Mouse brain body disability model construction method based on sleep deprivation combined with treadmill experiment

Abstract

The invention relates to the technical field of brain body disability model construction, and discloses a mouse brain body disability model construction method based on sleep deprivation combined with a treadmill experiment. The method comprises the following steps: randomly grouping mice and installing sensors to acquire basic values; carrying out three-day treadmill adaptation training and recording parameters; an individualized modeling scheme is determined; modeling according to a periodic structure and adjusting the speed in real time; obtaining functional data through a behavioral test; and determining serum biochemical indexes to verify the brain body disability model. According to the method, individualized parameter setting and real-time monitoring and regulation can be realized, and meanwhile, the scientificity and reliability of the model are ensured through multi-dimensional verification.

Description

Technical Field

[0001] The present application relates to the technical field of constructing a brain-body disability model, and in particular to a method for constructing a mouse brain-body disability model based on a sleep deprivation combined with a treadmill experiment. Background Art

[0002] Brain-body disability is a state of brain-body fatigue and disability caused by excessive physical and mental work. With the development of the aging population, this state is becoming more and more common. Brain-body disability can lead to a decline in activity and behavioral ability and cognitive ability, reduced work efficiency, induce chronic diseases and cause health problems. Current studies have shown that the occurrence of brain-body disability is related to the interaction between physical and mental fatigue, but its inherent neurophysiological mechanism is still unclear. In the field of animal experiments, researchers have tried simple sleep deprivation models or simple exercise fatigue models, but these models cannot fully simulate the complex state of human brain-body disability.

[0003] There are obvious deficiencies in existing animal research on brain-body disability: first, there is a lack of standardized animal models specifically targeting the characteristics of brain-body disability, which makes it difficult to compare and repeat research results; second, existing methods mostly rely on the subjective experience of researchers to set training parameters and lack objective scientific basis, resulting in a high failure rate in modeling; third, the inability to monitor the physiological state of animals in real time can easily lead to excessive fatigue or even accidental death of mice during the experiment, which not only does not meet animal ethical requirements, but also reduces experimental efficiency; fourth, there is a lack of individualized regulatory mechanisms, and it is impossible to accurately model according to the specificity of each mouse; fifth, the verification method is single, and it is difficult to comprehensively evaluate the multi-dimensional characteristics of brain-body disability. Summary of the invention

[0004] The present application provides a method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiments, which is used to achieve individualized parameter setting and real-time monitoring and regulation, while ensuring the scientificity and reliability of the model through multi-dimensional verification.

[0005] In the first aspect, the present application provides a method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiments, and the method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiments comprises: dividing the mice into a control group and a model group by random grouping, and installing micro-sensors on the mice in the model group to collect the basal values ​​of heart rate, body temperature and activity; performing three days of treadmill adaptation training on all mice according to preset exercise parameters, and recording the exercise tolerance and physiological response values ​​of each mouse; determining an individualized exercise speed threshold and a heart rate safety upper limit for each model group mouse according to the adaptation training records, and generating an individual A personalized brain-body disability modeling scheme was developed; brain-body disability modeling was performed on the model group mice according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest, and the treadmill speed was adjusted every ten minutes based on the real-time heart rate data to form a brain-body disability model; the constructed brain-body disability model mice were subjected to rotarod, maze, open field and novel object recognition tests to obtain exhaustion time, spatial memory ability, autonomous activity and cognitive function data; blood samples were collected from mice to determine the levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase to complete the construction of the mouse brain-body disability model.

[0006] In the technical solution provided in the present application, mice were randomly divided into a control group and a model group and microsensors were installed, which ensured the scientific nature of the experimental design and the objectivity of data collection. The application of microsensors made it possible to monitor physiological parameters such as heart rate, body temperature and activity level in real time, providing a data basis for subsequent individualized modeling. Secondly, the exercise tolerance and physiological response values ​​of each mouse were recorded during the three-day treadmill adaptation training, which not only reduced the stress response of the mice to the unfamiliar environment, but also accumulated a large amount of individual data for later analysis. Thirdly, based on the adaptation training records, the individualized exercise speed threshold and heart rate safety upper limit were determined for each model group mouse, generating a personalized brain and body disability modeling solution. This individualized design significantly improved the success rate and safety of model construction. The modeling method is simple and easy to use, avoiding the modeling failure or accidental death of mice caused by the traditional "one-size-fits-all" method; further, the modeling is carried out according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest, and the treadmill speed is dynamically adjusted every ten minutes based on the real-time heart rate data, ensuring that the mice are always within a safe and effective load range during the modeling process, greatly improving the accuracy of the modeling; at the same time, through multi-dimensional behavioral tests such as rotating rods, mazes, open fields, and new object recognition, the movement ability and cognitive function of mice are comprehensively evaluated, and multiple index data such as exhaustion time, spatial memory ability, autonomous activity, and cognitive function are obtained; finally, through the analysis of blood samples to determine a variety of biochemical indicators, the effectiveness of the brain-body disability model is verified at the molecular level, providing an in-depth basis for mechanism research. The organic combination of the above technical features has constructed a brain-body disability model method that comprehensively considers individual differences, real-time monitoring and regulation, and multi-dimensional verification, which not only improves the scientificity and repeatability of the model, but also greatly enhances animal welfare, providing a reliable experimental platform for mechanism research, prevention and control strategy evaluation, and drug screening of brain-body disability. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0008] Figure 1 This is a schematic diagram of an embodiment of a method for constructing a mouse brain-body disability model based on a sleep deprivation combined with a treadmill experiment in an embodiment of the present application; Figure 2 This is a schematic diagram showing the comparison of exhaustion time in the rotating rod fatigue test in the embodiment of the present application; Figure 3 Schematic diagram of activity time comparison of the rotating rod fatigue test in the embodiment of the present application Figure 4This is a schematic diagram showing the motion trajectory comparison of the rotating rod fatigue test in the embodiment of the present application; Figure 5 This is a schematic diagram of the comparison of the discrimination index of the rotating rod fatigue test in the embodiment of the present application; Figure 6 This is a schematic diagram of the serum biochemical index detection data in the examples of this application. DETAILED DESCRIPTION

[0009] The embodiment of the present application provides a method for constructing a mouse brain-body disability model based on a sleep deprivation combined treadmill experiment. The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not have to be used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments described here can be implemented in an order other than that illustrated or described here. In addition, the terms "including" or "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0010] For ease of understanding, the specific process of the embodiment of the present application is described below. Figure 1 In the embodiments of the present application, one embodiment of the method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment includes: Step S101, randomly dividing the mice into a control group and a model group, and installing microsensors on the mice in the model group to collect heart rate, body temperature and basic activity values; Step S102, performing treadmill adaptation training on all mice for three days according to preset exercise parameters, and recording the exercise tolerance and physiological response value of each mouse; Step S103, determining an individualized exercise speed threshold and a heart rate safety upper limit for each model group mouse according to the adaptation training record, and generating a personalized brain and body disability modeling plan; Step S104, the model group mice were subjected to brain-body disability modeling according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest, and the treadmill speed was adjusted every ten minutes according to the real-time heart rate data to form a brain-body disability model; Step S105, performing rotarod, maze, open field and novel object recognition tests on the constructed brain-body disability model mice to obtain exhaustion time, spatial memory ability, locomotor activity and cognitive function data; Step S106, collecting blood samples from mice, measuring the levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase, and completing the construction of the mouse brain-body disability model.

[0011] It is understandable that the execution subject of the present application can be a system for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment, or a terminal or a server, which is not limited here. The present application embodiment is described by taking the server as the execution subject as an example.

[0012] Specifically, the experimental mice were divided into a control group and a model group according to the random number table method, with 6-20 mice in each group, to ensure the consistency of the two groups of mice in age, weight and basic status. Subsequently, a microsensor weighing no more than 1 gram was installed on the mice in the model group. The sensor was fixed to the back of the mouse through a special vest, which can monitor and record the heart rate (in the range of 300-800 times / minute), body temperature (in the range of 35-39°C) and triaxial acceleration data (reflecting activity) in real time. During the recording period, the mice were placed in a constant temperature environment (22±2°C), maintained in a 12-hour light / 12-hour dark photoperiod, and continuously monitored for 48 hours to obtain basic physiological parameters.

