A method for constructing an animal model of acute stress

Abstract

The application provides a method for constructing an acute stress animal model. The method comprises the following steps: (1) on the first day to the next day, making the mice experience overnight illumination; (2) on the second day, making the mice experience a cold environment; (3) on the third day, making the mice experience underwater trauma; and (4) on the fourth day, making the mice experience electric shock. The acute stress animal model constructed by the application can stably induce various behavioral changes such as anxiety, fear, depression and enhanced startle reflex, and is accompanied by physiological index changes such as HPA axis activation, body weight reduction and food intake reduction, so that the multi-dimensional phenotype characteristics after acute stress can be more comprehensively reflected. The acute stress animal model constructed by the application is significantly superior to the traditional model in terms of the degree of coincidence with human complex trauma, the coverage range of symptoms and the stability of phenotype, and the expected goal of constructing a composite acute stress model is better achieved.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and more specifically, to a method for constructing an acute stress animal model. Background Technology

[0002] Acute stress response is a transient psychosomatic dysfunction triggered by a sudden and severe threat, causing serious damage to an individual's mental and physical health. Without timely intervention, it can easily develop into post-traumatic stress disorder (PTSD). Therefore, timely intervention is crucial for individual functional recovery and PTSD prevention. Currently, most acute stress responses are treated with psychotherapy, but the effects are unsatisfactory and the relapse rate is high, requiring medication as an adjunct. However, there are currently no approved drugs specifically for acute stress response, and existing drugs have many shortcomings. Finding safe and effective drugs has become an urgent need, and constructing animal models that simulate real trauma is a key prerequisite for drug screening.

[0003] Existing stress-related animal models are mainly divided into two categories: single stressor models and multiple stressor models. Single stressor models include foot shock (FS) models, underwater trauma (UT) models, acute restraint stress (ARS) models, predator-based stress models, social defeat models (SD) models, and social isolation models (SI) models. Multiple stressor models include single prolonged stress (SPS) models and chronic unpredictable mild stress (CUMS) models. However, each of the above models has its limitations. The main disadvantages include: (1) The stressor is singular and cannot simulate complex trauma situations: Most existing models use a single stressor, while trauma in real high-risk environments (such as fire rescue and military operations) has multi-dimensional characteristics: physical fatigue caused by long-term work, the impact of harsh environment, perception of direct threat to life, and physical pain. (1) Single stressor cannot fully reproduce these complex factors; (2) Narrow symptom coverage and lack of multidimensional phenotypes: Existing models have narrow symptom coverage, while human acute stress response is a combination of multidimensional symptoms such as anxiety, fear, depression, hypervigilance, HPA axis activation, weight loss, and reduced food intake. Incomplete symptom coverage makes it impossible to fully assess drug efficacy during drug screening; (3) Mismatched time course, affecting drug screening window: Symptoms of acute stress response usually appear within 1 hour after the trauma event. If it is a single trauma event, the symptoms usually subside within a few days. Existing models such as SPS show symptoms after 7 days, and CUMS requires several weeks to induce, which does not match the time course characteristics of acute stress and cannot meet the needs of rapid screening of drugs for acute stress; (4) Poor model stability and low reproducibility: The individual susceptibility of underwater trauma models varies greatly, and the individual differences of CUMS models are large, with some animals showing no obvious symptoms, resulting in poor experimental reproducibility and affecting the reliability of drug screening. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of existing acute stress animal models, the present invention aims to solve the following technical problems: 1. Addressing the issue of single stressors: Establishing a composite stress model that integrates multiple trauma elements (physical fatigue, harsh environment, life-threatening situations, and physical pain) to more realistically simulate traumatic situations in high-risk and complex environments.

[0005] 2. Addressing the issue of narrow symptom coverage: Constructing animal models that can simultaneously induce multi-dimensional stress phenotypes such as anxiety, fear, depression, hypervigilance, and HPA axis activation, comprehensively reflecting the core symptoms of acute stress response.

[0006] 3. Address the time-time mismatch issue: Establish a model where symptoms appear rapidly and remain stable within the acute time window (hours to days) to meet the time-time requirements for acute stress drug screening.

[0007] 4. Address the issue of poor model stability: By systematically optimizing the type, quantity, application order, frequency, and stimulation pattern of stressors, a standardized modeling scheme with clear parameters and high repeatability is established.

[0008] The main technical problem solved by this invention is to establish a multi-stressor acute stress model that integrates multiple trauma factors and can rapidly induce a comprehensive acute stress phenotype, for reliable screening and evaluation of drugs for acute stress response intervention.

[0009] The first aspect of this invention provides a method for constructing an acute stress animal model, the method comprising: Step (1): The mice were exposed to light overnight from the first day to the next day; Step (2): The mice were exposed to a cold environment on the second day; Step (3): On the third day, the mice experienced underwater trauma; Step (4): On the fourth day, the mouse experiences an electric shock.

[0010] In some implementations, the overnight lighting in step (1) lasts for 12 h to 24 h.

[0011] In some implementations, the overnight lighting in step (1) lasts for 12 hours.

[0012] In some implementations, the illuminance of the all-night lighting in step (1) is 10,000 Lux-12,000 Lux.

[0013] In some implementations, the illumination intensity of the all-night lighting in step (1) is 12000 Lux.

[0014] In some implementations, the overnight lighting time in step (1) is from 20:00 on the first day to 8:00 on the next day.

[0015] In some implementations, the overnight lighting time in step (1) is from 19:00 on the first day to 7:00 on the next day.

[0016] In some implementations, the temperature in step (1) is room temperature.

[0017] In some implementations, the temperature in step (1) is 24°C.

[0018] In some implementations, the cold environment in step (2) is 4°C.

[0019] In some implementations, step (2) is spaced 2 h to 5 h apart from step (1).

[0020] In some implementations, step (2) is 3.5 h apart from step (1).

[0021] In some implementations, step (2) lasts for 3 h to 8 h.

[0022] In some implementations, step (2) lasts for 5 hours.

[0023] In some implementations, step (2) is performed between 11:30 and 16:30 on the following day.

[0024] In some implementations, step (3) involves first allowing the mouse to swim freely, and then allowing the mouse to drown.

[0025] In some implementations, the drowning lasts for 5-15 seconds. The drowning time must be sufficient to prevent the mouse from drowning.

[0026] In some implementations, the drowning lasts for 10 seconds.

[0027] In some implementations, the free swimming lasts 10 min to 30 min.

[0028] In some implementations, the free swimming lasts for 15 minutes.

[0029] In some implementations, the interval between the start of step (3) and the start of step (2) is 16-17 hours.

[0030] In some implementations, step (3) is performed between 8:30 and 9:30 on the third day.

[0031] In some implementations, the electric shock in step (4) is receiving multiple electric shocks over 20 minutes.

[0032] In some implementations, the electric shock in step (4) is receiving 20 electric shocks within 20 minutes.

[0033] In some implementations, the electric shock current is 0.6 mA to 1 mA.

[0034] In some implementations, the electric shock current is 0.8 mA.

[0035] In some implementations, the duration of each electric shock is 8 s-15 s.

[0036] In some implementations, each electric shock lasts for 10 seconds.

[0037] In some implementations, the electric shocks are spaced 50-70 seconds apart.

[0038] In some implementations, the interval between the start of step (4) and the start of step (3) is 23-25 ​​hours.

[0039] In some implementations, step (4) is performed between 8:30 and 9:30 on the fourth day.

[0040] In some implementations, the mice include, but are not limited to, C57 mice, Balb / c mice, and ICR mice.

[0041] In some implementations, the mice include female mice and male mice.

[0042] In some implementations, the mouse is a male Balb / c mouse.

[0043] In some implementations, the method for constructing an acute stress animal model includes: Step (1): From the first day to the next day, the mice were kept under 24°C for 12 hours of continuous light, with a light intensity of 12000 Lux. Step (2): On the second day, the mice were subjected to a 4°C cold environment for 5 hours after experiencing overnight lighting, with an interval of 3.5 hours. Step (3): On the third day, the mice swam freely for 15 minutes, then drowned for 10 seconds; Step (4): On the fourth day, the mice received 20 electric shocks within 20 minutes. The current of the electric shock was 0.8 mA, the duration of each shock was 10 s, and the interval between each shock was randomized from 50 s to 70 s.

[0044] A second aspect of the present invention provides a system for constructing an acute stress animal model, the system comprising: First treatment unit: used to subject mice to overnight lighting from the first day to the next day; Second treatment unit: used to subject mice to a cold environment on the second day; Third treatment unit: used for mice to experience underwater trauma in the third day; Fourth processing unit: used to subject mice to electric shocks on the fourth day.

[0045] A third aspect of the present invention provides a construction apparatus for constructing an acute stress animal model, the construction apparatus comprising: one or more processors, and a memory for storing one or more computer programs, wherein the one or more computer programs are executed by the one or more processors to perform the following: Procedure 1: The mice were exposed to light overnight from day 1 to day 2; Procedure 2: The mice were exposed to a cold environment on the second day; Operation 3: On the third day, the mice experienced underwater trauma; Operation 4: On the fourth day, the mice were given an electric shock.

[0046] The fourth aspect of the present invention provides any of the following methods: (1) A method for screening drug candidates for treating acute stress response, the method comprising: a) Applying the reagent to be screened to the acute stress agonist constructed by the method described in the first aspect of the present invention; b) To test the therapeutic effect of the reagents to be screened on acute stress response; (2) A method for evaluating the therapeutic effect of a drug for treating acute stress response, the method comprising: a) Applying the drug to the acute stress agonist constructed by the method described in the first aspect of the present invention; b) To test the therapeutic effect of the drug on the acute stress response; (3) A method for studying the pathogenesis of acute stress response, wherein the method uses an acute stress animal constructed by the method described in the first aspect of the present invention to study the pathogenesis of acute stress response.

[0047] The fifth aspect of the present invention provides any of the following applications: (1) The application of the acute stress animal model constructed by the method of the first aspect of the present invention in screening drug candidates for the treatment of acute stress response; (2) The application of the acute stress animal model constructed by the method described in the first aspect of the present invention in evaluating the therapeutic effect of drugs for treating acute stress response; (3) Application of the acute stress animal model constructed by the method described in the first aspect of the present invention in the study of the pathogenesis of acute stress response.

