their expression levels reflect the inflammatory response in the hippocampal tissue under high-temperature environment. Excessive inflammatory response may lead to damage and death of nerve cells. Taking ATP5H as a molecular marker for mitochondrial damage in mouse hippocampal tissue, and TNF-α and IL-6 as inflammatory factors, it provides clear biomarkers for evaluating learning and cognitive impairment caused by high-temperature environment. By detecting the expression levels of these molecules, the present invention can accurately judge the degree of mitochondrial damage and inflammatory response in the hippocampal tissue of mice under high-temperature environment, so as to quantitatively evaluate learning and cognitive impairment, and more objectively and accurately judge whether the learning and cognitive impairment model is successfully established.

[0015] Furthermore, when a mouse meets the following four criteria simultaneously, it is judged that a learning and cognitive impairment model is successfully established;

[0016] ① The rectal temperature of the mouse rises to 38 - 42 °C; ② The plasma lactate concentration increases by 5.56% - 65.57%, and the lactate concentration in the hippocampal tissue increases by 10.7% - 55.2%; ③ Behaviorally, the spontaneous exploration ability of the mouse weakens, showing an anxious state, and the spatial memory ability and non-spatial memory ability decline; ④ The expression level of the mitochondrial damage molecule in the hippocampal tissue increases by 13.86% - 56.38%; the expression level of TNF-α increases by 22.43% - 212.80%, and the expression level of IL-6 increases by 13.58% - 234.80%.

[0017] According to the above technical means, when the monitoring indicators of the mouse meet the above four criteria simultaneously, the heat exposure ends, and it is judged that a learning and cognitive impairment model is successfully established. The above judgment method not only enhances the adaptability of experimental mice to periodic heat exposure, 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 stepwise temperature increase condition is to stepwise increase the temperature from the initial temperature of 25 °C to 39 °C, with a heating rate of 0.67 °C / min and a relative humidity of 60%; the time of the periodic heat exposure is 14 consecutive days, with 4 h of heat exposure per day.

[0019] According to the above technical means, the initial temperature is stepped up from room temperature of 25°C to 39°C at a rate of 0.67°C / min, with a relative humidity of 60%, and heat exposure for 4 h, approaching the human high-temperature warning temperature, simulating the temperature that the helicopter cockpit may reach in hot weather near the ground with poor heat dissipation, which is more in line with the actual thermal environment and can more realistically reflect the thermal stress state of flight personnel in the actual cockpit environment; and with the above stepped-up temperature conditions, heat exposure for 4 h every day and a heat exposure frequency of 14 consecutive days are more in line with the periodic high-temperature exposure situation faced by flight personnel in actual flight operations, can more accurately simulate the working mode of flight personnel, and precisely study the learning and cognitive damage after their long-term exposure to high-temperature environment, providing a scientific basis for the protection of flight personnel in high-temperature cockpit environment and improving flight safety and operation ability.

[0020] Further, the daily heat exposure time is any consecutive 4 h from 8:00 to 14:00.

[0021] According to the above technical means, heat exposure is selected within the time period from 8:00 to 14:00, which is convenient for different mice to receive periodic heat exposure in the same biological rhythm cycle, reducing the systematic error between the test indexes of different mice; at the same time, this stage is consistent with the regular flight operation time period of flight personnel, and is the main time period for the cockpit temperature to rise in the high-temperature season, which is more in line with the actual flight environment, ensuring the authenticity and reliability of the model.

[0022] Application of a learning and cognitive damage model obtained according to the above establishment method in the preparation of drugs for preventing or treating heat injury.

[0023] A method for screening drugs for preventing or treating heat injury, including the step of using the drug to be screened in the learning and cognitive damage model obtained according to the above establishment method.

[0024] An experimental system for the above establishment method, including:

[0025] An exposure chamber for controlling temperature and humidity to simulate a near-body-temperature cockpit environment;

[0026] A temperature measuring device for monitoring the rectal temperature of mice;

[0027] A behavioral test device for testing the anxiety behavior, spatial memory ability and non-spatial memory ability of mice;

[0028] A molecular detection device for analyzing the serum lactate concentration of mice, the expression levels of mitochondrial damage molecules and inflammatory factors in hippocampal tissue.