[0013] Both the control group and the model group mice were placed on a modified treadmill device for three days of adaptive training. The device separates the runway from the electric shock grid area by inserting a partition to prevent the mice from being stimulated by unnecessary electric shocks. The adaptive training adopts a progressive scheme: the treadmill speed is set to 10 meters / minute on the first day and lasts for 1200 seconds; the same speed is maintained on the second day and extended to 1800 seconds; the same speed is maintained on the third day and extended to 3600 seconds. The movement status of each mouse was recorded during the training process, including stable walking time, fatigue performance (such as staggering, number of prostrate times) and other indicators to quantify exercise tolerance. At the same time, micro-sensors are used to collect heart rate change curves, recovery time and body temperature fluctuations in real time to form a physiological response value data set.

[0014] According to the data collected during the adaptation training, a personalized modeling scheme was established for each model group mouse. The specific process is to extract the exercise tolerance change curve and heart rate fluctuation characteristic diagram from the adaptation training record, and calculate the slope of tolerance change during three days of training as the training adaptability index. When the exercise tolerance of mouse A on the third day was 35% higher than that on the first day, while mouse B only increased by 18%, the training adaptability index of mouse A was higher. According to this index, a relative tolerance coefficient was generated by comparing it with the average level within the group. At the same time, the heart rate basal level, exercise peak and recovery time were analyzed from the heart rate fluctuation characteristic diagram to determine the upper limit of heart rate safety, which is usually set to 95% of the peak heart rate. According to the relative tolerance coefficient, the standard low-speed exercise value (1 m / min) and medium-speed exercise value (5 m / min) were adjusted to generate an individualized exercise speed range, forming a complete personalized brain-body disability modeling scheme.

[0015] The model group mice were treated according to the cycle structure of "slow-speed exercise-rest-medium-speed exercise-rest". In the specific implementation, the treadmill was driven at an individualized low-speed exercise speed (usually in the range of 1-2 meters / minute) for 20 hours to cause continuous low-intensity fatigue; then the treadmill was stopped for 1 hour to allow the mice to rest for a limited time; then the treadmill was driven at an individualized medium-speed exercise speed (usually in the range of 4-6 meters / minute) for 2 hours to form acute medium-intensity fatigue superposition; finally, the treadmill was stopped again for 1 hour to complete a 24-hour cycle. The key technical point is to adjust the treadmill speed every 10 minutes according to the real-time heart rate data: when the heart rate is monitored to exceed the safety upper limit, the treadmill speed is automatically reduced by 10%; when the heart rate is 20% lower than the expected value, the speed is increased by 5% to ensure that the modeling intensity is both effective and safe. This cycle is carried out continuously for 7-10 days to form a stable brain-body disability model. The brain-body disability state is verified by four standardized behavioral tests. In the rotarod test, mice were placed on an accelerating rotarod (5-35 rpm, acceleration time 3 minutes), and the time point when the mice fell from the rotarod was recorded as the exhaustion time. In the Y-maze test, mice were allowed to explore freely in a three-arm maze for 8 minutes, and the proportion of alternating entries into different arms was calculated as an indicator of spatial memory ability. In the open field test, the total activity distance and the time spent in the central area of ​​the mice in a 50 cm square open field within 5 minutes were recorded to quantify the degree of autonomous activity. The new object recognition test was conducted in two stages: first, the mice were allowed to become familiar with two identical objects for 5 minutes, and one of them was replaced with a new object after resting for 1 hour. The difference in time for exploring the new and old objects was recorded, and the recognition index was calculated as cognitive function data. Blood samples were collected and serum was obtained after centrifugation. Seven biochemical indicators were measured using the corresponding detection methods: alkaline phosphatase and creatine kinase reflected muscle metabolism and liver function; glucose and urea indicated energy metabolism and protein decomposition; superoxide dismutase, malondialdehyde and glutathione peroxidase showed oxidative stress levels. For example, in one experiment, the average alkaline phosphatase activity of mice in the brain-body disability model group was 156.8±15.2U / L, significantly higher than 92.3±10.5U / L in the control group; while the superoxide dismutase activity was 102.5±9.8U / mg, significantly lower than 158.6±12.3U / mg in the control group, indicating that metabolic abnormalities and oxidative stress were aggravated under the brain-body disability state. Combining behavioral test data with biochemical index analysis results, it was confirmed that the brain-body disability model was successfully constructed, characterized by decreased motor ability, cognitive impairment, and metabolic disorders.

[0016] In the examples of the present application, mice were randomly divided into a control group and a model group and microsensors were installed to ensure the scientific nature of the experimental design and the objectivity of data collection. The application of microsensors made it possible to monitor physiological parameters such as heart rate, body temperature and activity in real time, providing a data basis for subsequent individualized modeling. Secondly, the exercise tolerance and physiological response values ​​of each mouse were recorded during the three-day treadmill adaptation training, which not only reduced the stress response of the mice to the unfamiliar environment, but also accumulated a large amount of individual data for later analysis. Thirdly, based on the adaptation training records, the individualized exercise speed threshold and heart rate safety upper limit were determined for each model group mouse, and a personalized brain and body disability modeling scheme was generated. This individualized design significantly improved the success rate and safety of model construction. The modeling failure or accidental death of mice caused by the traditional "one-size-fits-all" method was avoided; further, the modeling was carried out according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest, and the treadmill speed was dynamically adjusted every ten minutes based on the real-time heart rate data, ensuring that the mice were always in a safe and effective load range during the modeling process, greatly improving the accuracy of the modeling; at the same time, through multi-dimensional behavioral tests such as rotating rods, mazes, open fields, and new object recognition, the movement ability and cognitive function of mice were comprehensively evaluated, and multiple index data such as exhaustion time, spatial memory ability, autonomous activity, and cognitive function were obtained; finally, through blood sample analysis to determine a variety of biochemical indicators, the effectiveness of the brain-body disability model was verified at the molecular level, providing an in-depth basis for mechanism research. The organic combination of the above technical features has constructed a brain-body disability model method that comprehensively considers individual differences, real-time monitoring and regulation, and multi-dimensional verification, which not only improves the scientificity and repeatability of the model, but also greatly enhances animal welfare, providing a reliable experimental platform for mechanism research, prevention and control strategy evaluation, and drug screening of brain-body disability.

[0017] In a specific embodiment, the process of executing step S101 may specifically include the following steps: The male mice were randomly numbered and divided into a control group and a model group of equal size using a random number table method; The microsensor was fixed on the back of the mice in the model group through a preset vest; The mice were acclimated to the sensor weight in a constant temperature and light-controlled experimental environment; Using microsensors to continuously record the heart rate, body temperature, and activity level of mice at rest; According to the collected resting physiological parameters, the basic average value of physiological parameters of each mouse was calculated; The obtained heart rate basal values, body temperature basal values ​​and activity level basal values ​​were integrated into a basic data set of individualized modeling parameters.

[0018] Specifically, 12 8-week-old C57BL / 6 male mice with a weight range of 22±2g were selected. To ensure the randomness and reliability of the experiment, each mouse was assigned a unique number (M001-M012). These numbers were assigned to the control group and the model group by the random number table method. The specific operation was to use a random number generator to generate a random arrangement of 1-12, and the numbers corresponding to the first 6 digits were divided into the control group, and the numbers corresponding to the last 6 digits were divided into the model group, ensuring that the two groups were equal in number and there was no human intervention in the grouping process. The random number table method is a grouping method based on probability theory. It eliminates selection bias by introducing random factors and enhances the credibility of the experimental results. Installing microsensors on mice in the model group is a key link in constructing a brain-body disability model. The microsensor used in the present invention weighs only 0.8g, which is much lower than 5% of the mouse's body weight and will not cause a significant burden on the normal activities of the mouse. The sensor is fixed to the back of the mouse through a preset special vest. The vest is made of lightweight nylon material and has an elastic design to ensure that the sensor is stably fixed without affecting the freedom of movement of the mouse. The design of the vest takes into account the anatomical structure of mice. It is slightly narrow at the front end and slightly wider at the back end, which conforms to the body shape of mice and prevents them from slipping during treadmill exercise.

[0019] After installation, the mice were placed in a constant temperature and light-controlled experimental environment to adapt to the weight of the sensor. The constant temperature environment was controlled at 22±2℃, and the light cycle was 12 hours light / 12 hours dark, simulating the natural living environment of mice. The adaptation process usually takes 24 hours, and this time setting is based on the research data on the adaptability of mice to new objects. During the adaptation period, the mice's eating, drinking and general behavior were monitored to confirm that the sensor did not interfere with the mice's daily activities. After the adaptation period, the microsensor was used to continuously record the physiological parameters of the mice in a resting state. The sensor has a built-in heart rate detection module, a temperature sensor and a three-axis accelerometer. The sampling frequency is set to once every 10 seconds and the recording is continuous for 48 hours. Heart rate detection is based on electrocardiogram signal analysis, with a recording range of 300-800 times / minute, covering the normal heart rate fluctuation range of mice; the temperature sensor has a measurement range of 35-39℃ and an accuracy of ±0.1℃; the three-axis accelerometer detects the acceleration changes of the mouse in the three directions of X, Y and Z, with a sensitivity of ±2g, which is used to quantify the amount of activity. The data is transmitted wirelessly to the receiving device in real time, avoiding interference of wired connection on the mouse behavior.