[0048] The advantages and beneficial effects of this invention are as follows: Compared with existing classic models, the Multi-stressoracute stress model (MSAS) constructed in this invention is stable and reproducible, effectively solving the problems of single stressor and narrow symptom coverage in traditional models, and meeting the overall construction goals. First, the MSAS model has a high degree of fit with complex human trauma situations. Classic stress models, such as the foot shock model and acute restraint stress model, generally suffer from a single type of stress and difficulty in comprehensively simulating the multi-factor characteristics of real trauma. This invention can more comprehensively reproduce the key trauma features in high-risk environments, thus compensating for the deficiency of single stressor in classic models. Second, the MSAS model has a broader coverage of stress symptoms. The results of this invention suggest that in single stressor models such as restraint, foot shock, and underwater trauma, only foot shock can induce anxiety behavior, while having no significant effect on the core fear behavior of acute stress, making it difficult to simulate the diverse symptom manifestations after human acute stress. In contrast, the MSAS model stably induces a variety of behavioral changes, including anxiety, fear, depression, and enhanced startle reflex, accompanied by physiological changes such as HPA axis activation, weight loss, and reduced food intake, thus more comprehensively reflecting the multidimensional phenotypic characteristics after acute stress. Furthermore, the MSAS model exhibits phenotypic stability and strong reproducibility. While underwater trauma models show significant individual variability, and the CUMS model suffers from poor reproducibility and its stress duration does not conform to acute stress, this invention, through repeated validation, demonstrates that the MSAS model stably induces corresponding physiological and psychological behavioral abnormalities, with anxiety-like behaviors lasting for at least 24 hours, providing a stable and reliable time window for subsequent drug intervention studies. In summary, the MSAS model significantly outperforms traditional models in terms of fit with complex human trauma, symptom coverage, and phenotypic stability, effectively achieving the intended goal of constructing a composite acute stress model as described in this invention. Attached Figure Description

[0049] Figure 1This is a graph showing the results of anxiety behavior induced in mice by four stressors in the order of CL, CS, UT, and FS. A is a flowchart of the model; B, D, F, and H are plotted as line graphs of 30-minute open field tests 30 minutes after modeling, with 5-minute interval movement observed (mean ± SEM, n=8, Two-way ANOVA with Sidak's test); C, E, G, and I are plotted as bar graphs of 30-minute open field tests 30 minutes after modeling, with the total movement observed in the first 15 minutes (mean ± SEM, n=7 or 8, t-test, *P<0.05, **P<0.01); J, L, N, and P are plotted as line graphs of 30-minute open field tests 24 hours after modeling, with 5-minute interval movement observed (mean ± SEM, n=7 or 8, Two-way ANOVA with Sidak's test, *P<0.05); K, M, O, and Q are plotted as line graphs of 30-minute open field tests 24 hours after modeling, with 5-minute interval movement observed (mean ± SEM, n=7 or 8, Two-way ANOVA with Sidak's test, *P<0.05); K, M, O, and Q are plotted as line graphs of 30-minute open field tests 24 hours after modeling, with 5-minute interval movement observed (mean ± SEM, n=7 or 8, Two-way ANOVA with Sidak's test, *P<0.05); In the open field experiment, the total motion data for the first 15 minutes was plotted as a bar chart, Mean ± SEM, n=7 or 8, t test, *P<0.05.

[0050] Figure 2 Four stressors were intermittently stimulated (once each) in the order of CL, CS, UT, and FS to induce fear, depression, and startle behavior in mice. In mice with model A, fear behavior was evaluated using the conditioned fear test (Mean ± SEM, n=8, t test, *P<0.05); in mice with model C, depression behavior was evaluated using the tail suspension test (Mean ± SEM, n=8, t test, *P<0.05); and in mice with model D, startle reflex behavior was evaluated using the sound startle reflex test (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test, *P<0.05).

[0051] Figure 3The figures show the effects of the MSAS model on serum corticosterone, body weight, and food intake in mice. A) Blood samples were collected immediately after modeling and 30 minutes later. Serum corticosterone levels were measured using ELISA (Mean ± SEM, n=7, One-way ANOVA with Dunnett's test, ****P<0.0001). B) Body weight changes from Day 1 to Day 4 after modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test, *P<0.05, **P<0.01). C) Food intake from Day 1 to Day 4 after modeling (Mean ± SEM, n=4, Two-way ANOVA with Tukey's test, ****P<0.0001).

[0052] Figure 4 This is a graph showing the results of inducing stress symptoms using a pattern of one stimulation from each of the five single stressors. In the AD pattern, a 30-minute open field test was performed half an hour after stimulation with each of the five stressors; in the EF pattern, a conditioned fear test was performed after stimulation with each of the five stressors. Mean ± SEM, n=7 or 8, One-way ANOVA with Dunnett's test, *P<0.05, **P<0.01, ****P<0.0001, compared with the control group.

[0053] Figure 5 This is a graph showing the results of inducing stress symptoms by two stressors in a continuous stimulation pattern of UT, FS, UT, FS. Model A is a flowchart; Models B, D, F, and H are plotted as line graphs of 30-minute open field tests conducted half an hour later, with 5-minute interval movement observed at each test point (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test); Models C, E, G, and I are plotted as bar graphs of total movement observed within 30 minutes of the 30-minute open field test conducted half an hour later (Mean ± SEM, n=8, t-test); Models J and K are evaluated for fear behavior in mice using a conditioned fear test (Mean ± SEM, n=8, t-test, **P<0.01); Model L is evaluated for depressive behavior in mice using a tail suspension test (Mean ± SEM, n=8, t-test); Model M is evaluated for startle reflex behavior in mice using a sound startle reflex test (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test).

[0054] Figure 6This is a graph showing the results of inducing stress symptoms by three stressors in a sequential stimulation pattern of CL, FS, UT, FS. Model A is a flowchart; Models B, D, F, and H were tested 12 hours after modeling, and the 30-minute open field test was performed, with 5-minute interval movement observed as a line graph (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test, **P<0.01); Models C, E, G, and I were tested 30 minutes after modeling, and the total movement within 30 minutes was plotted as a bar graph (Mean ± SEM, n=8, t test, *P<0.05, **P<0.01); Models J and K were evaluated using a conditioned fear test (Mean ± SEM, n=8, t test, *P<0.05); Model L was evaluated using a tail suspension test (Mean ± SEM, n=8, t test); Model M was evaluated using a vocal startle reflex test (Mean ± SEM, n=8, t test). SEM, n=8, Two way ANOVA with Sidak's test.

[0055] Figure 7 The diagram shows the results of stress symptoms induced by four stressors in the order of UT, CS, FS, and CL. Among them, the flowchart of model A is shown. The anxiety behavior of mice was evaluated by the 30-minute open field test on the second day after BE modeling (Mean ± SEM, n=8, t test, *P<0.05). The fear behavior of mice was evaluated by the conditioned fear test after FG modeling (Mean ± SEM, n=8, t test, **P<0.01). The startle reflex test of mice was evaluated by the startle reflex test after H modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test).

[0056] Figure 8 This is a graph showing the results of inducing stress symptoms by stimulating five single stressors once daily for four consecutive days. In the AD group, the five stressors were stimulated four times, with a 30-minute open field test conducted half an hour after the last stimulation. In the EF group, a conditioned fear test was performed after stimulating the five stressors. Mean ± SEM, n=7 or 8, One-way ANOVA with Dunnett's test, #P<0.05, *P<0.05, **P<0.01, ****P<0.0001, compared with the control group.

[0057] Figure 9This is a graph showing the results of stress symptoms induced by two stressors in a pattern of intermittent stimulation (UT, FS, UT, FS) in the order of UT, FS (each stimulation twice). Model A is a flowchart; for models B, D, F, and H, the 30-minute open field test 24 hours later, with 5-minute interval movement data plotted as a line graph (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test, *P<0.05); for models C, E, G, and I, the 30-minute open field test 30 minutes later, with total movement data plotted as a bar graph (Mean ± SEM, n=8, t test, **P<0.01); for models J and K, the conditioned fear test was used to evaluate mouse fear behavior after modeling (Mean ± SEM, n=8, t test, ****P<0.0001); for model L, the tail suspension test was used to evaluate mouse depressive behavior after modeling (Mean ± SEM, n=8, t test); and for model M, the sound startle reflex test was used to evaluate mouse startle behavior (Mean ± SEM, n=8, t test). ±SEM, n=8, Two way ANOVA with Sidak's test.

[0058] Figure 10 The diagram shows the results of stress symptoms induced by four stressors in the order of UT, CS, FS, and CL. Among them, the flowchart of model A is shown. The anxiety behavior of mice was evaluated by the 30-minute open field test on the second day after BE modeling (Mean ± SEM, n=8, t test, *P<0.05). The fear behavior of mice was evaluated by the conditioned fear test after FG modeling (Mean ± SEM, n=8, t test, **P<0.01). The startle reflex test of mice was evaluated by the startle reflex test after H modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test).

[0059] Figure 11 The diagram shows the effect of the order of stressor application on the establishment of acute stress models. Model A flowchart: BE models were established within one day in the order of CL, CS, FS, UT. Anxiety behavior in mice was evaluated using a 30-minute open field test. Mean ± SEM, n=8, t test, *P<0.05. Model F flowchart: GJ models were established within one day in the order of overnight lighting, cold stress, underwater trauma, and foot shock. Anxiety behavior in mice was evaluated using a 30-minute open field test. Mean ± SEM, n=8, t test, *P<0.05.

[0060] Figure 12This chart shows the results of inducing stress symptoms using four stressors in intermittent stimulation patterns (UT / CS, FS / CL, UT / CS, FS / CL, each stimulated twice). A is the model flowchart; B is the evaluation of anxiety behavior using the 15-minute open field test 30 min after modeling (Mean ± SEM, n=7 or 8, t test, *P<0.05); C is the evaluation of anxiety behavior using the 5-minute elevated cruciate maze test 4 h after modeling (Mean ± SEM, n=7 or 8, t test, *P<0.05, **P<0.01); D is the evaluation of depressive behavior using the tail suspension test after modeling (Mean ± SEM, n=7, t test); E is the evaluation of fear behavior using the conditioned fear test after modeling (Mean ± SEM, n=6 or 7, t test, ****P<0.0001); F is the evaluation of startle reflex test after modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test). *P<0.05.