[0029] According to the above technical means, the exposure chamber is used to simulate the helicopter cockpit environment and, in combination with precise control of the temperature and humidity inside the exposure chamber, reproduce the complex thermal environment of the pilot during flight operations, providing a stable and experiment - requirement - compliant thermal exposure environment for the mice, ensuring the reliability and repeatability of the experimental results, being able to more realistically reflect the thermal stress state of flight personnel in the actual cockpit environment, and making the experimental results more valuable for practical reference;

[0030] The rectal temperature of the mice is monitored in real - time by a temperature - measuring device. The change in rectal temperature can intuitively reflect the thermoregulation situation and thermal stress level of the mice, providing important data for evaluating the physiological stress response of the mice in a high - temperature environment;

[0031] The anxiety behavior, spatial memory ability, and non - spatial memory ability of the mice are accurately tested by a behavioral testing device, which directly reflects the changes in the learning and cognitive functions of the mice and helps to deeply study the influence mechanism of the high - temperature environment on learning and cognition;

[0032] The lactic acid concentrations in the plasma and hippocampal tissues of the mice, and the expression levels of mitochondrial damage molecule (ATP5H) and inflammatory factors (TNF - α and IL - 6) in the hippocampal tissues are analyzed by a molecular detection device. It reveals the mechanism of learning and cognitive damage caused by the high - temperature environment at the molecular level, and comprehensively understands the physiological state of the mice in the high - temperature environment in combination with the rectal temperature of the mice, providing key data for deeply understanding the damage of the high - temperature environment to the nervous system and helping to discover potential therapeutic targets and protective measures;

[0033] In summary, by integrating the exposure chamber, temperature - measuring device, behavioral testing device, and molecular detection device into a system, the automation and integration of the experimental process are realized, greatly saving the experimental time and labor costs and improving the research efficiency.

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

[0035] According to the above technical means, the present invention can monitor the temperature and humidity inside the exposure chamber in real - time through the temperature - regulation feedback device and the humidity - regulation feedback device, and according to the preset stepped temperature - rising condition (stepped temperature - rising from the initial temperature of 25°C to 39°C, the temperature - rising speed is 0.67°C / min, the relative humidity is 60%, 4 hours of thermal exposure per day for 14 consecutive days), conduct precise regulation. Once the temperature or humidity inside the exposure chamber deviates from the set value, the feedback device will immediately start the regulation mechanism to ensure that the temperature and humidity are always kept within the target range, thus more accurately simulating the near - body - temperature cockpit environment, making the experimental results closer to the actual situation, and ensuring the reliability and accuracy of the experiment.

[0036] Furthermore, the ethology testing device 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. The novel object recognition test device is used to test the non-spatial memory ability of mice.

[0037] According to the above technical means, the open field test chamber effectively evaluates the anxiety behavior of mice by recording their activities in an open environment, such as the residence time in the central area, movement distance, and speed. Anxious mice usually tend to move more at the edge of the open field and enter the central area less frequently. The Y-maze device takes advantage of the exploratory nature of mice in a new environment and accurately tests the spatial memory ability of mice by recording the number of entries and time spent in each arm of the maze. The novel object recognition test device evaluates the non-spatial memory ability of mice based on the difference in exploration time between new and old objects. These three devices evaluate the behavior of mice from different perspectives, comprehensively covering multiple aspects such as anxiety behavior, spatial memory ability, and non-spatial memory ability, providing rich ethological data for in-depth research on learning and cognitive impairment. Moreover, they can obtain the behavior data of mice in a relatively short time, improving the efficiency, reliability, and effectiveness of the experiment.

[0038] Beneficial effects achieved by the present invention:

[0039] 1. By subjecting mice to periodic heat exposure in a stepwise temperature-rising environment, the present invention has a high frequency of heat exposure, which can more realistically simulate the characteristics of gradually increasing and periodically changing temperature in a helicopter cockpit environment. Compared with traditional heat exposure models, it is closer to the thermal environment faced by flight personnel during actual operations, helping to deeply study the influence mechanism of near-body temperature cockpit environment on the learning and cognition of flight personnel.