[0020] The collected raw data are processed and converted into meaningful basic average values ​​of physiological parameters. The heart rate data processing first filters out abnormal values ​​to exclude abnormal fluctuations caused by sudden activity of mice or temporary poor contact of sensors, and then calculates the average heart rate in a resting state within 48 hours (the judgment standard is that the triaxial accelerometer data shows that the mice are in a relatively static state) as the heart rate basic value. The body temperature data is also calculated as the 48-hour average value as the body temperature basic value after the abnormal values ​​are eliminated. The activity calculation is more complicated. It is necessary to first vector synthesize the original data of the triaxial acceleration to obtain the synthetic acceleration, and then count the number of times the synthetic acceleration change rate exceeds the threshold per unit time, quantify it into the activity value, and finally calculate the 48-hour average value as the activity basic value. The three basic values ​​are integrated into a basic data set of individualized modeling parameters. The specific approach is to establish a data table containing mouse numbers, heart rate basic values, body temperature basic values, and activity basic values, and add statistical analysis data such as standard deviation and coefficient of variation to form a complete individualized parameter basic set. For example, the mouse numbered M003 had a 48-hour heart rate basal value of 485 beats / minute with a standard deviation of 28 beats / minute; a body temperature basal value of 37.2°C with a standard deviation of 0.3°C; and an activity basal value of 125 units / hour with a standard deviation of 35 units / hour. These data directly reflect the physiological baseline state of the mouse. Individual differences can be found by comparing with other mice in the group. For example, the heart rate basal value of M003 is 15 beats / minute higher than the group average, while the body temperature basal value is at the group average level. This individualized data set provides a scientific basis for the subsequent modeling parameter setting, allowing experimenters to adjust the specific parameters of sleep deprivation and treadmill experiments according to the individual characteristics of each mouse, thereby ensuring the scientificity and safety of the modeling process and improving the success rate of the construction of the brain-body disability model.

[0021] For example, in a specific embodiment of the present application, the brain-body fatigue modeling mice were grouped: the experimental mice were randomly divided into a control group (NC group, 6-20 mice) and a brain-body fatigue model group (SD group, 6-20 mice); on the basis of the existing instrument (small animal treadmill, model: ZS-PT-IV), each runway and electric shock grid area were separated by inserting a partition, and holes were punched on the cover to facilitate the mice to eat and drink water and drive the mice to exercise; Before formal modeling, both the control group and model group mice were placed on a treadmill for 3 days of adaptation. During the adaptation phase, the treadmill parameters were set to 10 m / min, with an acceleration time of 60 s, including 1200 s on the first day, 1800 s on the second day, and 3600 s on the third day. Parameter setting for the brain-body fatigue modeling phase of mice: During the modeling phase, the control group mice were placed in the modeling device and raised normally, and the treadmill parameters were not set to be in a static state; during the modeling phase of the model group mice, the treadmill parameters were set in sequence as follows: 1) 1 m / min, 72000 s; 2) 0 m / min, 3600 s; 3) 5 m / min, 7200 s; 4) 0 m / min, 3600 s.

[0022] The four parameters were used as a cycle, and the mice were trained for 7-10 days continuously. After observing the fatigue state of the mice, the control group and model group mice were subjected to behavioral experiments such as rotating rod, Y maze, open field and new object recognition to detect whether this modeling method has an effect on the motor behavior and cognitive ability of the mice; serum biochemical detection after brain and body fatigue modeling of mice: ALP (alkaline phosphatase), CK (creatine kinase), Glu (glucose), UREA (urea), T-SOD (total superoxide dismutase), MDA (malondialdehyde) and GPX (glutathione peroxidase) in the serum of the two groups of mice were detected to further verify the effect of this modeling method on the physiological indicators of mice.

[0023] Specifically, the results of the rotarod fatigue test showed that after 8 days of sleep deprivation combined with treadmill exercise, the exhaustion time of the SD group mice was significantly reduced compared with that of the NC group (p < 0.05). Figure 2 As shown in the Y maze test results, the activity of SD group mice in the maze was reduced compared with that of NC group (p < 0.05). Figure 3 Compared with the NC group, the movement trajectory of the SD group mice in the open field was significantly reduced (p < 0.05), and the movement distance (p < 0.05) and activity time (p < 0.05) in the central area were significantly reduced, see Figure 4 In the new object recognition experiment, compared with the NC group, the discrimination index of the SD group mice in terms of distance (p < 0.05) and time (p < 0.05) of contacting the new object showed a significant decrease, see Figure 5 The above results show that after sleep deprivation combined with treadmill modeling, the motor and cognitive performance of mice were reduced, and the brain-body disability model of mice was successfully established.

[0024] Behavioral specific operations: (1) Rotarod test: The test was conducted using a rotarod fatigue instrument, with the following conditions: rotation speed 5-35 r / min, acceleration time 3 min. When the mouse is exhausted, it cannot keep up with the speed of the rotarod and falls off the rotarod. The body of the mouse touches the bottom partition, and the instrument automatically records the exhaustion time. Each test lasts 10 min. The experiment is repeated 3 times, with an interval of 10 min between each experiment. The average value is taken as the rotarod exhaustion time of the mouse. If the mouse does not fall off the rod within 10 min, the exhaustion time is recorded as 10 min.

[0025] (2) Y-maze: The test mice were placed in a Y-shaped maze device with a dark inner wall and a three-pronged channel. They were allowed to explore freely under the detection of the animal behavior monitoring system. The software recorded data such as their walking route, speed, total distance, and number of arm entries. At the beginning of the experiment, the test mice were placed at the end of one arm of the Y-maze and explored for 8 minutes. After each mouse was tested, the feces and urine in the maze were cleaned and wiped with 75% ethanol to eliminate the influence of mouse odor. The behavioral performance of the Y-maze was calculated by the alternation percentage, which was as follows: alternation percentage = [total number of non-repeated entries into the three arms / (total number of arm entries-2)] × 100%.

[0026] (3) Open field test: This test is used to evaluate the autonomous behavior, exploratory behavior and tension of experimental animals in a novel environment. The experimental device consists of an open field reaction box and an automatic data acquisition and processing system. The open field reaction box is a closed plane area that is divided into multiple small blocks and is divided into a central area and an edge area. A digital camera is mounted directly above the open field reaction box to record the activities of the animals. The automatic data acquisition and processing system is used to analyze the behavioral data of the animals. At the beginning of the experiment, the mouse is placed in the center of the bottom surface of the open field reaction box to adapt for 30 seconds, and then the mouse's movement trajectory is recorded within 5 minutes. Between each experiment, the inner wall and bottom of the reaction box are wiped with alcohol to prevent the remaining information of the animal from the last time (such as the animal's urine, feces, and odor) from affecting the results of the next test.

[0027] (4) Novel object recognition experiment: The animals' natural curiosity about novel things is used to evaluate their learning and memory abilities. Before the test, mice are placed in the test room to adapt to the test environment. During the training phase, two identical objects (A and B) are placed at the left and right ends of one side wall of the experimental box. The mice are placed in the arena with their backs facing the two objects. The distance between the tip of the mouse's nose and the two objects is the same. The mice are allowed to explore freely for 5 minutes, during which the instrument automatically records the number of times and time the mice touch the two objects. After the training, the mice are returned to the cage to rest for 1 hour before the test begins. During the detection phase, the object B in the arena is replaced with a new object (C). The mice are placed in the arena again and allowed to move freely for 5 minutes. The exploration of the new object (C) is observed. The learning and memory abilities of the mice are evaluated by recording and analyzing the time the mice spend exploring the new and old objects.

[0028] 2. Serum biochemical index test data: Brain-body dysfunction is not only a subjective feeling, but also objectively causes changes in physiological indicators. When brain-body dysfunction occurs, the body's oxidative stress is unbalanced, metabolism is abnormal, and the immune system is affected. In order to better test whether mice have brain-body dysfunction, the ALP, CK, Glu, UREA, T-SOD, MDA and GPX levels in the two groups of mice were tested in serum. The results of serum biochemical index test showed that compared with the NC group, the expression levels of ALP (p < 0.05), CK (p < 0.05), UREA (p < 0.05) and MDA (p < 0.05) in the SD group mice were increased, and the expression levels of Glu (p < 0.05), T-SOD (p < 0.05) and GPX (p < 0.05) were decreased, see Figure 6 The above results showed that sleep deprivation combined with treadmill modeling changed the expression levels of body-related metabolism and oxidative stress-related markers, further verifying the successful construction of the mouse brain-body disability model.