[0061] Figure 13 The diagram shows the results of stress symptoms induced by four stressors in the order of UT, CS, FS, and CL. Among them, the flowchart of model A is shown. The anxiety behavior of mice was evaluated by the 30-minute open field test on the second day after BE modeling (Mean ± SEM, n=8, t test, *P<0.05). The fear behavior of mice was evaluated by the conditioned fear test after FG modeling (Mean ± SEM, n=8, t test, **P<0.01). The startle reflex test of mice was evaluated by the startle reflex test after H modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test).

[0062] Figure 14The diagram shows the results of inducing stress symptoms by four stressors in the order of UT, CS, FS, and RS. Model A is a flowchart. After BE modeling, anxiety behavior in mice was evaluated using a 30-minute open field test (Mean ± SEM, n=7 or 8, t test, **P<0.01). FG modeling was evaluated using a conditioned fear test on the first day after modeling (Mean ± SEM, n=8, t test, **P<0.01). HI modeling was evaluated using a conditioned fear test on the first day after modeling (Mean ± SEM, n=8, t test, **P<0.01). J modeling was evaluated using a sound startle reflex test (Mean ± SEM, n=8), using a two-way ANOVA with Sidak's test.

[0063] Figure 15 The diagram shows the results of inducing stress symptoms by four stressors in a sequential stimulation pattern of CL, CS, UT, and FS. Among them, A is the model flowchart; B is the evaluation of fear behavior through the conditioned fear test, Mean ± SEM, n=8, t test, **P<0.01; C is the evaluation of startle behavior through the sound startle reflex test, Mean ± SEM, n=8, Two-way ANOVA with Sidak's test.

[0064] Figure 16 This chart shows the results of inducing stress symptoms using four stressors in intermittent stimulation patterns (UT / CS, FS / CL, UT / CS, FS / CL, each stimulated twice). A is the model flowchart; B is the evaluation of anxiety behavior using the 15-minute open field test 30 min after modeling (Mean ± SEM, n=7 or 8, t test, *P<0.05); C is the evaluation of anxiety behavior using the 5-minute elevated cruciate maze test 4 h after modeling (Mean ± SEM, n=7 or 8, t test, *P<0.05, **P<0.01); D is the evaluation of depressive behavior using the tail suspension test after modeling (Mean ± SEM, n=7, t test); E is the evaluation of fear behavior using the conditioned fear test after modeling (Mean ± SEM, n=6 or 7, t test, ****P<0.0001); F is the evaluation of startle reflex test after modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test). *P<0.05.

[0065] Figure 17The results are shown in the diagrams for the induction of stress symptoms by four stressors in an intermittent stimulation pattern in the order of CL, CS, UT, FS, CL, CS, UT, FS. A is a flowchart of the model. B, D, F, and H were tested in an open field experiment the day after modeling, and the movement at 5-minute intervals was plotted as a line graph (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test, *P<0.05). C, E, G, and I were tested in an open field experiment the day after modeling, and the movement within 30 minutes was plotted as a bar graph (Mean ± SEM, n=8, t test, *P<0.05, **P<0.01). J and K were evaluated for fear behavior using a conditioned fear test after modeling (Mean ± SEM, n=7 or 8, t test, **P<0.01). L was evaluated for startle reflex behavior using a sound startle reflex test after modeling (Mean ± SEM, n=8, Two-way ANOVA with Sidak's test).

[0066] Figure 18 This figure shows the results of assessing the sensitivity of C57, Balb / c, and ICR mice using the sound startle reflex test. After establishing the combined stressor model, the sensitivity of male and female C57, Balb / c, and ICR mice was assessed using the sound startle reflex test. Mean ± SEM, n=6-8, *P<0.05, Two-way ANOVA with Sidak's test.

[0067] Figure 19 The graph shows the results of sensitivity assessment of three mouse strains (C57, Balb / c, and ICR) using the conditioned fear test. A represents the percentage of rigidity in the situational fear test for the three mouse strains, Mean ± SEM, n=8, Two-way ANOVA with Sidak's test; B represents the percentage of rigidity in the situational fear test for the three mouse strains, Mean ± SEM, n=8, t test. Detailed Implementation

[0068] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0069] Example 1: Influence of the MSAS Model on Psychological Indicators 1. Experimental Materials (1) Experimental animals: Balb / c mice, male, 6 - 8 weeks old, weighing 20 - 22 g, purchased from Beijing Huafukang Biotechnology Co., Ltd., certificate batch number: SCXK (Beijing) 2024 - 0003. 5 mice per cage. 12 h day-night alternation, room temperature (22 ± 2 °C), humidity controlled at (40 ± 20)%, free access to water and food. Before the formal experiment, the animals were allowed to adapt to the饲养 environment for at least 3 days, and the mice were stroked every day to make them adapt and get used to the experimental operation. In addition, all experimental procedures were carried out in accordance with the animal ethics usage guidelines formulated by the national research institute, and all animal studies were approved by the Animal Care and Use Committee of Beijing Institute of Pharmacology and Toxicology.

[0070] (2) Experimental instruments: The constant temperature light incubator was purchased from Shanghai Shangpu Instrument Equipment Co., Ltd.; the spontaneous activity tester, conditioned fear system, and startle reflex system were purchased from AniLab Scientific Instruments Co., Ltd.; the tail suspension tester was purchased from Beijing Zhongshi Dichuang Technology Development Co., Ltd. grating type; the 1800 mL beaker was purchased from Sichuan Shubo (Group) Co., Ltd.

[0071] 2. Experimental Methods (1) Modeling method: On the first day, constant light exposure (CL): The mice were placed in a constant temperature incubator for 12 h, from 20:00 to 8:00 the next day, at a temperature of 24 °C and a light intensity of 12000 Lux. On the second day, cold stress (CS): The mice were placed in a 4 °C constant temperature incubator for 5 h (11:30 - 16:30). On the third day, underwater trauma (UT): 1200 mL of water was added to an 1800 mL beaker, and the mice were placed in the water to swim freely for 15 min, and then gently submerged with an inverted funnel for 10 s, carried out from 8:30 - 9:30. On the fourth day, foot shock (FS): The mice were placed in an electric shock box and randomly received 20 mild foot shocks within 20 min, each time 0.8 mA, 10 s, randomly spaced 50 - 70 s, carried out from 8:30 - 9:30. During the空档期 of the experimental treatment, the mice were returned to the mouse house, and the饲养 environment was kept the same as that of the control group. The overall method is as Figure 1 A.

[0072] Control group: 12 h day-night alternation, room temperature (22 ± 2 °C), humidity controlled at (40 ± 20)%, free access to water and food.

[0073] 2. Open field test: The open field test was conducted 30 min and 24 h after the last stressor (foot shock). The total distance of movement and the movement of the central area of ​​the mice were observed every 5 min within 30 min and a line graph was drawn. The movement of the mice in the first 15 min was statistically analyzed and a bar graph was drawn.

[0074] 3. Sound startle reflex test: The sound startle reflex test was conducted the day after the model was established. After the mice adapted to the background noise (70 dB) environment for 5 minutes, they were subjected to 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random interval of 10-30 s). The maximum startle amplitude of each sound was recorded.

[0075] 4. Tail Suspension Test: The tail suspension test was conducted the day after modeling. The end of the mouse's tail was fixed, so that it was suspended upside down and could not touch the ground or other objects. The mouse's behavior was recorded for 6 minutes, and the immobility time in the last 4 minutes was recorded.

[0076] 5. Conditioned Fear Experiment: Six hours after modeling, mice were placed in an electric shock box (context A) for one instance of conditioned-unconditioned stimulus pairing training, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling was different from that for conditioned-unconditioned stimulus pairing training. Twenty-four hours after pairing training, fear testing was performed. Mice were placed in context A for 5 minutes without sound cues or electric shock stimulation to test contextual fear. After changing the scenario, mice were placed in context B and given 5 sound cues to test cued fear.

[0077] 3. Experimental Results To assess anxiety behavior in mice after modeling, an open field test (30 min test duration) was performed at two time points: 30 min and 24 h after modeling. The line graph within the 30 min period showed a significant trend of behavioral differences between the two groups of mice in the first 15 min; therefore, data from the first 15 min were selected for analysis. The results showed that the total movement distance of the mice did not change significantly in the first 15 min, but the percentage of movement distance in the central region, the percentage of time spent in the central region, and the number of times the mice traversed the central region were all significantly reduced, indicating that the mice exhibited significant anxiety behavior. Figure 1BI). To observe the duration of anxious behavior, an open field test (test duration 30 min) was performed again 24 h after modeling, and the data from the first 15 min were analyzed. The results showed that there was no difference in the total distance of movement between the two groups of mice, but the number of times the model group crossed the central area was significantly reduced, indicating that the mice still had anxiety 24 h after modeling. Figure 1 (JQ). The above results indicate that this version of the model can successfully induce anxiety behavior in mice, and this anxiety behavior can last for at least 24 hours.

[0078] After modeling, the mice's fear behavior, startle reflex behavior, and depressive behavior were further evaluated using the conditioned fear test, the vocal startle reflex test, and the tail suspension test, respectively. The conditioned fear test evaluated fear behavior, the vocal startle reflex test evaluated startle behavior, and the tail suspension test evaluated depressive behavior. In the conditioned fear test, this version of the model could induce situational fear behavior in mice. Figure 2 A), but it cannot produce auditory cue fear behavior ( Figure 2 B); In the tail suspension test, the immobility time of mice in the model group was significantly reduced, indicating that this version of the model can induce depressive behavior in mice (B). Figure 2 C); In the sound startle reflex experiment, the shaking amplitude of mice in the model group was significantly increased at the 14th and 24th 115 dB sound stimuli, indicating that this version of the model can induce startle behavior in mice (C). Figure 2 D). This part of the results suggests that the MSAS model can induce fear, fright, and depression behaviors in mice.

[0079] Example 2: Effects of the MSAS model on physiological indicators 1. Experimental materials (1) The experimental animals are the same as in Example 1.