[0040] 2. By continuously monitoring multiple indicators such as the rectal temperature of mice, the lactate concentration in plasma and hippocampal tissue, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in hippocampal tissue, the present invention comprehensively evaluates the state changes of mice during heat exposure from multiple levels including physiology, behavior, and tissue. Among them, the rectal temperature and lactate concentration can reflect the degree of physiological stress response of mice. Behavioral changes can directly indicate whether the learning and cognitive functions of mice are affected. 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 comprehensively evaluating learning and cognitive impairment.

[0041] 3. In the present invention, by strictly and comprehensively establishing the judgment criteria for the successful establishment of the learning and cognitive impairment model, it is required that the rectal temperature and lactic acid concentration of the mice increase within a preset range, there are changes in anxiety behavior, spatial memory ability and non-spatial memory ability in terms of behavior, and the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampal tissue also change. And the above four criteria need to exist simultaneously, ensuring that the established learning and cognitive impairment model has a high degree of consistency and accuracy, avoiding the problem of unreliable models caused by vague criteria, and providing a reliable experimental object for subsequent research.

[0042] 4. The successful establishment of the learning and cognitive impairment model in the present invention helps to deeply understand the influence mechanism of the high-temperature environment on the learning and cognition of flight personnel, provides a scientific basis for the construction and improvement of the flight safety protection measures and early warning system in the high-temperature environment. Through the research on the learning and cognitive impairment model, effective protection measures can be explored specifically to reduce the risk of learning and cognitive impairment of flight personnel in the high-temperature cockpit environment and improve flight safety and operation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a comparison chart of the rectal temperatures of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically for the 2nd - 5th days in the present invention;

[0044] Figure 2 It is a comparison chart of the rectal temperatures of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically for the 6th - 9th days in the present invention;

[0045] Figure 3 It is a comparison chart of the rectal temperatures of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically for the 10th - 14th days in the present invention;

[0046] Figure 4 It is a comparison chart of the rectal temperatures of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically with the change of exposure time on a single day in the present invention;

[0047] Figure 5 It is a detection chart of the body weight and food intake of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically in the present invention;

[0048] Figure 6 It is a detection chart of the plasma lactic acid concentration and hippocampal lactic acid concentration of the normal group, heat exposure group and heat exposure + lactic acid inhibition group of mice exposed to the near-body temperature cockpit environment periodically in the present invention;

[0049] Figure 7Graph showing the assessment of anxiety behavior of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0050] Figure 8 Graph showing the assessment of spatial memory and exploratory behavior of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0051] Figure 9 Graph showing the assessment of non-spatial learning and memory ability of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0052] Figure 10 Graph showing the detection of mitochondrial damage molecular ATP5H, 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 of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0053] Figure 11 Graph showing hematoxylin-eosin staining of the hippocampal tissues of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0054] Figure 12 Graph showing Nissl staining of the hippocampal tissues of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention;

[0055] Figure 13 Graph showing Golgi staining of the hippocampal tissues of mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group of mice periodically exposed to a near-body temperature cockpit environment in the present invention. Detailed implementation manners

[0056] To better understand the technical solution, objectives, and beneficial effects of the present invention, the present invention is further specifically described through the following embodiments. However, it should not be construed as a limitation to the present invention. For some non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention content, they are also considered to fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the reagent manufacturers. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the technical field.

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

[0058] In the present application Figure 5 - Figure 13 involves *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns: P > 0.05.

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

[0060] 1. Animal selection and preparation

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

[0062] 2) Let the above-mentioned healthy C57 / BL6 male mice drink water, eat food and sleep normally, and set aside for use.

[0063] 2. Model establishment

[0064] Randomly divide the above-mentioned healthy C57 / BL6 male mice into equal amounts and randomly divide them into a normal group and a heat exposure group, with 18 mice in each group. Place each group in the exposure chamber, and use a temperature regulation feedback device and a humidity regulation feedback device to adjust the temperature and humidity in the exposure chamber respectively, and process them according to the following methods:

[0065] Normal group: As a blank control, each mouse is in an environment with a constant temperature of 25°C and a relative humidity of 60%, and is continuously heat-exposed for 4 hours at any time during the period from 8:00 to 14:00 every day for 14 consecutive days;

[0066] Heat exposure group: Each mouse is stepwise heated from an initial temperature of 25°C to 39°C at a heating rate of 0.67°C / min in an environment with a relative humidity of 60%, and is continuously heat-exposed for 4 hours at any time during the period from 8:00 to 14:00 every day for 14 consecutive days.