[0029] In a specific embodiment, the process of executing step S102 may specifically include the following steps: Both the control group and the model group mice were placed in a modified treadmill apparatus for gradient adaptation training; Set the first day's training speed to 10 m / min and the duration to 1200 s through the treadmill control panel; Set the training speed for the second day to 10 m / min and the duration to 1800 s through the treadmill control panel; Set the training speed for the third day to 10 m / min and the duration to 3600 s through the treadmill control panel; Quantify the exercise tolerance of mice based on their behavioral performance and posture changes during the adaptation training process; Micro-sensors were used to collect the heart rate change curve and body temperature fluctuation data of mice throughout the adaptation training to obtain physiological response values.

[0030] Specifically, the mice in the control group and model group that had completed grouping and sensor installation were placed in a modified treadmill device for gradient adaptation training. The modified treadmill device is a special device modified on the basis of a standard small animal treadmill (model: ZS-PT-IV). The main improvements include: inserting a transparent plexiglass partition between each runway to prevent mice from interfering with each other; physically isolating the runway and the electric shock grid area to prevent mice from being stimulated by unnecessary electric shocks due to stopping exercise; evenly punching holes on the lid to facilitate mice to eat, drink and observe; adding a soft brush drive device to replace the traditional electric shock method to drive mice to exercise and reduce stress response interference. These improvements ensure the safety of the training environment and the accuracy of data collection. The adaptation training adopts a progressive scheme, and the exercise parameters are accurately set through the treadmill control panel. The speed was set to 10 meters / minute on the first day of training, and the duration was 1200 seconds (20 minutes). This initial setting takes into account the unfamiliarity of mice to the treadmill environment, with a moderate speed and a short time to reduce the stress response of the first contact. The setting process is operated through the touch screen interface of the treadmill control panel. Enter "10" in the "Speed ​​Setting" column and "1200" in the "Time Setting" column, then press the "Confirm" and "Start" buttons, and the treadmill starts to run according to the preset parameters. The second day of training maintains the same speed of 10 meters / minute, but extends the duration to 1800 seconds (30 minutes) to increase the training load of the mice. The setting method is the same as the first day, only the "Time Setting" parameter is modified. The third day of training continues to maintain a speed of 10 meters / minute, and the duration is further extended to 3600 seconds (60 minutes) to fully adapt the mice to long-term treadmill exercise. This design of gradually increasing the training time is in line with the physiological principle of gradual progression, avoiding stress reactions caused by a sudden increase in training intensity, and ensuring the adaptability of mice to treadmill exercise.

[0031] During the three-day adaptation training, it is necessary to quantify and record the exercise tolerance of mice. This indicator reflects the adaptability of mice to treadmill exercise. The quantification method includes observing and recording the following parameters: percentage of stable walking time (the proportion of the time that the mouse can keep up with the treadmill speed and walk normally in the total training time), number of falls (the number of times the mouse loses balance due to fatigue or maladaptation), number of prone rests (the number of times the mouse stops running and lies on the treadmill on its own initiative), number of expulsions (the number of times a soft brush is needed to drive the mouse to continue exercising), and exercise posture score (based on the coordination and smoothness of the mouse's running posture, 1-5 points). These parameters are obtained through manual observation combined with video recording, and then the comprehensive score of exercise tolerance is calculated according to the weighted formula. The formula is: Exercise tolerance = a×percentage of stable walking time + b×(1-number of falls / standard value) + c×(1-number of prone rest times / standard value) + d×(1-number of expulsion times / standard value) + e×exercise posture score / 5 Among them, a, b, c, d, and e are the weight coefficients of each indicator, which are set to 0.4, 0.15, 0.15, 0.15, and 0.15 respectively; the standard value is the preset benchmark number, which is determined according to the training duration. The multi-dimensional behavioral observation data are converted into a quantitative score of 0-100 points. At the same time, the heart rate change curve and body temperature fluctuation data of the mice were collected throughout the whole process using the micro-sensors previously installed on the backs of the mice in the model group. The sensor records the heart rate and body temperature data at a preset frequency (usually once every 10 seconds) to form a time series. The heart rate change curve records the complete heart rate change process of the mouse from the resting state to the exercise state, and then to the recovery period after the exercise, including three key indicators: basal heart rate, peak exercise heart rate, and recovery time. The body temperature fluctuation data reflects the dynamic changes in the body temperature of the mouse during exercise. After filtering these raw data (removing abnormal values ​​caused by poor sensor contact or vigorous activity of mice), the following indicators are calculated: heart rate rise rate (rate at which the heart rate reaches the peak value at the beginning of exercise), heart rate peak duration, heart rate recovery rate (rate at which the heart rate returns to the base value after exercise), body temperature rise amplitude, body temperature recovery time, etc. These indicators constitute the physiological response value, which directly reflects the physiological adaptation state of mice to exercise load.

[0032] For example, in one experiment, a male mouse numbered M005 showed obvious adaptation during three days of adaptation training. On the first day of training, the mouse first came into contact with the treadmill and was nervous. The percentage of stable walking time was only 65%, fell 17 times, crouched to rest 8 times, and needed to be driven away 12 times. The movement posture score was 2 points, and the calculated exercise tolerance was 62 points. The heart rate data showed that the basal heart rate was 475 beats / minute, reaching a maximum of 695 beats / minute during exercise, and it took 8 minutes to return to the basal value after exercise; the body temperature rose from 37.1℃ to 38.3℃. On the second day of training, at the same speed but longer time, the mouse showed signs of adaptation, with stable walking time increased to 78%, falls reduced to 9 times, crouched to rest 6 times, and driven away 8 times. The posture score increased to 3 points, and the exercise tolerance increased to 73 points. The peak heart rate dropped to 670 beats / minute, and the recovery time was shortened to 6.5 minutes. During the 60-minute long training on the third day, although the training time doubled, the mice continued to show improved adaptability: stable walking time reached 85%, fell only 5 times, crouched to rest 4 times, drove away 5 times, posture score reached 4 points, and exercise tolerance increased to 82 points. The peak heart rate further dropped to 650 beats / minute, and the recovery time was shortened to 5 minutes. By comparing the data of these three days, it is clear to see the adaptation process of M005 mice to treadmill exercise.

[0033] In a specific embodiment, the process of executing step S103 may specifically include the following steps: The exercise tolerance change curve and heart rate fluctuation characteristic diagram of each model group mouse during the three-day treadmill adaptation training were extracted from the adaptation training records; The training adaptability index of mice was calculated by the slope of the three-day exercise tolerance change curve, and the relative tolerance coefficient was obtained by comparing it with the baseline value; According to the heart rate fluctuation characteristic diagram, the mouse's heart rate basal level, exercise peak level and recovery time were determined, and a scatter plot of heart rate tolerance was drawn; The boundary curve fitting method was used on the heart rate tolerance scatter plot to identify the critical point of heart rate, and the critical point was increased by 10% as the upper limit of heart rate safety. The standard low-speed exercise value is linearly adjusted according to the relative tolerance coefficient to calculate the individualized low-speed exercise speed value range; The standard medium-speed exercise value is nonlinearly adjusted according to the relative tolerance coefficient to calculate the individualized medium-speed exercise speed value range; The individualized low-speed exercise speed value, individualized medium-speed exercise speed value, heart rate safety upper limit and duration parameters of each stage are integrated to form a personalized brain-body disability modeling plan.

[0034] Specifically, the exercise tolerance change curve and heart rate fluctuation characteristic diagram of each model group mouse during the three-day treadmill adaptation training were extracted from the adaptation training records. The exercise tolerance change curve is to draw the exercise tolerance value (0-100 points) calculated every day during the three-day adaptation training into a time series curve, with the horizontal axis being the number of training days (1, 2, 3) and the vertical axis being the corresponding exercise tolerance score. The heart rate fluctuation characteristic diagram is to draw the heart rate data collected during the adaptation training into a continuous time series diagram, with the horizontal axis being the time point and the vertical axis being the heart rate value (times / minute), including the complete heart rate change process before, during and after exercise. During the data extraction process, it is necessary to first eliminate abnormal values, such as drastic fluctuations in heart rate caused by loose sensors or sudden behavior of mice, to ensure the authenticity and reliability of the data.