[0080] (2) Experimental instruments: The microplate reader was purchased from Meigu Molecular Instruments (Shanghai) Co., Ltd.; the rest are the same as in Example 1.

[0081] (3) Experimental reagents: Mouse corticosterone (CORT) ELISA kit, purchased from Shanghai Jianglai Biotechnology Co., Ltd., catalog number JL11918.

[0082] 2. Experimental Methods (1) Body weight and food intake: The body weight and food weight of mice were measured before modeling. On the first, second, third and fourth days after modeling, the body weight and remaining food of mice were measured, and the food intake of each group of mice was calculated.

[0083] (2) ELISA was used to detect serum corticosterone levels.

[0084] 3. Experimental Results Further investigation was conducted into the effects on physiological indicators in mice, specifically changes in serum corticosterone levels, body weight, and food intake. To detect serum corticosterone levels, blood samples were collected from mice immediately after modeling and 30 minutes after modeling. Enzyme-linked immunosorbent assay (ELISA) results showed that serum corticosterone levels were significantly elevated at both time points. Figure 3 (A) In addition, for four consecutive days after the modeling process, two physiological indicators—weight changes and total food intake—were continuously monitored in the mice. Weight monitoring results showed that within two days after modeling, the weight of the model group mice was significantly lower than that of the control group, while two days after modeling, the weight of the model group mice showed a gradual recovery trend. Figure 3 B); Feeding monitoring results showed that, both during the modeling period and within four days after the modeling period, the total food intake of the model group mice was significantly lower than that of the control group (B). Figure 3 C). The above results confirm that this version of the model can not only induce stress-related behavioral symptoms in mice at the psychological level, but also induce significant stress-related physiological changes in mice at the physiological level, further verifying the effectiveness of the model.

[0085] Example 3: The effect of increasing or decreasing the number of stressors on the establishment of an acute stress model 1. Experimental Methods I. Continuous stimulation A. Each of the five single stressors was stimulated once. 1. Five methods for modeling single stressors: a. Underwater trauma (UT): 1200 mL of water was poured into an 1800 mL beaker, and mice were allowed to swim freely for 15 min, then gently submerged for 10 s using an inverted funnel. b. Constant light exposure (CL): Mice were placed in a constant temperature incubator for 12 h (20:00-8:00 the next day) at 24 ℃ with a light intensity of 12000 Lux. c. Cold stress (CS): Mice were placed in a constant temperature incubator at 4 ℃ for 5 h. d. Footshock (FS): Mice were placed in a shock chamber and received 20 mild foot shocks over 20 min (0.8 mA, 10 s, with random intervals of 50-70 s). e. Restraint stress (RS): Mice were restrained in a restraint tube for 12 h (20:00-8:00 the next day).

[0086] 2. Open Field Test: Thirty minutes after establishing the five single stressor models, an open field test was conducted to observe the total distance of movement and the movement of the central area in mice within 30 minutes. 3. Conditioned Fear Test: Thirty minutes after establishing the five single stressor models, mice were placed in an electric shock box (context A) for one instance of conditioned-unconditioned stimulus pairing training, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned pairing training. The day after pairing training, fear testing was performed. Mice were placed in context A for 5 minutes without sound cues or electric shock stimulation to test contextual fear. After changing the scenario, mice were placed in context B and given five sound cues to test cued fear.

[0087] B. Two stressors are applied sequentially in the order of UT, FS, UT, FS. 1. Modeling Methods: 1) Underwater Trauma: Add 1200 mL of water to an 1800 mL beaker, immerse the mouse in the water for 15 min, then gently submerge it for 10 s using an inverted funnel. 2) Foot Shock: 2.5 h after underwater immersion, place the mouse in an electric shock chamber and randomly administer 20 mild foot shocks over 20 min, each at 0.8 mA for 10 s, with random intervals of 50-70 s. 3) Underwater Trauma: Repeat step 1) 2.5 h after foot shock. 4) Foot Shock: Repeat step 2) 2.5 h after underwater trauma. 2. Open Field Experiment: Conduct an open field experiment half an hour after modeling. Observe the total distance of movement and the movement of the central area of ​​the mouse every 5 min over 30 min and plot a line graph. Also, plot a bar graph of the movement over the first 30 min. 3. Startle Reflex Experiment: Conduct a startle reflex experiment 15 h after modeling. The experimental method is the same as above.

[0088] C. Three stressors are applied sequentially in the order of CL, FS, UT, FS. 1. Modeling methods: 1) Overnight illumination: Mice were placed in a constant temperature incubator for 12 hours, from 20:00 to 8:00 the next day, at a temperature of 24℃ and a brightness of 12000 Lux. 2) Foot shock: 2.5 hours after overnight illumination, mice were placed in an electric shock chamber and randomly received 20 mild foot shocks within 20 minutes, each at 0.8 mA for 10 seconds, with random intervals of 50-70 seconds. 3) Underwater trauma: 2.5 hours after foot shock, 1200 mL of water was added to an 1800 mL beaker, and the mice were allowed to swim freely for 15 minutes, then gently submerged for 10 seconds using an inverted funnel. 4) Foot shock: 2.5 hours after underwater trauma, the foot shock procedure in step 2) was repeated.

[0089] 2. Open Field Test: 12 hours after modeling, conduct the open field test and record movement over 30 minutes. The experimental procedure is the same as above. 3. Sound Startle Reflex Test: 12 hours after modeling, conduct the sound startle reflex test. The experimental method is the same as above. 4. Conditioned Fear Test: 12 hours after modeling, conduct conditioned-unconditioned stimulus pairing training, followed by a fear test the next day. The experimental method is the same as above. 5. Tail Suspension Test: 20 hours after modeling, conduct the tail suspension test (13:00-13:30).

[0090] D. Four stressors are applied sequentially in the order of UT, CS, FS, CL. 1. Model Establishment: 1) Underwater Trauma: Add 1200 mL of water to an 1800 mL beaker, immerse the mice in the water for free swimming for 15 min, and then gently submerge them for 10 s using an inverted funnel. 2) Cold Stress: Place the mice in a 4 ℃ constant temperature incubator for 5 h. 3) Foot Shock: Place the mice in an electric shock chamber and randomly administer 20 mild foot shocks over 20 min, each at 0.8 mA for 10 s. 4) Overnight Illumination: Place the mice in a constant temperature incubator for 12 h, from 19:00 to 7:00 the next day, at a temperature of 24 ℃ and maximum brightness.

[0091] 2. Open Field Test: The open field test was conducted the morning after modeling, recording the total distance traveled by the mice and the distance traveled in the central area within 30 minutes. 3. Auditory Startle Reflex: The auditory startle reflex test was conducted the afternoon after modeling. 4. Conditioned Fear Test: The day after modeling, the mice were placed in an electric shock box (context A) for two cycles of conditioned-unconditioned stimulus pairing training, i.e., auditory-electric shock association training. Auditory parameters: 7000 Hz, 80 dB, 20 s; Electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned stimulus pairing training. The day after pairing training, fear testing was conducted. The mice were placed in context A for 5 minutes without auditory cues or electric shock stimulation to test contextual fear. After changing the scenario, the mice were placed in context B and given 5 auditory cues to test cued fear.

[0092] II. Intermittent Stimulation A. Five single stressors were applied once daily for four days. 1. Five methods for modeling single stressors: a. Underwater trauma: Pour 1200 mL of water into an 1800 mL beaker, immerse the mouse in the water for 15 min of free swimming, then gently submerge it for 10 s using an inverted funnel. Repeat for four consecutive days. b. Overnight illumination: Place the mouse in a constant temperature incubator for 12 h (20:00-8:00 the next day) at 24℃ with a light intensity of 12000 Lux. Repeat for four consecutive days. c. Cold stress: Place the mouse in a constant temperature incubator at 4℃ for 5 h. Repeat for four consecutive days. d. Foot shock: Place the mouse in an electric shock chamber and administer 20 mild foot shocks over 20 min (0.8 mA, 10 s, with random intervals of 50-70 s). Repeat for four consecutive days. e. Restraint stress: Restrain the mouse in a restraint tube for 12 h (20:00-8:00 the next day). Repeat for four consecutive days.

[0093] 2. Open Field Test: Thirty minutes after establishing the five single stressor models, an open field test was conducted to observe the total distance the mice traveled and the movement of the central area within 30 minutes. 3. Conditioned Fear Test: Thirty minutes after establishing the five single stressor models, mice were placed in an electric shock box (context A) for one instance of conditioned-unconditioned stimulus pairing training, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned stimulus pairing training. The day after pairing training, fear testing was performed. Mice were placed in context A for 5 minutes without sound cues or electric shock stimulation to test contextual fear. After changing the scenario, mice were placed in context B and given five sound cues to test cued fear.

[0094] B. Two stressors are applied intermittently in the order of UT, FS, UT, FS. 1. Modeling Method: Day 1: Underwater Trauma (8:30-9:30): Add 1200 mL of water to an 1800 mL beaker, immerse the mice in the water for free swimming for 15 min, then gently submerge them for 10 s using an inverted funnel. Day 2: Foot Shock (14:30-15:30): Place the mice in an electric shock chamber and randomly administer 20 mild foot shocks over 20 min, each at 0.8 mA for 10 s, with an interval of 50-70 s. Day 3: Underwater Trauma (10:00-11:00): Repeat the procedure from Day 1. Day 4: Foot Shock: Repeat the procedure from Day 2 (17:00-17:30).

[0095] 2. Open Field Test: On the morning of the fifth day, a 30-minute open field test was conducted. The total distance traveled by the mice every 5 minutes and the movement in the central area were observed and plotted as a line graph. A bar chart was also created to summarize the total movement over the 30 minutes. 3. Conditioned Fear Test: On the afternoon of the fifth day, conditioned-unconditioned stimulus pairing training was conducted. Fear behavior testing was performed on the morning of the sixth day. 4. Tail Suspension Test: On the afternoon of the fifth day, the tail suspension test was conducted, following the same procedures as before. 5. Sound Startle Reflex Test: On the afternoon of the fifth day, the sound startle reflex test was conducted, following the same procedures as before.

[0096] C.MSAS model: Four stressors are intermittently stimulated according to CL, CS, UT, and FS.