[0067] 3. Index monitoring

[0068] 1) Monitoring of the rectal temperature of mice

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

[0070] 1.2 Before monitoring, the environmental temperature is stabilized at 22 ± 1°C, the relative humidity is maintained at 45 - 55%, and the pre-adaptation duration ≥ 30 minutes (the metabolic stable period of mice);

[0071] 1.3 Use a special fixator to fix the body position (45° supine position), insert the probe into the anus of the mouse to a depth of 2 - 2.5 cm (standard for adult ICR mice), measurement duration: maintain the probe stable for ≥ 10 seconds, monitor and record the changes in the rectal temperature of each group of mice.

[0072] 2) Monitoring of the lactic acid concentration of mice

[0073] Experiments were conducted on the normal group and the heat exposure group. During the process of continuous periodic heat exposure modeling, a molecular detection device was used to monitor the lactic acid levels in the plasma and hippocampal tissues of mice in each group on the 7th and 14th days, and the changes in lactic acid in the plasma and hippocampal tissues of mice in each group were recorded.

[0074] 3) Observation of behavioral changes in mice

[0075] 3.1 Evaluation of mouse anxiety behavior through the Open field test

[0076] Each group of mice was placed with their backs to the experimenter in the central area of the open field. The experimenter quickly left, and the mice were allowed to move freely in the open field test box. The video acquisition software or the video recording system of the camera was quickly turned on to record the activities of each group of mice in the open field for 5 - 10 minutes (the software acquisition can also be turned on in advance, and then the mice are placed in. The preparation time in the early stage can be set to be deducted during the software analysis process); after the monitoring time ended, the mice were taken out and put back into the breeding cage. The open field box was thoroughly cleaned with 75% alcohol or experimental disinfectant water to prevent the information left by the current mice (such as animal urine, feces, and odor) from affecting the next test results. After waiting to dry, the mice were replaced and the above experiment was repeated, and the anxiety conditions of each group of mice were recorded.

[0077] 3.2 Evaluation of mouse spatial memory and exploratory behavior through the Y maze test

[0078] Each group of mice was placed in the laboratory to adapt to the environment for at least one day to reduce environmental stress; then on the 7th and 14th days 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 the mice were 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 Evaluation of mouse non - spatial learning and memory ability through the Novel Object Recognition Test (NORT)

[0080] Before the experiment, the unfamiliarity of each group of mice was eliminated by stroking them every day to prevent irritation to the mice during the operation; then on the 7th and 14th days of continuous periodic heat exposure, the experiment was carried out through the following 3 stages:

[0081] The first stage was the adaptation period: Each mouse was placed in the Novel Object Recognition Test device (without objects) and allowed to move freely for 10 minutes;

[0082] The second stage is the familiarity period: Place 2 identical objects (A and B, ensuring that the objects have no odor and are fixed and immovable) in the novel object recognition experimental device. The objects are approximately 10 cm away from the two side walls (giving the experimental animals exploration space). Place the mouse with its back facing the objects into the device at an equal distance from the objects, and use a camera device and software to record the exploration time of the mouse on each object (contacting the object with the mouth or nose and approaching the object within a range of about 2 - 3 cm are both considered exploration of the object). Measure and record the number of times, time, and distance of the mouse exploring each object within 5 minutes;

[0083] The third stage is the testing period: Select 1 hour after the completion of the second stage as the time interval for detecting memory. Replace one of the two identical objects with a different object (AC or BC) and place it in the device. Similarly, place the mouse with its back facing the object into the device at an equal distance from the object for 5 minutes and record the memory ability of the mouse;

[0084] Among them, to quantify the memory ability, calculate the discrimination index = (exploration time of the old object - exploration time of the new object / [exploration time of the old object]) × 100%; recognition index = (exploration time of the new object / [exploration time of the new object + exploration time of the old object]) × 100%;

[0085] Three objects are used in the experiment: A and B are completely identical, and C is significantly different from A and B. The diameter of the objects in the mouse experiment is generally 3 cm.