[0035] Next, the training adaptability index of the mice was calculated by the slope of the three-day exercise tolerance change curve. The specific calculation method is: linear regression analysis is performed on the exercise tolerance change curve to obtain the slope k value of the curve, that is: , in, represents the i-th day, represents the exercise tolerance score on day i, and The k value represents the rate of improvement of the mouse's adaptability. The larger the k value, the stronger the mouse's adaptability to training. The calculated k value is compared with the average k value of all mice in the model group (i.e., the baseline value) to obtain the relative tolerance coefficient. The relative tolerance coefficient reflects the difference in adaptability of individual mice relative to the average level of the group. A value greater than 1 indicates that the adaptability is higher than the average, and a value less than 1 indicates that the adaptability is lower than the average.

[0036] At the same time, the heart rate basal level, exercise peak level and recovery time of the mice were determined based on the analysis of the heart rate fluctuation characteristic graph. The basal heart rate level is defined as the stable heart rate in the resting state before exercise, the exercise peak level is the highest heart rate value reached during exercise, and the recovery time refers to the time required for the heart rate to return to near the basal level after exercise (usually defined as no more than 10% of the basal level). These three indicators are used as coordinate points to draw a scatter plot of heart rate tolerance, with the horizontal axis being the exercise duration and the vertical axis being the heart rate value. The graph contains heart rate data points at multiple time points.

[0037] The boundary curve fitting method was applied to the heart rate tolerance scatter plot to identify the critical point of heart rate. The boundary curve fitting method is a data boundary detection algorithm that identifies the boundary of data distribution by connecting and smoothing the extreme points on the scatter plot. The specific operation is: first find the highest heart rate points in different time periods, connect these points with a smooth curve to form the upper boundary curve of heart rate; then find the lowest heart rate point and connect them to form the lower boundary curve. The intersection trend point of the two boundary curves is the critical point of heart rate, which represents the limit value that the heart rate of mice may reach during long-term exercise. The critical point is floated up by 10% as the upper limit of heart rate safety to ensure that the heart rate will not exceed the physiological safety range during the modeling process.

[0038] According to the relative tolerance coefficient calculated above, the standard low-speed motion value is linearly adjusted to calculate the individualized low-speed motion speed value range. The standard low-speed motion value is set to 1 meter / minute, and the individualized adjustment adopts the linear transformation formula: ,in The standard low speed is 1 m / min. In this way, mice with a higher relative tolerance coefficient will obtain higher low-speed exercise values, while mice with a lower tolerance coefficient will obtain lower speed values, ensuring that the modeling intensity matches the individual tolerance capacity.

[0039] For medium-speed exercise, considering that the load increase of high-speed exercise on mice is nonlinear, a nonlinear adjustment method is needed to calculate the individual medium-speed exercise speed value range. The standard medium-speed exercise value is set to 5 meters / minute, and the nonlinear adjustment uses a power function transformation: , where V_{medium speed standard} is the standard medium speed value of 5 m / min, and the exponent 0.8 is a nonlinear factor determined based on the physiological characteristics of mice, which can avoid excessive exercise intensity in mice with high tolerance coefficients while maintaining individual differences.

[0040] The individualized low-speed exercise speed value, the individualized medium-speed exercise speed value, the heart rate safety upper limit value, and the duration parameters of each stage (20 hours of low-speed exercise, 1 hour of rest, 2 hours of medium-speed exercise, and 1 hour of rest) are integrated to form a complete personalized brain-body disability modeling scheme. During the integration process, a modeling scheme data table containing the following fields needs to be created: mouse number, relative tolerance coefficient, heart rate safety upper limit value, individualized low-speed exercise speed value, individualized medium-speed exercise speed value, and duration of each stage. This modeling scheme will guide the subsequent construction process of the brain-body disability model.

[0041] Taking the mouse numbered M007 as an example, the exercise tolerance of the mouse in the three-day adaptation training was 67 points, 74 points and 83 points respectively. The slope k=8 points / day was calculated by linear regression, while the average slope of all mice in the model group was 7 points / day, so its relative tolerance coefficient α=1.14. Heart rate data analysis showed that M007's basal heart rate was 465 beats / minute, and its peak heart rate was 710 beats / minute. The critical point of its heart rate was determined to be 650 beats / minute by the boundary curve fitting method. After floating up by 10%, the upper limit of the heart rate safety was 715 beats / minute. The relative tolerance coefficient was used to calculate its individualized low-speed exercise speed: 1 meter / minute×1.14=1.14 meters / minute; individualized medium-speed exercise speed: 5 meters / minute×1.14^0.8=5.56 meters / minute. The final personalized brain-body disability modeling scheme for M007 included: low-speed exercise at 1.14 m / min for 20 hours, rest for 1 hour, medium-speed exercise at 5.56 m / min for 2 hours, rest for 1 hour, and a safe upper limit of heart rate of 715 beats / minute. This personalized scheme ensures that the modeling process can achieve sufficient intensity to induce brain-body disability without exceeding the physiological tolerance range of the mouse, thereby improving the success rate and safety of model construction.

[0042] In a specific embodiment, the process of executing step S104 may specifically include the following steps: The mice in the model group were placed in a modified treadmill device according to the personalized brain-body disability modeling scheme, and the sleep deprivation combined with exercise modeling procedure was started; The treadmill is driven at an individualized low-speed motion speed for 20 hours continuously, resulting in continuous low-intensity fatigue accumulation; By stopping the driving of the treadmill, the mice entered a 1-hour rest phase, forming an incomplete recovery state; Drive the treadmill at an individualized medium-speed exercise speed for 2 hours to form a medium-intensity acute fatigue superposition; The treadmill was stopped again to allow the mice to enter a 1-hour rest phase, completing a full cycle; The heart rate safety upper limit was compared with the real-time heart rate data every ten minutes. When the heart rate exceeded the safety upper limit, the treadmill speed was reduced by ten percent. When the heart rate was twenty percent below the standard, the treadmill speed was increased by five percent. This was repeated for seven to ten days, and the treadmill speed was continuously adjusted to form a brain-body disability model while ensuring the safety of the experiment.

[0043] Specifically, after completing the design of the personalized modeling scheme, the model group mice were placed in a modified treadmill device according to the personalized brain-body disability modeling scheme, and the sleep deprivation combined with exercise modeling program was started. The modified treadmill device is a special equipment modified on the basis of the standard ZS-PT-IV small animal treadmill. It is equipped with a transparent partition to separate the runway, and a soft brush is added instead of the electric shock grid. The top is punched for easy observation and feeding. The sleep deprivation combined with exercise modeling procedure refers to a modeling method that restricts the sleep of mice by long-term low-speed exercise and combines short-term medium-speed exercise to form a composite fatigue. This method simulates the brain-body disability state caused by alternating long-term low-intensity mental work with short-term medium-intensity physical work in humans. In the first stage of modeling, the treadmill is driven to run continuously for 20 hours at an individualized low-speed movement speed to form a continuous low-intensity fatigue accumulation. The individualized low-speed movement speed is calculated for each mouse in the early stage based on the performance of adaptive training, usually in the range of 1-2 meters per minute. 20 hours of continuous low-speed exercise can keep the mice in a passive awake state, achieve sleep deprivation, and will not cause excessive physical fatigue. During this stage, the mice's activities are passively maintained, similar to the low-intensity mental work of humans maintaining a single posture for a long time, which leads to decreased attention and cognitive fatigue. The treadmill control panel sets the corresponding speed according to the individual parameters of each mouse and runs for 20 hours. During this period, the mice can only get extremely limited sleep time, mainly manifested as a short micro-sleep state.

[0044] After 20 hours of low-speed exercise, the treadmill was stopped to allow the mice to enter a 1-hour rest phase, forming an incomplete recovery state. This 1-hour rest time is the optimal setting verified by multiple experiments. It is shorter than the time required for normal mice to fully recover, but it is enough for the mice to obtain partial relief from extreme fatigue. The incomplete recovery state refers to the state in which the physiological and cognitive functions of the mice only recover to a sub-healthy level, similar to the state in which humans take a short rest after high-intensity work but do not completely eliminate fatigue. At this time, the heart rate of the mouse will gradually decrease from the exercise state, but usually will not completely return to the basal level, and the activity level will increase but not as active as normal.

[0045] After resting for 1 hour, the treadmill was driven to run continuously for 2 hours at an individualized medium-speed exercise speed, forming a medium-intensity acute fatigue superposition. The individualized medium-speed exercise speed is usually in the range of 4-6 meters / minute, which is enough to keep the mice in an active running state and significantly increase their exercise load. Medium-intensity acute fatigue superposition refers to the re-application of a higher intensity exercise load on the basis of incomplete recovery in the previous period, which multiplies the cumulative effect of fatigue. This is similar to the situation in which humans engage in physical activity after long-term mental work, resulting in double fatigue of the brain and body. 2 hours of medium-speed exercise will significantly increase the heart rate of mice, close to the safe upper limit of their heart rate, accelerate breathing, and have obvious fatigue behavior such as unstable gait and frequent prostration.