[0097] 2. Experimental Results This part of the experiment set up different combinations of single, two, three, and four stressors. Through behavioral experiments such as open field and conditioned fear, stress-related behaviors in mice were detected to explore the effect of different numbers of stressors on model construction. The experimental results are described below.

[0098] I. Continuous stimulation A. Each of the five single stressors was stimulated once. In Group A, five stressors were selected: CL, UT, CS, RS, and FS. Each stressor was stimulated only once. Anxiety-like behaviors were then assessed using an open field test, and fear behaviors were evaluated using a conditioned fear test to determine whether a single stressor could independently induce significant stress symptoms. Results showed that in the open field test, compared to the control group, only the plantar electric shock induced a significant decrease in the percentage of total movement distance to central region movement distance among the five stressors, suggesting that plantar electric shock can induce anxiety behavior in mice; while the other four stressors showed no significant differences. Figure 4 AD). In the conditioned fear experiment, there were no significant differences in fear behaviors induced by the five stressors compared to the control group. Figure 4 EF).

[0099] B. Two stressors are applied sequentially in the order of UT, FS, UT, FS. In Group B of the experiment, the two stressors were applied twice a day in the order of UT, FS, UT, FS. The experimental flowchart is as follows. Figure 5 As shown in Figure A, anxiety behavior was subsequently evaluated using the open field test, fear behavior using the conditioned fear test, startle reflex test using the vocalization startle reflex test, and depression behavior using the tail suspension test. The results showed that in the conditioned fear test, this version of the model could induce situational fear behavior in mice. Figure 5 J) but cannot produce auditory cue fear behavior ( Figure 5 K); however, in the open field test, the tail test, and the sound startle reflex test, there were no significant differences between the two groups ( Figure 5 BI, LM).

[0100] C. Three stressors are applied sequentially in the order of CL, FS, UT, FS. In Group C, the three stressors were administered in the following order within one day: CL, FS, UT, FS. CL and UT were stimulated once, and FS was stimulated twice. The experimental flowchart is shown in Figure 6A. After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, startle reflex test using the vocalization startle reflex test, and depression behavior using the tail suspension test. The results showed that, compared with the control group, the total movement distance of the model group mice remained unchanged, but the percentage of central region movement distance, the percentage of central region movement time, and the number of times the central region was crossed were significantly reduced, suggesting that this version of the model can induce anxiety behavior in mice. Figure 6 BI); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice ( Figure 6 J) but cannot produce auditory cue fear behavior ( Figure 6 K); In the tail suspension test and the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 6 LM).

[0101] D. Four stressors are applied sequentially in the order of UT, CS, FS, CL. In Group D of the experiment, the four stressors were applied once a day in the order of UT, CS, FS, and CL. The experimental procedure was as follows: Figure 7 As shown in Figure A. After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the vocal startle reflex test. Results showed that in the open field test, the model group mice only exhibited a significant decrease in total movement distance, which was attributed to a decline in physical fitness. Therefore, we concluded that this version of the model failed to induce anxiety behavior in the mice. Figure 7 BE); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice ( Figure 7 F) but cannot produce auditory cue fear behavior ( Figure 7 G); In the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 7 H).

[0102] II. Intermittent Stimulation A. Five single stressors were applied once daily for four days. Five stressors (CL, UT, CS, RS, and FS) were applied once daily for four days. Anxiety behavior was then evaluated using an open field test, and fear behavior was evaluated using a conditioned fear test. Results showed that, compared to the control group, only the foot shock induced a significant decrease in total movement distance, percentage of central region movement distance, and number of central region crossings among the five stressors. This suggests that foot shock can induce anxiety behavior in mice, restraint induced an increase in total movement distance, while the other three stressors showed no significant differences. Figure 8(AF). In the conditioned fear experiment, there were no significant differences in fear behaviors induced by the five stressors compared to the control group.

[0103] B. The two stressors were intermittently stimulated in the order of UT, FS, UT, FS.

[0104] Two stressors were applied twice daily in the order of UT, FS, UT, FS (Figure 9A). After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, startle reflex test using the sound startle reflex test, and depressive behavior using the tail suspension test. Results showed that, compared with the control group, the model group mice had significantly reduced time spent in the central region and fewer times they passed through it, indicating that this version of the model could induce anxiety behavior in mice. Figure 9 BI); In the conditioned fear experiment, the percentage of situational fear rigidity in the model group mice was significantly increased, indicating that this version of the model can induce situational fear behavior in mice. Figure 9 J), but there was no significant difference in fear of auditory cues (J). Figure 9 K); In the tail suspension test and the sound startle reflex test, there were no significant differences between the two groups of mice ( Figure 9 LM).

[0105] C.MSAS model: Four stressors are intermittently stimulated in the order of CL, CS, UT, FS.

[0106] The experimental results are the same as those in Examples 1 and 2.

[0107] The above results indicate that the number of stressors is key to regulating the phenotype of acute stress models. During continuous stimulation on a single day, the combination of three stressors induced the most comprehensive symptoms (fear, anxiety, and shock), but failed to induce depressive behavior compared to the MSAS model. Concentrated stimulation of four stressors was less effective, suggesting that the single-day model needs to appropriately reduce the number of stressor types and increase repetitive stimulation. During intermittent stimulation over four days, applying one of the four stressors daily (MSAS model) induced a more comprehensive range of symptoms. Complex stress models require optimization of the number and application method of stressors based on the duration of stimulation to comprehensively simulate acute stress responses.

[0108] Example 4: The effect of changing the order of stressors on the establishment of an acute stress model 1. Experimental Methods A. Four stressors are applied sequentially in the order of UT, CS, FS, CL. Model establishment: 1) Underwater trauma: 1200 mL of water was added to an 1800 mL beaker, and mice were allowed to swim freely for 15 min, then gently submerged for 10 s using an inverted funnel. 2) Cold stress: Mice were placed in a 4 ℃ constant temperature incubator for 5 h. 3) Foot shock: Mice were placed in an electric shock chamber and randomly received 20 mild foot shocks over 20 min, each at 0.8 mA for 10 s. 4) Overnight lighting: Mice were placed in a constant temperature incubator for 12 h, from 19:00 to 7:00 the next day, at a temperature of 24 ℃ and maximum brightness. The following morning after model establishment, an open field experiment was conducted, recording the total distance traveled by the mice over 30 min, the distance traveled in the central area, etc.

[0109] B. The four stressors are applied sequentially in the order of CL, CS, FS, UT. Within a single day, mice were subjected to a series of stresses in the following order: overnight lighting (20:00-8:00 the next day), cold stress (8:30-13:30), foot shock (14:30-15:00), and underwater trauma (16:00-16:30). Half an hour after the modeling was completed, an open field experiment was conducted to record the total distance of movement and the distance of movement in the central area within 30 minutes.

[0110] C. Four stressors are applied sequentially in the order of CL, CS, UT, FS. Within a single day, mice were subjected to a series of stresses in the following order: overnight lighting (20:00-8:00 the next day), cold stress (8:30-13:30), underwater trauma (14:30-15:00), and foot shock (16:00-16:30). Half an hour after the modeling was completed, an open field experiment was conducted to record the total distance of movement and the distance of movement in the central area within 30 minutes.

[0111] 2. Experimental Results: In this part of the study, the type, number, and frequency of stressors were used as control conditions. Only the order of single application of the four stressors was adjusted. The anxiety-like behavior of mice was detected by open field test to clarify the regulatory role of the stressor application order variable in the construction of the acute stress model. The experimental results are as follows.

[0112] A. Four stressors are applied sequentially in the order of UT, CS, FS, CL. In Group A of the experiment, the four stressors were applied once a day in the order of UT, CS, FS, and CL. The experimental procedure was as follows: Figure 10 As shown in Figure A. After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the vocal startle reflex test. Results showed that in the open field test, the model group mice only exhibited a significant decrease in total movement distance, which was attributed to a decline in physical fitness. Therefore, we concluded that this version of the model failed to induce anxiety behavior in the mice. Figure 10BE); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice ( Figure 10 F) but cannot produce auditory cue fear behavior ( Figure 10 G); In the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 10 H).

[0113] B. The four stressors are applied sequentially in the order of CL, CS, FS, UT. In Group B, the four stressors were applied once a day in the order of CL, CS, FS, and UT. Figure 11 A), a 30-minute open field test was conducted half an hour after modeling. The results showed that in the open field test, the model in this sequence could only induce a decrease in the total distance of movement in mice, with no significant difference in the movement of the central area. Therefore, this version of the model failed to induce anxiety behavior in mice. Figure 11 BE).

[0114] C. Four stressors are applied sequentially in the order of CL, CS, UT, FS. In Group C, the four stressors were applied once each in the order of CL, CS, UT, and FS. Figure 11 F), a 30-minute open field experiment was conducted half an hour after modeling. The results showed that in the open field experiment, the percentage of central region movement distance and the percentage of central region dwell time induced by the model under this sequence were significantly increased, contrary to the expected results. Figure 11 GJ).

[0115] The results indicate that adjusting the order of application of the four stressors significantly affects the anxiety-like behavior phenotype. Orders ending in UT or CL showed no significant anxiety-inducing effect, while orders ending in FS actually improved anxiety-like behavior, contrary to the expected trend. This suggests that the order of stressor application plays an important role in the induction of anxiety-like behavior.

[0116] Example 5: The effect of changing the frequency of stressor stimulation on the establishment of an acute stress model 1. Experimental Methods A.MSAS model: Four stressors were intermittently stimulated in the order of CL, CS, UT, FS (each stimulus was given once), as in Examples 1 and 2.

[0117] B. The four stressors were intermittently stimulated in the morning / afternoon according to the following schedule: UT / CS, FS / CL, UT / CS, FS / CL (each stimulation was performed twice). 1. Modeling method: On the first day, underwater trauma was applied in the morning (8:00-8:30), followed by cold stress in the afternoon (11:30-16:00); on the second day, foot electric shock was applied in the morning (9:00-10:00), followed by all-night lighting (20:00-8:00 the next day); the first day's procedure was repeated on the third day; the second day's procedure was repeated on the fourth day. Experimental parameters were the same as above.