[0086] 4) Analysis of the cognitive impairment mechanism in mice

[0087] 4.1 Detection of ATP5H, TNF-α, and IL-6 protein molecules in the hippocampal tissue of mice

[0088] During the process of continuous periodic heat exposure modeling, take the mice in each group on the 7th and 14th days. After intraperitoneal injection anesthesia with sodium pentobarbital (40 mg / kg), open the skull to dissect the hippocampal tissue. After extracting the total hippocampal protein and quantitatively measuring the concentration, use Western blotting to detect the protein expression levels of ATP5H, TNF-α, and IL-6;

[0089] 4.2 Staining analysis of the hippocampal tissue of mice

[0090] Hematoxylin-eosin (HE) staining: In the assessment of tissue damage after heat exposure, in this example, HE staining is used to stain the hippocampal tissue of each group to compare the basic morphological characteristics of the hippocampal tissue of each group. Through the changes in the nucleus and cytoplasm, it provides an intuitive basis for evaluating the degree of cell damage caused by heat exposure. Among them, the hematoxylin staining in HE staining makes the nucleus appear blue or purple, and the 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 example, Nissl staining was used to stain the hippocampal tissues of each group to observe the shape, size, and quantity of Nissl bodies, and these characteristics are closely related to the functional state of neurons;

[0092] Golgi staining: Golgi staining was used to evaluate the damage of mouse hippocampal tissues after heat exposure. In this example, Golgi staining was used to stain the hippocampal tissues of each group, which can clearly show the dendrites of hippocampal neurons, including dendritic spines, to reveal the subtle morphological changes of nerve cells in the animal hippocampus caused by heat exposure.

[0093] 4. Criteria for judging the successful establishment of the model

[0094] ① The rectal temperature of the mice increased to 38 - 42°C; ② The lactic acid concentration in the plasma increased by 5.56% - 65.57%, and the lactic acid concentration in the hippocampal tissues increased by 10.7% - 55.2%; ③ Anxiety state appeared behaviorally, and the spontaneous exploration ability of the mice weakened, specifically manifested as a decrease in the moving distance and staying time in the central area during the open field behavioral assessment; the spatial memory ability and non-spatial memory ability decreased, specifically manifested as a decrease in the discrimination index and recognition index; ④ The expression level of the mitochondrial damage molecule ATP5H protein in the hippocampal tissues increased by 13.86% - 56.38%; the protein expression level of the 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 of the above four criteria need to be present simultaneously to confirm the successful establishment of the learning and cognitive impairment model.

[0096] 5. Experimental results

[0097] 1) Monitoring of the rectal temperature of the mice, as Figure 1 - Figure 4 shown:

[0098] Normal group: As shown in Table 1, the average rectal temperature of each mouse in the normal group after 4 hours of heat exposure for 14 consecutive days was 37.39°C;

[0099] Heat exposure group: As shown in Table 2, the average rectal temperature of each mouse in the heat exposure group after 4 hours of heat exposure for 14 consecutive days was 39.28°C, meeting the judgment criterion ①;

[0100] Table 1 Monitoring results of the rectal temperature of mice in the normal group

[0101] Exposure time 0h 1h 2h 3h 4h Mean / °C 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 Monitoring results of the rectal temperature of mice in the heat exposure group

[0103] Exposure time 0h 1h 2h 3h 4h Mean / °C 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 of lactic acid concentration in mice, as Figure 6 shown:

[0105] Normal group: As can be seen from Table 3, the lactic acid concentrations in the plasma and hippocampal tissues of mice in the normal group were at normal levels;

[0106] Heat exposure group: As can be seen from Table 4, on the 7th day of heat exposure, the lactic acid concentration in the plasma of mice in the heat exposure group increased by 65.57% compared with the normal group, and the lactic acid concentration in the hippocampal tissue increased by 42.74% compared with the normal group; on the 14th day of heat exposure, the lactic acid concentration in the plasma of mice in the heat exposure group increased by 55.02% compared with the normal group, and the lactic acid concentration in the hippocampal tissue increased by 12.73% compared with the normal group. The increase amplitudes of the lactic acid concentrations in the plasma and hippocampal tissues monitored on the 7th and 14th days of heat exposure in the heat exposure group all met the judgment criterion ②;