[0046] After 2 hours of moderate-speed exercise, the treadmill was stopped again, allowing the mice to enter a second 1-hour rest phase, completing a full cycle. The second rest was also an incomplete recovery, but unlike the first rest, the mice were now in a state of compound fatigue, and their recovery ability was further reduced. A complete cycle lasted a total of 24 hours, including 20 hours of slow-speed exercise, 1 hour of rest, 2 hours of moderate-speed exercise, and 1 hour of rest. This cycle setting simulates the fatigue accumulation and incomplete recovery process of humans in a working day.

[0047] During the entire modeling process, precise regulation based on real-time heart rate data is crucial. The microsensor collects heart rate data every 10 seconds, transmits it to the data processing module, and compares the heart rate with the safety upper limit every ten minutes. When the heart rate is detected to exceed the safety upper limit, the treadmill speed is immediately reduced by 10% to prevent the mouse from excessive fatigue and physiological damage; when the heart rate is 20% lower than the standard (the standard is usually 120% of the resting heart rate), the treadmill speed is increased by 5% to ensure sufficient modeling intensity. This dynamic adjustment mechanism makes the modeling process safe and effective, and can accurately respond to the real-time status of each mouse. The above cycle is performed continuously for 7 to 10 days, and the treadmill speed is continuously adjusted to form a brain-body disability model while ensuring experimental safety. The 7 to 10-day duration setting is based on the previous research findings that this duration is sufficient to induce a stable brain-body disability phenotype without causing irreversible pathological changes. After completing a 24-hour cycle every day, the sleep deprivation and exercise fatigue effects continue to accumulate, and the mice gradually show brain-body disability characteristics such as decreased activity and cognitive impairment.

[0048] Taking the mouse numbered M008 as an example, the personalized modeling scheme for this mouse includes: low-speed exercise at 1.22 m / min for 20 hours, rest for 1 hour, medium-speed exercise at 5.73 m / min for 2 hours, rest for 1 hour, and the upper limit of heart rate safety is 705 beats / minute. During the low-speed exercise phase on the first day of modeling, the initial heart rate of M008 was 470 beats / minute. As time went on, the heart rate rose to 580 beats / minute after 10 hours, which was still below the upper limit of safety and continued to run at the original speed. After 18 hours, the heart rate rose to 695 beats / minute, close to the upper limit of safety, and the system reduced the speed by 10% to 1.10 m / minute. After the first rest phase, the heart rate dropped to 520 beats / minute, and the heart rate was still higher than the baseline value before the start of medium-speed exercise. After 1 hour of medium-speed exercise, the heart rate reached 700 beats / minute, close to the upper limit of safety, and the speed was reduced again to 5.16 m / minute. After 8 consecutive days of modeling, M008 showed obvious characteristics of brain-body dysfunction, such as decreased activity, slow reaction, and decreased memory. The exhaustion time in the rotarod test dropped from 280 seconds at baseline to 120 seconds, and spatial working memory was significantly impaired in the Y-maze test. Serum biochemical indicators also showed typical changes in brain-body dysfunction, confirming that the brain-body dysfunction model was successfully constructed.

[0049] In a specific embodiment, the process of executing step S105 may specifically include the following steps: The exercise endurance of the brain-body dysfunction model mice was tested by using an accelerating rotarod device, with the speed gradually increasing from 5 rpm to 35 rpm, and the time when the mice fell from the rotarod was recorded as the exhaustion time; The spatial exploration behavior of brain-body dysfunction model mice was recorded using a three-arm Y-shaped maze. The ratio of the total number of non-repeated entries into the three arms to the total number of entries within 8 minutes was measured to obtain the spatial memory ability index. The brain-body disability model mice were placed in an open field box, and the movement trajectory, total activity distance and central area residence time of the mice were recorded by an infrared tracking system within 5 minutes, and the spontaneous activity index was calculated; During the training phase, two identical objects were placed in the test box to allow the brain-body dysfunction model mice to freely explore for 5 minutes, and then rest for 1 hour. One of the objects was then replaced with a new object, and the time difference ratio of exploring the new and old objects was recorded to obtain cognitive function data. The performance data of the control group and the brain-body dysfunction model group mice in the four tests were compared and analyzed to generate a behavioral performance difference map; The dual decline in motor and cognitive abilities of the brain-body disability model was verified through behavioral performance difference maps, and the validity of exhaustion time, spatial memory ability, autonomous activity and cognitive function data was confirmed.

[0050] Specifically, the exercise endurance test of brain-body disability model mice was carried out by an accelerated rotating rod device. The accelerated rotating rod device is an instrument specially used to evaluate the motor coordination and endurance of mice. The model is ZS-PT-2, which consists of a rotating round rod with a diameter of 3 cm and a speed control unit. During the test, the mouse was placed on a stationary round rod. After starting the device, the speed started from 5 revolutions per minute and linearly accelerated to 35 revolutions per minute within 3 minutes. As the speed gradually increased, the mouse needed to keep speeding up to maintain balance, and eventually fell from the rotating rod due to muscle fatigue or decreased coordination. The bottom of the instrument is equipped with a sensing pad that can automatically detect the time when the mouse falls and record it as exhaustion time. Each mouse was tested 3 times with an interval of 10 minutes, and the average value was taken as the final exhaustion time data. If the mouse does not fall within 10 minutes, it is recorded as 600 seconds. The exhaustion time directly reflects the exercise endurance and coordination ability of the mouse, and is an important indicator for evaluating the motor ability of the brain-body disability model. Next, the spatial exploration behavior of the brain-body disability model mice was recorded using a three-arm Y-shaped structure maze. The Y-maze consists of three arms of equal length, each arm is 35 cm long, 8 cm wide, and 15 cm high, and the angle between the three arms is 120 degrees. The inner wall of the maze is painted black to reduce visual interference. During the test, the mouse was placed at the end of one of the arms, facing the center, and allowed to explore the maze freely for 8 minutes. The whole process was recorded by the top camera, and the position and route of the mouse were tracked in real time by animal behavior analysis software. Healthy mice have spontaneous alternating behavior characteristics and tend to explore different arms in turn rather than repeatedly entering the same arm. When processing data, the total number of times the mouse entered the arm was first recorded, and then the total number of non-repeated entries into the three forks (i.e., the number of behaviors of continuously entering three different arms) was counted. The spatial memory ability index was calculated by the following formula: alternation rate = (total number of non-repeated entries into the three forks) / (total number of arm entries-2)×100%. This index reflects the spatial working memory ability of mice, and mice with brain-body disability model usually show a significantly reduced alternation rate.

[0051] The third test is to place the brain-body disability model mice in an open open field box to evaluate their autonomous exploratory behavior. The open field box is a square box with a side length of 50 cm and a height of 40 cm. The bottom is divided into 16 small squares, and the central area is defined as the middle 4 squares. Before the test, the bottom and side walls of the open field box were wiped clean with 75% alcohol to eliminate the odor interference left by the mice in the previous test. During the test, the mice were placed in the center of the open field, and all the activities of the mice within 5 minutes were recorded by the infrared tracking system installed on the top. The infrared tracking system consists of an infrared camera and professional animal behavior analysis software, which can automatically identify the position of the mouse and track its movement trajectory. Data processing includes calculating parameters such as the total activity distance (the total distance the mouse moves in 5 minutes, in centimeters), the central area residence time (the total time the mouse stays in the central 4 squares, in seconds), and the number of upright times (the number of times the mouse's hind limbs stand and the forelimbs are raised). These parameters are calculated comprehensively to form the index of voluntary activity, and the calculation formula is: voluntary activity = (total activity distance / standard value A) × 0.6 + (central area stay time / standard value B) × 0.3 + (number of upright times / standard value C) × 0.1, where standard values ​​A, B, and C are the average performance values ​​of mice in the healthy control group. Mice with brain-body disability models usually show a significant decrease in voluntary activity.