[0118] 2. Open Field Test: The open field test was conducted half an hour after modeling. The total distance of movement and the movement of the central area of ​​the mice were observed within 15 minutes. 3. Sound Startle Reflex Test: The sound startle reflex test was conducted the day after modeling. After the mice adapted to a background noise environment (70 dB) for 5 minutes, they underwent 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random intervals of 10-30 s). The maximum startle amplitude of each sound was recorded. 4. Conditioned Fear Test: The day after modeling, the mice were placed in an electric shock box (context A) for one conditioned-unconditioned stimulus pairing training session, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling was different from that for conditioned-unconditioned stimulus pairing training. 24 hours later, a fear test was conducted. Mice were placed in context A for 5 minutes without auditory cues or electric shock stimulation to test context fear. After changing the scene, mice were placed in context B and given 5 auditory cues to test cue fear. 5. Elevated cross maze test: The elevated cross maze test was conducted 4 hours after modeling. Mice were placed in the center of the maze with their heads facing any open arm. Mouse behavior was recorded for 10 minutes, and the proportion of activity on the open arms was analyzed. 6. Tail suspension test: The tail suspension test was conducted 24 hours after modeling. The tail of the mouse was fixed at the end, making it hang upside down and unable to touch the ground or other objects. Mouse behavior was recorded for 6 minutes, and the immobility time in the last 4 minutes was calculated.

[0119] 2. Experimental Results The experimental results of the MSAS model in Group A are the same as those in Examples 1 and 2.

[0120] In Group B of the experiment, the four stressors were applied twice each day in the following order: morning / afternoon: UT / CS, FS / CL, UT / CS, FS / CL. Figure 12A). After modeling, anxiety behavior was evaluated using the open field test, elevated cross maze test, fear behavior using the conditioned phobia test, startle reflex test using the vocalization startle reflex test, and depressive behavior using the tail suspension test. Results showed that in the open field test, compared with the control group, the model group mice had a significantly increased time spent in the central region, and the percentage of movement distance and number of times the central region was traversed showed an increasing trend, contrary to the expected trend. Figure 12 B): In the elevated cross maze test, compared with the control group, the percentage of time spent in the open arm, the percentage of time spent in the closed arm, and the percentage of time spent in the central region were significantly increased in the model group mice, which was also contrary to the expected trend. Figure 12 C); In the tail suspension experiment, the percentage of immobile time in the model group mice showed a decreasing trend, contrary to the expected trend. Figure 12 D); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice but cannot induce auditory cue fear behavior. Figure 12 E); In the sound startle reflex experiment, the shaking amplitude of mice in the model group was significantly increased at the 14th and 24th 115 dB sound stimuli, indicating that this version of the model can induce startle behavior in mice (E); Figure 12 F).

[0121] For four stressors (CL, CS, UT, and FS), different stimulation frequencies were set, and the results showed that the two stress programs induced different stress-related symptoms. The MSAS model, with one stressor per day, induced fear, anxiety, fright, and depressive behaviors. Group B, which increased the stimulation frequency compared to Group A, actually improved anxiety behaviors, while there was no significant difference in depressive behaviors. This suggests that in a complex stress model, a higher stimulation frequency is not necessarily better; it needs to be optimized by considering the number of stressors and the way they are applied to obtain a stable and comprehensive stress behavior phenotype. In addition, the MSAS model induced a more comprehensive range of stress symptoms, making this version of the model more ideal.

[0122] Example 6: The effect of changing the type of stressor on the establishment of an acute stress model 1. Experimental Methods A. Four stressors are applied sequentially in the order of UT, CS, FS, CL. 1. Model Establishment: 1) Underwater Trauma: Add 1200 mL of water to an 1800 mL beaker, immerse the mice in the water for free swimming for 15 min, and then gently submerge them for 10 s using an inverted funnel. 2) Cold Stress: Place the mice in a 4 ℃ constant temperature incubator for 5 h. 3) Foot Shock: Place the mice in an electric shock chamber and randomly administer 20 mild foot shocks over 20 min, each at 0.8 mA for 10 s. 4) Overnight Illumination: Place the mice in a constant temperature incubator for 12 h, from 19:00 to 7:00 the next day, at a temperature of 24 ℃ and a maximum brightness of 12000 Lux.

[0123] 2. Open Field Test: The open field test was conducted the morning after modeling, recording the total distance traveled by the mice and the distance traveled in the central area within 30 minutes. 3. Auditory Startle Reflex: The auditory startle reflex test was conducted the afternoon after modeling. 4. Conditioned Fear Test: The day after modeling, the mice were placed in an electric shock box (context A) for two cycles of conditioned-unconditioned stimulus pairing training, i.e., auditory-electric shock association training. Auditory parameters: 7000 Hz, 80 dB, 20 s; Electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned stimulus pairing training. The day after pairing training, fear testing was conducted. The mice were placed in context A for 5 minutes without auditory cues or electric shock stimulation to test contextual fear. After changing the scenario, the mice were placed in context B and given 5 auditory cues to test cued fear.

[0124] B. Four stressors were applied sequentially in the order of UT, CS, FS, and RS. 1. Modeling methods: 1) Underwater trauma: Add 1200 mL of water to an 1800 mL beaker, immerse the mice in the water for free swimming for 15 min, and then gently submerge them for 10 s using an inverted funnel. 2) Cold stress: Place the mice in a 4 ℃ constant temperature incubator for 5 h. 3) Foot shock: Place the mice in an electric shock chamber and randomly administer 20 mild foot shocks within 20 min, each at 0.8 mA for 10 s. 4) Restraint stress: Restrain the mice in a restraint tube for 12 h.

[0125] 2. Open Field Test: The open field test was conducted the morning after modeling, recording the total distance traveled and the distance traveled in the central area within 30 minutes. 3. Sound Startle Reflex Test: The sound startle reflex test was conducted the afternoon after modeling. 4. Conditioned Fear Test: The day after modeling, mice were placed in an electric shock box (context A) for one instance of conditioned-unconditioned stimulus pairing training, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned stimulus pairing training. Fear tests were conducted on the second and third days after pairing training. Mice were placed in context A for 5 minutes without sound cues or electric shock stimulation to test contextual fear. After changing the scenario, mice were placed in context B and given 5 sound cues to test cued fear.

[0126] 2. Experimental Results A. Four stressors are applied sequentially in the order of UT, CS, FS, CL. In Group A of the experiment, the four stressors were applied once a day in the order of UT, CS, FS, and CL. The experimental procedure was as follows: Figure 13 As shown in Figure A. After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the vocal startle reflex test. Results showed that in the open field test, the model group mice only exhibited a significant decrease in total movement distance, which was attributed to a decline in physical fitness. Therefore, we concluded that this version of the model failed to induce anxiety behavior in the mice. Figure 13 BE); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice ( Figure 13 F) but cannot produce auditory cue fear behavior ( Figure 13 G); In the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 13 H).

[0127] B. Four stressors were applied sequentially in the order of UT, CS, FS, and RS. In Group B, "CL" was replaced with "RS," a stressor that appears more frequently in the literature. The four stressors were applied once a day in the order of UT, CS, FS, and RS. Figure 14 A), after modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the vocal startle reflex test. Results showed that after replacing "CL" with "RS", the percentage of total movement distance to central area movement distance in the model group mice significantly increased in the open field test, contrary to the ideal trend. Figure 14BE); In the conditioned fear experiment, this version of the model induced situational fear behavior in mice that lasted for two days ( Figure 14 F, H), but cannot produce auditory cue fear behavior ( Figure 14 G, I); In the vocal startle reflex test, there was no significant difference between the two groups of mice (G, I); Figure 14 J). Therefore, this version of the model can only induce situational fear behavior.

[0128] The above results indicate that both groups A and B, with their different stressors, can significantly induce fear behavior, but have no significant effect on the sound startle reflex test. After replacing CL with RS, the mice's activity level in the open field showed a significant upward trend, suggesting that changes in the type of stressor can have a significant regulatory effect on anxiety-like behavior results related to the open field test.

[0129] Example 7: The Effect of Changing Stress Stimulation Patterns on the Establishment of an Acute Stress Model 1. Experimental Methods I. A total of 4 stimulations A. Four stressors are applied sequentially in the order of CL, CS, UT, FS. Within a single day, mice were subjected to a series of stresses in the following order: overnight lighting (20:00-8:00 the next day), cold stress (8:30-13:30), underwater trauma (14:30-15:00), and foot shock (16:00-16:30). Half an hour after the modeling was completed, an open field experiment was conducted to record the total distance of movement and the distance of movement in the central area within 30 minutes.

[0130] B.MSAS model: Four stressors are intermittently applied in the order of CL, CS, UT, FS. 1. Modeling Method: Day 1: Overnight Illumination: Mice were placed in a constant temperature incubator for 12 hours (20:00-8:00 the next day) at 24 ℃ with a light intensity of 12000 Lux. Day 2: Cold Stress: Mice were placed in a 4 ℃ constant temperature incubator for 5 hours (11:30-16:30). Day 3: Underwater Trauma: 1200 mL of water was added to an 1800 mL beaker, and mice were allowed to swim freely for 15 minutes, then gently submerged for 10 seconds using an inverted funnel. Day 4: Foot Shock: Mice were placed in an electric shock chamber and randomly received 20 mild foot shocks within 20 minutes, each at 0.8 mA for 10 seconds, with random intervals of 50-70 seconds.

[0131] 2. Open Field Test: The open field test was conducted 30 minutes and 24 hours after the last stressor (foot shock). The total distance of movement and the movement of the central area of ​​the mice were observed every 5 minutes within 30 minutes and plotted as a line graph. The movement in the first 15 minutes was also plotted as a bar graph. 3. Auditory Startle Reflex Test: The auditory startle reflex test was conducted the day after modeling. After the mice adapted to a background noise environment (70 dB) for 5 minutes, they underwent 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random intervals of 10-30 s). The maximum startle amplitude of each sound was recorded. 4. Tail Suspension Test: The tail suspension test was conducted the day after modeling. The tail of the mouse was fixed at the end, making it hang upside down and unable to touch the ground or other objects. The behavior of the mouse was recorded for 6 minutes, and the immobility time in the last 4 minutes was recorded. 5. Conditioned Fear Experiment: Six hours after modeling, mice were placed in an electric shock box (context A) for one instance of conditioned-unconditioned stimulus pairing training, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling differed from that for conditioned-unconditioned stimulus pairing training. Twenty-four hours after pairing training, fear testing was performed. Mice were placed in context A for 5 minutes without sound cues or electric shock stimulation to test contextual fear. After changing the scenario, mice were placed in context B and given 5 sound cues to test cued fear.