[0107] Table 3 Monitoring results of lactic acid concentration in mice in the normal group

[0108] Tissue Day 7 Day 14 Plasma mean / mmol / L 6.10 5.18 Hippocampus mean / mmol / g 1.24 1.10

[0109] Table 4 Monitoring results of lactic acid concentration in mice in the heat exposure group

[0110] Tissue Day 7 Day 14 Plasma mean / mmol / L 10.10 8.03 Hippocampus mean / mmol / g 1.77 1.24

[0111] 3) Observation of behavioral changes in mice, as Figure 7 - Figure 9 shown:

[0112] Normal group: There were no abnormalities in the open field, Y-maze, and novel object recognition tests;

[0113] Heat exposure group: Abnormalities were found in the open field, Y-maze, and novel object recognition tests. Specifically, during the open field behavioral assessment, the moving distance and staying time in the central area decreased, the spontaneous exploration ability of mice weakened, and an anxious state appeared; during the Y-maze and novel object recognition test assessments, the discrimination index and recognition index decreased, and the spatial memory ability and non-spatial memory ability declined, meeting the judgment criterion ③;

[0114] 4) Analysis of the cognitive impairment mechanism in mice, as Figure 10 - 13 shown:

[0115] Normal group: As can be seen from Table 5, the proteins of ATP5H, TNF-α, and IL-6 were normally expressed in the hippocampal tissues of mice, and no abnormalities were found in the staining of the hippocampal tissues;

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

[0117] Moreover, the staining of the hippocampal tissue showed obvious damage to the neuronal structure and abnormal number of Nissl bodies, and the number of dendritic branches and dendritic spine density of neurons were reduced compared with the control group;

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

[0119] Table 5 Protein expression results of ATP5H, TNF-α, and IL-6 in normal group mice

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

[0121] Table 6 Protein expression results of ATP5H, TNF-α, and IL-6 in heat exposure group mice

[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 improvement of the heat exposure model by intervening in lactate levels

[0124] 1. Animal selection and preparation

[0125] Same as Example 1.

[0126] 2. Model establishment

[0127] The above-mentioned healthy C57 / BL6 male mice were equally and 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 a temperature regulation feedback device and a humidity regulation feedback device were used to adjust the temperature and humidity in the exposure chamber respectively, and the following methods were used for treatment:

[0128] Pre-treatment: Each mouse in the heat exposure + lactate inhibition group was intraperitoneally injected with a sodium oxalate saline solution with a dose of 300 mg / kg 1 h before heat exposure; at the same time, each mouse in the normal group was intraperitoneally injected with an equal amount of normal saline;

[0129] Normal group: As a blank control, the mice after each treatment were exposed to heat continuously for 4 hours at any time during the period from 8:00 to 14:00 every day in an environment with a constant temperature of 25°C and a relative humidity of 60% for 14 consecutive days;

[0130] Heat exposure + lactate inhibition group: Each of the treated mice was heated stepwise to 39°C at an initial temperature of 25°C with a heating rate of 0.67°C / min and a relative humidity of 60%. They were exposed to heat continuously for 4 hours at any time during the period from 8:00 to 14:00 every day for 14 consecutive days.

[0131] 3. Index monitoring

[0132] Same as Example 1.

[0133] 4. Experimental results

[0134] 1) Monitoring of the rectal temperature of mice, as Figure 1 - Figure 4 shown:

[0135] Normal group: As shown in Table 7, the average rectal temperature of each mouse in the normal group was 37.39°C after 4 hours of heat exposure for 14 consecutive days;

[0136] Heat exposure + lactate inhibition group: As shown in Table 8, the average rectal temperature of each mouse in the heat exposure group was 38.63°C after 4 hours of heat exposure for 14 consecutive days;

[0137] Table 7 Monitoring results of rectal temperature of mice in the normal group

[0138] Exposure time 0h 1h 2h 3h 4h Mean / °C 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 Monitoring results of rectal temperature of mice in the heat exposure + lactate inhibition group