[0052] The fourth test is a new object recognition experiment, which is used to evaluate the cognitive function of mice. The test uses a square box with a side length of 40 cm and a height of 30 cm, which is divided into a training phase and a test phase. In the training phase, two identical objects (usually solid plastic cylinders with a diameter of 3 cm, marked as object A and object B) are placed in the diagonal position of the test box, 10 cm away from the side wall. The mouse is placed in the venue with its back facing the two objects, and the mouse is allowed to explore freely for 5 minutes, while the behavior of the mouse is recorded by the top camera. Exploration behavior is defined as the mouse's nose being no more than 2 cm away from the object, or directly touching the object. After the training, the mouse is returned to the cage to rest for 1 hour before entering the test phase. In the test phase, the original object B is replaced with a new object C (different in shape and texture from A), the position remains unchanged, and the mouse is allowed to explore freely for 5 minutes again. Data processing requires calculating the exploration time of the mouse for the new object C and the old object A (recorded as TC and TA respectively), and then calculating the recognition index: recognition index = TC / (TA+TC). Healthy mice have a natural curiosity about novel things, and their recognition index is usually greater than 0.6. However, due to impaired cognitive function, the brain-body disability model mice have a decreased ability to distinguish between new and old objects, and their recognition index is significantly reduced. After completing the above four tests, the performance data of the control group and the brain-body disability model group mice in each test were compared and analyzed to generate a behavioral performance difference map. Data processing First, the normality test was performed on each test result. The data that conformed to the normal distribution were compared between groups using the independent sample t test, and the non-normal distribution data were compared using the Mann-WhitneyU test. The significance level of the difference was set at P<0.05. The behavioral performance difference map includes four dimensions: exercise endurance (exhaustion time), spatial memory ability (Y maze alternation rate), autonomous activity (comprehensive index of open field test) and cognitive function (new object recognition index). In each dimension, the two groups of data were standardized and drawn into a radar chart to intuitively display the behavioral characteristics of the brain-body disability model.

[0053] The behavioral performance difference map can verify the dual decline characteristics of motor ability and cognitive ability of the brain-body disability model and confirm the validity of various behavioral data. The complete behavioral evaluation system can comprehensively reflect the multi-dimensional manifestations of brain-body disability and provide a scientific basis for the effectiveness of the model. Taking the mouse numbered M010 as an example, before modeling, the exhaustion time of the mouse in the rotating rod test was 285 seconds, the Y maze alternation rate was 72%, the open field autonomous activity index was 0.92, and the new object recognition index was 0.68. After 8 days of sleep deprivation combined with treadmill modeling, the test results under the same conditions became 142 seconds, 51%, 0.47 and 0.52 respectively. The data comparison clearly shows that the mouse has a significant decline in many aspects such as motor coordination, spatial memory, autonomous exploration behavior and object recognition ability, which is in line with the typical characteristics of brain-body disability. At the same time, the test results of the control group mouse M003 remained basically stable within the same time span, with the exhaustion time changing from 272 seconds to 265 seconds, the Y-maze alternation rate changing from 70% to 68%, the autonomous activity changing from 0.95 to 0.93, and the novel object recognition index changing from 0.70 to 0.69, indicating the stability of the test conditions and the reliability of the data.

[0054] In a specific embodiment, the process of executing step S106 may specifically include the following steps: Blood samples were collected from the control group and the brain-body dysfunction model mice; The blood samples were centrifuged and the serum supernatant was extracted to prepare biochemical test specimens; The activities of serum alkaline phosphatase and creatine kinase were measured using an enzyme-labeled instrument to obtain data on muscle metabolism and liver function status; The glucose and urea levels in serum were measured by glucose oxidase method and urea nitrogen method to obtain data on energy metabolism and protein decomposition status; Chemical colorimetry was used to measure the superoxide dismutase activity, malondialdehyde content, and glutathione peroxidase activity in serum to obtain the data on oxidative stress levels; The measured levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase were integrated and analyzed with the behavioral data to confirm the metabolic and oxidative stress characteristics of the brain-body disability state and complete the construction of the mouse brain-body disability model.

[0055] Specifically, blood samples were collected from the control group and the mice with brain-body disability model by the orbital venous plexus blood collection method, which is a fast and effective mouse blood collection technique. In the specific operation, 24 hours after the completion of the behavioral test, the mice were lightly anesthetized, and the capillary was gently pierced into the medial orbital venous plexus of the mice to collect about 0.5 ml of blood in a sterile anticoagulant tube. The advantages of the orbital venous plexus blood collection method are simple operation, stable blood collection volume, little harm to mice, and suitable for one-time terminal blood collection. The collected blood samples need to be centrifuged immediately. The blood samples are placed in a centrifuge and centrifuged at 3000 rpm for 10 minutes to separate the serum supernatant. During the centrifugation process, the formed elements such as red blood cells settle to the bottom of the tube, while the serum supernatant is located in the upper layer. Use a pipette to carefully absorb the upper transparent serum liquid and transfer it to a new sterile centrifuge tube to avoid absorbing the cell components in the lower layer. The extracted serum supernatant is the biochemical test specimen, which can be tested immediately after aliquoting or stored in a -80℃ refrigerator for testing. The standard operating procedure requires that blood be centrifuged within two hours after collection to ensure that the biochemical indicators do not deteriorate due to prolonged storage.

[0056] The activity of alkaline phosphatase and creatine kinase in serum was measured by microplate reader to obtain muscle metabolism and liver function status data. Microplate reader is a precision instrument capable of quantitative analysis of enzyme activity. It calculates enzyme activity by measuring the absorbance change of light of a specific wavelength. Alkaline phosphatase (ALP) is a hydrolase mainly distributed in the liver, bones and small intestine. Its increased activity usually indicates abnormal liver or bone metabolism. Creatine kinase (CK) is mainly present in myocardial and skeletal muscles. It is a key enzyme in muscle energy metabolism. Its increased activity usually reflects muscle tissue damage or metabolic changes after strenuous exercise. The determination method uses a commercial kit. The specific steps are: mix the serum sample, substrate solution and buffer according to the instructions of the kit, react at a specific temperature for a specified time, and then measure the absorbance change at 405 nanometers. The enzyme activity is calculated according to the standard curve in U / L.

[0057] Glucose and urea content in serum were determined by glucose oxidase method and urea nitrogen method to obtain data on energy metabolism and protein decomposition status. Glucose oxidase method is a classic method for measuring blood glucose. Its principle is to use glucose oxidase to catalyze glucose oxidation to produce hydrogen peroxide, which reacts with a color developer under the action of peroxidase to generate a colored substance. The glucose concentration is calculated by measuring the absorbance. Urea nitrogen method is a standard method for measuring urea content in serum. Urea is the end product of protein metabolism, and its level reflects the body's protein decomposition status. The determination adopts urease-glutamate dehydrogenase method. Urea is hydrolyzed into ammonia and carbon dioxide under the action of urease. Ammonia and α-ketoglutarate are reduced to glutamate in the presence of NADH. The urea concentration is calculated by measuring the reduction of NADH. Both indicators were determined using commercial kits and operated according to the instructions. The final concentration was calculated by the standard curve. The unit of glucose is mmol / L and the unit of urea is mg / dL.

[0058] Chemical colorimetry was used to determine the activity of superoxide dismutase, malondialdehyde content and glutathione peroxidase in serum to obtain data on oxidative stress levels. Superoxide dismutase (T-SOD) is an important antioxidant enzyme in the body that can scavenge superoxide anion free radicals. A decrease in its activity indicates a weakened antioxidant capacity. Malondialdehyde (MDA) is a product of lipid peroxidation, and its increase indicates that cell membrane lipid peroxidation damage has worsened. Glutathione peroxidase (GPX) is a key enzyme that scavenge hydrogen peroxide and lipid peroxides. A decrease in its activity also reflects a decrease in antioxidant capacity. All three indicators were determined by chemical colorimetry. The principle is to form a colored product through a specific color development reaction, and to calculate the target concentration or enzyme activity by measuring the absorbance at different wavelengths. The standard operating procedure requires strict control of temperature, time and reagent purity, and the setting of positive and negative controls to ensure the accuracy and reliability of the experimental results.

[0059] The seven biochemical indexes and behavioral data were integrated and analyzed to confirm the metabolic and oxidative stress characteristics of the brain-body disability state and complete the construction of the mouse brain-body disability model. The data integration analysis first tested the normality of each index to confirm the data distribution characteristics. For data that conformed to the normal distribution, the independent sample t test was used to compare the differences between the control group and the brain-body disability model group; for data that did not conform to the normal distribution, non-parametric test methods such as the Mann-Whitney U test were used. The significance level of the difference was set at P < 0.05. In addition to the comparison between groups, the correlation between each biochemical index and the behavioral index was analyzed. Pearson correlation analysis (normal distribution data) or Spearman rank correlation analysis (non-normal distribution data) was used to calculate the correlation coefficient r value and determine the significance level. This multi-dimensional data integration analysis can comprehensively evaluate the effectiveness of the brain-body disability model and reveal the intrinsic connection between behavioral phenotypes and biochemical abnormalities.