[0132] II. A total of 8 stimulations A. Four stressors were intermittently applied in the following order: morning / afternoon: UT / CS, FS / CL, UT / CS, FS / CL. 1. Modeling method: On the first day, underwater trauma was applied in the morning (8:00-8:30), followed by cold stress in the afternoon (11:30-16:00); on the second day, foot electric shock was applied in the morning (9:00-10:00), followed by all-night lighting (20:00-8:00 the next day); the first day's procedure was repeated on the third day; the second day's procedure was repeated on the fourth day. Experimental parameters were the same as above.

[0133] 2. Open Field Test: The open field test was conducted half an hour after modeling. The total distance of movement and the movement of the central area of ​​the mice were observed within 15 minutes. 3. Sound Startle Reflex Test: The sound startle reflex test was conducted the day after modeling. After the mice adapted to a background noise environment (70 dB) for 5 minutes, they underwent 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random intervals of 10-30 s). The maximum startle amplitude of each sound was recorded. 4. Conditioned Fear Test: The day after modeling, the mice were placed in an electric shock box (context A) for one conditioned-unconditioned stimulus pairing training session, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling was different from that for conditioned-unconditioned stimulus pairing training. 24 hours later, a fear test was conducted. Mice were placed in context A for 5 minutes without auditory cues or electric shock stimulation to test context fear. After changing the scene, mice were placed in context B and given 5 auditory cues to test cue fear. 5. Elevated cross maze test: The elevated cross maze test was conducted 4 hours after modeling. Mice were placed in the center of the maze with their heads facing any open arm. Mouse behavior was recorded for 10 minutes, and the proportion of activity on the open arms was analyzed. 6. Tail suspension test: The tail suspension test was conducted 24 hours after modeling. The tail of the mouse was fixed at the end, making it hang upside down and unable to touch the ground or other objects. Mouse behavior was recorded for 6 minutes, and the immobility time in the last 4 minutes was calculated.

[0134] B. The four stressors were intermittently stimulated in the morning / afternoon according to the following schedule: UT / CS, FS / CL, UT / CS, FS / CL (each stimulation was performed twice). 1. Modeling method: On the first day, underwater trauma was applied in the morning (8:00-8:30), followed by cold stress in the afternoon (11:30-16:00); on the second day, foot electric shock was applied in the morning (9:00-10:00), followed by all-night lighting (20:00-8:00 the next day); the first day's procedure was repeated on the third day; the second day's procedure was repeated on the fourth day. Experimental parameters were the same as above.

[0135] 2. Open Field Test: The open field test was conducted half an hour after modeling. The total distance of movement and the movement of the central area of ​​the mice were observed within 15 minutes. 3. Sound Startle Reflex Test: The sound startle reflex test was conducted the day after modeling. After the mice adapted to a background noise environment (70 dB) for 5 minutes, they underwent 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random intervals of 10-30 s). The maximum startle amplitude of each sound was recorded. 4. Conditioned Fear Test: The day after modeling, the mice were placed in an electric shock box (context A) for one conditioned-unconditioned stimulus pairing training session, i.e., sound-electric shock association training. Sound parameters: 7000 Hz, 80 dB, 20 s; electric shock parameters: 0.8 mA, 1 s. The scenario for foot shock modeling was different from that for conditioned-unconditioned stimulus pairing training. 24 hours later, a fear test was conducted. Mice were placed in context A for 5 minutes without auditory cues or electric shock stimulation to test context fear. After changing the scene, mice were placed in context B and given 5 auditory cues to test cue fear. 5. Elevated cross maze test: The elevated cross maze test was conducted 4 hours after modeling. Mice were placed in the center of the maze with their heads facing any open arm. Mouse behavior was recorded for 10 minutes, and the proportion of activity on the open arms was analyzed. 6. Tail suspension test: The tail suspension test was conducted 24 hours after modeling. The tail of the mouse was fixed at the end, making it hang upside down and unable to touch the ground or other objects. Mouse behavior was recorded for 6 minutes, and the immobility time in the last 4 minutes was calculated.

[0136] 2. Experimental Results This study kept the number of stressors and the total number of stimuli constant, and explored the impact of stressor stimulation patterns on the phenotype of an acute stress model by setting up two modes: continuous stimulation on a single day and intermittent stimulation over multiple days. At the same time, it combined various behavioral experiments to comprehensively evaluate the validity of the model. The results are presented in two parts.

[0137] I. A total of 4 stimulations A. Four stressors are applied sequentially in the order of CL, CS, UT, FS. In Group A, the four stressors were applied once a day in the order of CL, CS, UT, FS. Figure 15A). After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the sound startle reflex test. Results showed that in the open field test, the model in this sequence significantly increased the percentage of central region movement distance and the percentage of central region dwell time in mice, contrary to expectations (Figure 11 GJ); in the conditioned fear test, this version of the model induced situational fear behavior in mice but not auditory cue fear behavior (…). Figure 15 B); In the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 15 C).

[0138] B.MSAS model: Four stressors are intermittently applied in the order of CL, CS, UT, FS. The experimental results are the same as those in Examples 1 and 2.

[0139] Combining data from groups A and B, and targeting four stressors (CL, CS, UT, and FS), with the same total number of stimuli (4 times), different stimulation durations were set. The results showed that the two stress protocols induced different stress-related symptoms. In group A, the four stressors were concentrated in one day, inducing only fear behavior; there was no significant difference in startle and depressive behaviors, but anxiety behavior was improved in the open field test. In group B (MSAS model), the four stressors were distributed over four days. Compared with group A, this group induced significant anxiety, startle, and depressive behaviors simultaneously, in addition to fear behavior, resulting in more comprehensive stress symptoms. This suggests that in complex stress models, under the same number of stimuli, the stimulation pattern has a certain influence on the model phenotype, and multi-day stimulation is more conducive to comprehensively inducing various core stress symptoms.

[0140] II. A total of 8 stimulations A. Four stressors were intermittently applied in the following order: morning / afternoon: UT / CS, FS / CL, UT / CS, FS / CL. In Group A, the four stressors were applied twice each morning / afternoon in the following order: UT / CS, FS / CL, UT / CS, FS / CL (Figure 16 A). After modeling, anxiety behavior was evaluated using the open field test, elevated cruciate maze test, fear behavior using the conditioned phobia test, startle reflex test using the vocalization startle reflex test, and depressive behavior using the tail suspension test. Results showed that in the open field test, compared to the control group, the model group mice had a significantly increased time spent in the central region, and the percentage of movement distance and number of times the central region was traversed showed an increasing trend, contrary to the expected trend. Figure 16 B): In the elevated cross maze test, compared with the control group, the percentage of time spent in the open arm, the percentage of time spent in the closed arm, and the percentage of time spent in the central region were significantly increased in the model group mice, which was also contrary to the expected trend. Figure 16C); In the tail suspension experiment, the percentage of immobile time in the model group mice showed a decreasing trend, contrary to the expected trend. Figure 16 D); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice but cannot induce auditory cue fear behavior. Figure 16 E); In the sound startle reflex experiment, the shaking amplitude of mice in the model group was significantly increased at the 14th and 24th 115 dB sound stimuli, indicating that this version of the model can induce startle behavior in mice (E); Figure 16 F).

[0141] B. The four stressors are intermittently applied in the order of CL, CS, UT, FS, CL, CS, UT, FS. In Group B of the experiment, the four stressors were applied twice daily over eight days in the following order: CL, CS, UT, FS, CL, CS, UT, FS. The model flowchart is as follows: Figure 17 As shown in Figure A. After modeling, anxiety behavior was evaluated using the open field test, fear behavior using the conditioned fear test, and startle reflex test using the vocal startle reflex test. Results showed that in the open field test, compared to the control group, while the total movement distance remained the same, the percentage of movement distance in the central region, the percentage of time spent in the central region, and the number of times the mice passed through the central region were all significantly reduced in the model group mice, indicating that the model group mice exhibited anxiety behavior. Figure 17 BI); In conditioned fear experiments, this version of the model can induce situational fear behavior in mice but cannot induce auditory cue fear behavior. Figure 17 JK); In the vocal startle reflex test, there was no significant difference between the two groups of mice ( Figure 17 L). The results above show that this version of the model can induce anxiety and fear behaviors in mice.

[0142] For four stressors (CL, CS, UT, and FS), under the condition of the same total number of stimuli (8 times), different durations and methods of stimulus administration were set. The results showed that the two stress protocols induced different stress-related symptoms. In group A, the 8 stimuli were distributed over four days, which significantly induced fear and startle behaviors, but anxiety behaviors were improved in the open field test. In group B, the 8 stimuli were distributed over 8 days. Compared with group A, it significantly induced anxiety behaviors, but had no significant effect on startle reflex. This suggests that in the compound stress model, under the same number of stimuli, the stressor stimulation pattern has an important regulatory role on the model phenotype, and long-term dispersed stimulation is more conducive to inducing anxiety-like behaviors.

[0143] Example 8: Investigating the stress sensitivity of different species of male mice using a sound startle reflex experiment. 1. Experimental Methods Model Establishment: 1) Overnight Illumination: Mice were placed in a constant temperature incubator for 12 hours, from 19:00 to 7:00 the next day, at a temperature of 24℃ and maximum brightness. 2) Cold Environment: Mice were placed in a constant temperature incubator at 4℃ for 4 hours. 3) Underwater Trauma: 2000mL of water was added to a 3000mL beaker, and mice were allowed to swim freely for 40 seconds, then gently submerged for 10 seconds using an inverted funnel. 4) Body Pain: Mice were placed in an electric shock chamber and randomly received 20 mild foot shocks within 20 minutes, each at 0.8 mA and 1 second. Freezing time was recorded for the control and experimental groups within 20 minutes. The weight of each mouse was measured before modeling and again the morning after modeling. Food intake from the end of modeling to the morning of the following day was also recorded.