[0140] Exposure time 0h 1h 2h 3h 4h Mean / °C 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 of the lactate concentration of mice, as Figure 6 shown:

[0142] Normal group: As shown in Table 9, the lactate concentrations in the plasma and hippocampal tissues of the 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 tissue lactate concentration of the mice in the heat exposure + lactate inhibition group were significantly lower than those of the heat exposure group in Example 1 on the 7th and 14th days, and the differences compared with the normal group were significantly reduced;

[0144] Table 9 Monitoring results of lactate concentration of mice in the normal group

[0145] Tissue Day 7 Day 14 Plasma mean / mmol / L 6.10 5.18 Hippocampus mean / mmol / g 1.24 1.10

[0146] Monitoring results of lactic acid concentration in mice in the heat exposure + lactic acid inhibition group

[0147] Tissue Day 7 Day 14 Plasma mean / mmol / L 8.67 7.47 Hippocampus mean / mmol / g 1.42 0.91

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

[0149] Normal group: There were no abnormalities in the open field, Y-maze, and novel object recognition experiments;

[0150] Heat exposure + lactic acid inhibition group: The abnormalities in the open field, Y-maze, and novel object recognition experiments were significantly improved. The spontaneous exploration ability of the mice was improved, and the moving distance and staying time in the central area during the open field behavioral test were increased compared with those of the mice in the heat exposure group, with statistical differences. The spatial memory ability and non-spatial memory ability were improved, and the discrimination index and recognition index were increased compared with those of the mice in the heat exposure group;

[0151] 4) Analysis of the mechanism of cognitive impairment in mice

[0152] Normal group: As shown in Table 11, the proteins of ATP5H, TNF-α, and IL-6 were normally expressed in the hippocampal tissue of the mice, and there was no abnormality in the staining of the hippocampal tissue;

[0153] Heat exposure + lactic acid inhibition group: As shown in Table 12, on the 7th and 14th days, the normal expression increases of ATP5H, TNF-α, and IL-6 proteins in the hippocampal tissue of the mice were significantly inhibited. Specifically, on the 7th day, the protein expression level of ATP5H in the hippocampal tissue of the mice was reduced by 12.20% compared with that of the heat exposure group in Example 1, the protein expression level of TNF-α was reduced by 32.20% compared with that of the heat exposure group in Example 1, and the protein expression level of IL-6 was reduced by 38.32% compared with that of the heat exposure group in Example 1; on the 14th day, the protein expression level of ATP5H in the hippocampal tissue of the mice was reduced by 21.42% compared with that of the heat exposure group in Example 1, the protein expression level of TNF-α was reduced by 33.52% compared with that of the heat exposure group in Example 1, and the protein expression level of IL-6 was reduced by 29.42% compared with that of the heat exposure group in Example 1; and the staining of the hippocampal tissue showed that the neuronal structure damage and the abnormal number of Nissl bodies were reduced compared with those of the heat exposure group in Example 1, and the number of dendritic branches and dendritic spine density of the hippocampal neurons were increased compared with those of the heat exposure group in Example 1;

[0154] In summary, sodium oxalate saline solution, as a lactic acid synthesis inhibitor, inhibits the conversion of pyruvate to lactic acid through lactate dehydrogenase. After the lactic acid synthesis inhibition measure was adopted in the heat exposure group of mice, compared with the heat exposure group in Example 1, the lactic acid concentrations in the plasma and hippocampal tissue of the mice in the heat exposure + lactic acid inhibition group in this example were significantly reduced, and the learning and cognitive impairment were significantly improved;

[0155] And from Figure 5 it can be seen that before the experiment, there were no significant differences in the body weights and daily food intakes of the mice in the normal group, heat exposure group, and heat exposure + lactate inhibition group. After 14 days of heat exposure, the body weight of the mice in the normal group increased by about 1.5 g, while the body weight of the mice in the heat exposure group only increased by about 0.5 g, indicating that heat exposure significantly inhibited the body weight gain of the mice. In contrast, the body weight of the mice in the heat exposure + lactate inhibition group increased by about 1.2 g, which was significantly higher than that of the heat exposure group but lower than that of 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 the 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 Results of ATP5H, TNF-α, and IL-6 protein expressions in mice of the normal group

[0157]

[0158]

[0159] Table 12 Results of ATP5H, TNF-α, and IL-6 protein expressions in mice of 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 the heat exposure model

[0162] Take 18 healthy mice identical to those in Example 1, and under the conditions of the heat exposure group in Example 1, obtain the learning and cognitive impairment model again. Use the same indicators as in Example 1 to determine whether the model is successfully obtained, so as to verify the stability of the method adopted in the present invention.