[0060] Taking the brain-body disability model mouse No. M012 as an example, after 8 days of sleep deprivation combined with treadmill modeling, its serum ALP activity was 168U / L, significantly higher than 93U / L measured in the control group during the same period; CK activity was 532U / L, higher than 285U / L in the control group; blood glucose was 4.2mmol / L, lower than 5.6mmol / L in the control group; urea was 28mg / dL, higher than 19mg / dL in the control group; T-SOD activity was 98U / mg, lower than 156U / mg in the control group; MDA content was 3.8nmol / mL, higher than 2.1nmol / mL in the control group; GPX activity was 42U / L, lower than 68U / L in the control group. Through correlation analysis, it was found that serum CK activity was significantly negatively correlated with the time to exhaustion in the rotating rod, with a correlation coefficient of r=-0.72, indicating that abnormal muscle energy metabolism is closely related to decreased exercise capacity; T-SOD activity was positively correlated with the new object recognition index, with a correlation coefficient of r=0.68, suggesting that decreased antioxidant capacity is associated with cognitive impairment. This high consistency between the changes in serum biochemical indicators and behavioral data confirms the effectiveness of the construction of the brain-body disability model from the perspective of metabolism and oxidative stress.

[0061] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment, characterized in that: The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment includes: The mice were randomly divided into a control group and a model group, and microsensors were installed in the mice in the model group to collect the basal values ​​of heart rate, body temperature and activity; All mice were trained on a treadmill for three days according to the preset exercise parameters, and the exercise tolerance and physiological response values ​​of each mouse were recorded; According to the adaptive training records, the individualized exercise speed threshold and heart rate safety upper limit were determined for each model group of mice, and a personalized brain and body disability modeling scheme was generated; The model group mice were subjected to brain-body dysfunction modeling according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest. The treadmill speed was adjusted every ten minutes based on real-time heart rate data to form a brain-body dysfunction model. The constructed brain-body dysfunction model mice were subjected to rotarod, maze, open field and novel object recognition tests to obtain exhaustion time, spatial memory ability, locomotor activity and cognitive function data; Blood samples were collected from mice to measure the levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase to complete the construction of the mouse brain-body disability model.

2. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The mice were randomly divided into a control group and a model group, and microsensors were installed on the mice in the model group to collect the heart rate, body temperature and activity basal values, including: The male mice were randomly numbered and divided into a control group and a model group of equal size using a random number table method; The microsensor was fixed on the back of the mice in the model group through a preset vest; The mice were acclimated to the sensor weight in a constant temperature and light-controlled experimental environment; Using microsensors to continuously record the heart rate, body temperature, and activity level of mice at rest; According to the collected resting physiological parameters, the basic average value of physiological parameters of each mouse was calculated; The obtained heart rate basal values, body temperature basal values ​​and activity level basal values ​​were integrated into a basic data set of individualized modeling parameters.

3. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The treadmill adaptation training was performed on all mice for three days according to the preset exercise parameters, and the exercise tolerance and physiological response values ​​of each mouse were recorded, including: Both the control group and the model group mice were placed in a modified treadmill apparatus for gradient adaptation training; Set the first day's training speed to 10 m / min and the duration to 1200 s through the treadmill control panel; Set the training speed for the second day to 10 m / min and the duration to 1800 s through the treadmill control panel; Set the training speed for the third day to 10 m / min and the duration to 3600 s through the treadmill control panel; Quantify the exercise tolerance of mice based on their behavioral performance and posture changes during the adaptation training process; Micro-sensors were used to collect the heart rate change curve and body temperature fluctuation data of mice throughout the adaptation training to obtain physiological response values.

4. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The method of determining an individualized exercise speed threshold and a heart rate safety upper limit for each model group mouse according to the adaptive training records, and generating a personalized brain and body disability modeling scheme, includes: The exercise tolerance change curve and heart rate fluctuation characteristic diagram of each model group mouse during three days of treadmill adaptation training were extracted from the adaptation training records; The training adaptability index of mice was calculated by the slope of the three-day exercise tolerance change curve, and the relative tolerance coefficient was obtained by comparing it with the baseline value; According to the heart rate fluctuation characteristic diagram, the mouse's heart rate basal level, exercise peak level and recovery time were determined, and a heart rate tolerance scatter plot was drawn; The boundary curve fitting method was used on the heart rate tolerance scatter plot to identify the critical point of heart rate, and the critical point was increased by 10% as the upper limit of heart rate safety. The standard low-speed exercise value is linearly adjusted according to the relative tolerance coefficient to calculate the individualized low-speed exercise speed value range; The standard medium-speed exercise value is nonlinearly adjusted according to the relative tolerance coefficient to calculate the individualized medium-speed exercise speed value range; The individualized low-speed exercise speed value, individualized medium-speed exercise speed value, heart rate safety upper limit and duration parameters of each stage are integrated to form a personalized brain-body disability modeling plan.

5. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The brain-body disability model is formed for the model group mice according to the cycle structure of low-speed exercise, rest, medium-speed exercise, and rest, and the treadmill speed is adjusted every ten minutes according to the real-time heart rate data to form a brain-body disability model, including: The mice in the model group were placed in a modified treadmill device according to the personalized brain-body disability modeling scheme, and the sleep deprivation combined with exercise modeling procedure was started; The treadmill is driven at an individualized low-speed motion speed for 20 hours continuously, resulting in continuous low-intensity fatigue accumulation; By stopping the driving of the treadmill, the mice entered a 1-hour rest phase, forming an incomplete recovery state; Drive the treadmill at an individualized medium-speed exercise speed for 2 hours to form a medium-intensity acute fatigue superposition; The treadmill was stopped again to allow the mice to enter a 1-hour rest phase, completing a full cycle; The heart rate safety upper limit was compared with the real-time heart rate data every ten minutes. When the heart rate exceeded the safety upper limit, the treadmill speed was reduced by ten percent. When the heart rate was twenty percent below the standard, the treadmill speed was increased by five percent. This was repeated for seven to ten days, and the treadmill speed was continuously adjusted to form a brain-body disability model while ensuring the safety of the experiment.

6. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The constructed brain-body dysfunction model mice were subjected to rotarod, maze, open field and novel object recognition tests to obtain exhaustion time, spatial memory ability, autonomous activity and cognitive function data, including: The exercise endurance of the brain-body dysfunction model mice was tested by using an accelerating rotarod device, with the speed gradually increasing from 5 rpm to 35 rpm, and the time when the mice fell from the rotarod was recorded as the exhaustion time; The spatial exploration behavior of brain-body dysfunction model mice was recorded using a three-arm Y-shaped maze. The ratio of the total number of non-repeated entries into the three arms to the total number of entries within 8 minutes was measured to obtain the spatial memory ability index. The brain-body disability model mice were placed in an open field box, and the movement trajectory, total activity distance and central area residence time of the mice were recorded by an infrared tracking system within 5 minutes, and the spontaneous activity index was calculated; During the training phase, two identical objects were placed in the test box to allow the brain-body dysfunction model mice to freely explore for 5 minutes, and then rest for 1 hour. One of the objects was then replaced with a new object, and the time difference ratio of exploring the new and old objects was recorded to obtain cognitive function data. The performance data of the control group and the brain-body dysfunction model group mice in the four tests were compared and analyzed to generate a behavioral performance difference map; The dual decline in motor and cognitive abilities of the brain-body disability model was verified through behavioral performance difference maps, and the validity of exhaustion time, spatial memory ability, autonomous activity and cognitive function data was confirmed.

7. The method for constructing a mouse brain-body disability model based on sleep deprivation combined with treadmill experiment according to claim 1, characterized in that: The method comprises collecting blood samples from mice, measuring the levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase, and completing the construction of a mouse brain-body dysfunction model, including: Blood samples were collected from the control group and the brain-body dysfunction model mice; The blood samples were centrifuged and the serum supernatant was extracted to prepare biochemical test specimens; The activities of serum alkaline phosphatase and creatine kinase were measured using an enzyme-labeled instrument to obtain data on muscle metabolism and liver function status; The glucose and urea levels in serum were measured by glucose oxidase method and urea nitrogen method to obtain data on energy metabolism and protein decomposition status; Chemical colorimetry was used to measure the superoxide dismutase activity, malondialdehyde content, and glutathione peroxidase activity in serum to obtain the data on oxidative stress levels; The measured levels of alkaline phosphatase, creatine kinase, glucose, urea, superoxide dismutase, malondialdehyde and glutathione peroxidase were integrated and analyzed with the behavioral data to confirm the metabolic and oxidative stress characteristics of the brain-body disability state and complete the construction of the mouse brain-body disability model.

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