[0144] The next afternoon after modeling, the startle reflex was assessed by the following experiment: After mice had adapted to a background noise (70 dB) environment for 5 minutes, they were subjected to 30 repeated auditory stimulation tests (40 ms each time, 115 dB each time; random intervals of 10-30 seconds). The maximum startle amplitude of each sound was recorded.

[0145] Grouping: (1) control: C57, Balb / c, ICR (2) UPCS: C57, Balb / c, ICR.

[0146] 2. Experimental Results Under the same combined stressor modeling conditions, the sensitivity of C57, Balb / c, and ICR mice was assessed using a vocal startle reflex test. Results showed that in male mice, the startle amplitude in the Balb / c strain model group was significantly higher than that in the control group, while there was no significant difference between C57 and ICR strains; in female mice, there were no significant differences between the two groups in any of the three strains. Figure 18 The above results indicate that, under the same combined stressor modeling conditions, male Balb / c mice are more sensitive to stress stimuli.

[0147] Example 9: Investigating the stress sensitivity of different species of male mice using a conditioned fear experiment. 1. Experimental Methods Mice were placed in an electric shock chamber for 2 minutes to acclimatize, and then underwent 4 CS-US paired training sessions in context A. Each test consisted of 20 seconds of 80 dB auditory cues followed by 1 second of 0.8 mA foot shocks, with a 39-second interval between each shock test.

[0148] Modeling of a composite stressor model after training. 1) Overnight lighting: Mice were placed in a constant temperature incubator for 12 hours, from 19:00 to 7:00 the next day, at a temperature of 24℃ and maximum brightness. 2) Cold environment: Mice were placed in a constant temperature incubator at 4℃ for 4 hours. 3) Underwater trauma: 1200mL of water was added to an 1800mL beaker, and mice were allowed to swim freely for 40 seconds, then gently submerged for 10 seconds using an inverted funnel. 4) Body pain: Mice were placed in context B and randomly given 20 mild plantar electric shocks within 20 minutes, each 0.8 mA for 1 second. Fear behavior testing was conducted the day after modeling. Mice were exposed to context A for 3 minutes (without a sound cue), and the percentage of freezing time in the total test time was measured. Afterwards, mice were moved to context C and given 12 doses of 20 stone, and the percentage of freezing time in the total test time was measured.

[0149] 2. Experimental Results This study assessed the differences in sensitivity to combined stress in three mouse strains—C57, Balb / c, and ICR—through a conditioned fear experiment. Figure 19 The results showed that in male mice, the Balb / c strain exhibited the strongest stress sensitivity: its model group showed an upward trend in both situational fear and cue-based fear tests. In contrast, C57 mice only showed an upward trend in the percentage of rigidity in the situational fear test, while the cue-based fear response was lower than that of the control group, with the two trends being opposite. ICR mice showed smaller changes in the percentage of rigidity in both fear tests, with a lower overall response level, suggesting that they had the lowest sensitivity to this modeling protocol. In female mice, the Balb / c mouse model group showed an upward trend in both situational fear and cue-based fear tests; C57 mice did not show the downward trend observed in cue-based fear responses in males, but cue-based fear did show a downward trend; ICR mice showed a downward trend in the percentage of rigidity in the cue-based fear test. Combining the vocal startle reflex test and the conditioned fear test, male Balb / c mice had the highest sensitivity to the combined stress modeling in this experiment, followed by male C57 mice, while ICR mice (both male and female) had the lowest sensitivity.

[0150] Example 10: Pharmacodynamic differences of ketamine in MSAS and classic acute stress response animal models Using ketamine as the instrumental drug, the pharmacodynamic differences of ketamine were compared in the MSAS model, the classical conditioned fear model, and the acute restraint stress model. The results showed that: First, comparing the intervention effect of ketamine on anxiety-like behavior, intraperitoneal injection of 5 mg / kg ketamine significantly reversed model-induced anxiety-like behavior in the MSAS model; however, in the acute restraint stress model, a dose of 15 mg / kg was required to produce an anti-anxiety effect. This suggests that the effective dose of the same drug varies in different acute stress animal models, reflecting differences in drug sensitivity among different models, which may be related to differences in the stress modeling mechanism and the physiological state of the animals. Second, comparing the intervention effect of ketamine on fear behavior, ketamine did not show a significant extinction effect of fear memory in either the MSAS model or the classical conditioned fear model. These results suggest that the same drug can produce an anti-stress effect consistent with the classical model in the MSAS model; therefore, the MSAS model can be used to evaluate the intervention effect of drugs on acute stress responses. Furthermore, different stress models respond differently to the same drug, and the acute stress effect of ketamine intervention may focus more on alleviating anxiety-like behaviors.

[0151] Example 11 Evaluation of the intervention effect of drugs on acute stress response based on MSAS model Using the established MSAS model, the intervention effect of candidate compounds on acute stress response was evaluated, and the results are as follows. (1) Xelosiben (5-HT 2A / 1A (1) Receptor partial agonists: Intraperitoneal administration of 1 mg / kg significantly reduced model-induced fear behavior; 0.5 mg / kg significantly alleviated anxiety-like behavior. (2) Fluvoxamine (5-HT reuptake inhibitor, σ1 receptor agonist): Intraperitoneal administration of 30 mg / kg significantly alleviated fear behavior; 10–30 mg / kg doses effectively improved anxiety-like behavior. (3) Phenylezil (monoamine oxidase inhibitor) and OMDM-2 (endocannabinoid uptake inhibitor) did not show significant anti-fear or anti-anxiety effects at the tested doses. The above results indicate that selocillin and fluvoxamine can effectively intervene in acute stress-induced fear and anxiety-like symptoms in the MSAS model, providing a dose basis for subsequent mechanism research and drug development.

[0152] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing an acute stress animal model, characterized in that, The method includes: Step (1): The mice were exposed to light overnight from the first day to the next day; Step (2): The mice were exposed to a cold environment on the second day; Step (3): On the third day, the mice experienced underwater trauma; Step (4): On the fourth day, the mouse experiences an electric shock.

2. The method according to claim 1, characterized in that, The overnight lighting in step (1) lasts for 12 hours; Preferably, the illuminance of the all-night lighting in step (1) is 12000 Lux; Preferably, the overnight lighting time in step (1) is from 20:00 on the first day to 8:00 on the next day; Preferably, the overnight lighting time in step (1) is from 19:00 on the first day to 7:00 on the next day; Preferably, the temperature in step (1) is room temperature; Preferably, the temperature in step (1) is 24°C.

3. The method according to claim 1, characterized in that, The cold environment in step (2) is 4℃; Preferably, step (2) is spaced 3.5 h apart from step (1); Preferably, step (2) lasts for 5 hours; Preferably, step (2) is performed between 11:30 and 16:30 on the second day.

4. The method according to claim 1, characterized in that, Step (3) involves first allowing the mouse to swim freely, and then causing the mouse to drown. Preferably, the drowning lasts for 10 seconds; Preferably, the free swimming lasts for 15 minutes; Preferably, step (3) is spaced 16-17 hours apart from step (2); Preferably, step (3) is performed between 8:30 and 9:30 on the third day.

5. The method according to claim 1, characterized in that, In step (4), the electric shock is to receive 20 electric shocks within 20 minutes; Preferably, the electric shock current is 0.8 mA; Preferably, each electric shock lasts for 10 seconds; Preferably, the electric shocks are spaced 50-70 seconds apart. Preferably, step (4) is spaced 23-25 ​​hours apart from step (3); Preferably, step (4) is performed between 8:30 and 9:30 on the fourth day.

6. The method according to any one of claims 1-5, characterized in that, The mice were selected from C57 mice, Balb / c mice, and ICR mice; Preferably, the mice include female mice and male mice; Preferably, the mouse is a male Balb / c mouse; Preferably, the method for constructing an acute stress animal model includes: Step (1): From the first day to the next day, the mice were kept under 24°C for 12 hours of continuous light, with a light intensity of 12000 Lux. Step (2): On the second day, the mice were subjected to a 4°C cold environment for 5 hours after experiencing overnight lighting, with an interval of 3.5 hours. Step (3): On the third day, the mice swam freely for 15 minutes, then drowned for 10 seconds; Step (4): On the fourth day, the mice received 20 electric shocks within 20 minutes. The current of the electric shock was 0.8 mA, the duration of each shock was 10 s, and the interval between each shock was randomized from 50 s to 70 s.

7. A system for constructing an acute stress animal model, characterized in that, The system includes: First treatment unit: used to subject mice to overnight lighting from the first day to the next day; Second treatment unit: used to subject mice to a cold environment on the second day; Third treatment unit: used for mice to experience underwater trauma in the third day; Fourth processing unit: used to subject mice to electric shocks on the fourth day.

8. A construction apparatus for constructing an acute stress animal model, characterized in that, The construction apparatus includes: one or more processors, and a memory for storing one or more computer programs, which, when executed by the one or more processors, implement: Procedure 1: The mice were exposed to light overnight from day 1 to day 2; Procedure 2: The mice were exposed to a cold environment on the second day; Operation 3: On the third day, the mice experienced underwater trauma; Operation 4: On the fourth day, the mice were given an electric shock.

9. Any one of the following methods: (1) A method for screening drug candidates for treating acute stress response, characterized in that, The method includes: a) Applying the reagent to be screened to the acute stress agonist constructed by the method of any one of claims 1-6; b) To test the therapeutic effect of the reagents to be screened on acute stress response; (2) A method for evaluating the therapeutic effect of a drug for treating acute stress response, characterized in that the method comprises: a) Applying the drug to the acute stress agonist constructed by the method of any one of claims 1-6; b) To test the therapeutic effect of the drug on the acute stress response; (3) A method for studying the pathogenesis of acute stress response, characterized in that the method uses an acute stress animal constructed by the method described in any one of claims 1-6 to study the pathogenesis of acute stress response.

10. Any of the following applications: (1) The use of the acute stress animal model constructed by the method of any one of claims 1-6 in screening drug candidates for the treatment of acute stress response; (2) The application of the acute stress animal model constructed by the method of any one of claims 1-6 in evaluating the therapeutic effect of drugs for treating acute stress response; (3) The application of the acute stress animal model constructed by the method of any one of claims 1-6 in the study of the pathogenesis of acute stress response.

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