[0163] The results show that: during the continuous 14-day exposure of the mice with periodic near-body temperature heat exposure, the rectal temperature of the mice showed the same change pattern within 4 hours of heat exposure every day; the plasma and hippocampal lactate concentrations increased significantly; the experimental indicators of the open field, Y maze, and novel object recognition were abnormal; the normal expressions of ATP5H, TNF-α, and IL-6 proteins in the hippocampal tissue increased, indicating that the method of establishing the model in the present invention can stably establish the learning and cognitive impairment model caused by periodic exposure to the near-body temperature cockpit environment.

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

Claims

1. A method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cockpit environment, characterized in that, It includes the following steps: Place the mice in a stepwise temperature-rising environment for periodic heat exposure, and continuously monitor the state of the mice to obtain a learning and cognitive impairment model. The monitored indicators include the rectal temperature of the mice, the lactate concentrations in plasma and hippocampal tissue, behavioral changes, and the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampal tissue. The criteria for judging whether the learning and cognitive impairment model is successfully established include: ① The rectal temperature of the mice increases; ② The lactate concentrations in plasma and hippocampal tissue increase; ③ Anxiety behaviors, changes in spatial memory ability and non-spatial memory ability occur behaviorally; ④ The expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampal tissue increase. All of the above four criteria need to exist simultaneously to confirm the successful establishment of the learning and cognitive impairment model.

2. The method for establishing a learning and cognitive impairment model caused by periodic exposure to a near-body temperature cockpit environment according to claim 1, characterized in that The mitochondrial damage molecules in the hippocampal tissue of the mice include ATP5H; the inflammatory factors include 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 cockpit environment according to claim 2, wherein, When the mice meet the following four criteria simultaneously, it is judged that the learning and cognitive impairment model is successfully established; ① The rectal temperature of the mice rises to 38-42 °C; ② The lactate concentration in plasma increases by 5.56%-65.57%, and the lactate concentration in hippocampal tissue increases by 10.7%-55.2%; ③ Behaviorally, the spontaneous exploration ability of the mice weakens, showing an anxious state, and the spatial memory ability and non-spatial memory ability decline; ④ The expression level of mitochondrial damage molecules in the hippocampal tissue increases by 13.86%-56.38%; the expression level of TNF-α increases by 22.43%-212.80%, and the expression level of IL-6 increases 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 cockpit environment according to claim 1, wherein, The stepwise temperature-rising condition is to stepwise rise from the initial temperature of 25 °C to 39 °C, with a temperature-rising speed of 0.67 °C / min and a relative humidity of 60%; the time of the periodic heat exposure is 14 consecutive days, with 4 h of heat exposure every day.

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

00.

6. Use of a learning and cognitive impairment model obtained by the establishment method according to any one of claims 1-5 in the preparation of a drug for preventing or treating heat injury.

7. A method for screening a drug for preventing or treating thermal injury, characterized in that, It includes the step of using the drug to be screened in a learning and cognitive impairment model obtained by the establishment method according to 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, It includes: An exposure chamber for controlling temperature and humidity to simulate a near-body temperature cockpit environment; A temperature measurement device for monitoring the rectal temperature of the mice; A behavioral test device for testing the anxiety behavior, spatial memory ability and non-spatial memory ability of the mice; A molecular detection device for analyzing the serum lactate concentration of the mice, the expression levels of mitochondrial damage molecules and inflammatory factors in the hippocampal tissue.

9. The experimental system according to claim 8, wherein The exposure chamber is connected with a temperature regulation feedback device and a humidity regulation feedback device for regulating the temperature and humidity in the exposure chamber.

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

Images