Establishment method and application of anxiety disorder model based on pressure stimulation of peers

Abstract

The invention discloses a construction method and application of an anxiety disorder model based on peer pressure stimulation, and belongs to the field of animal models. The model construction method comprises the following steps: (1) experimental animal adaptation; (2) introducing resources and social elements; and (3) establishing a peer pressure scene. According to the method, through the steps of same nest adaptation-resource and social contact introduction-resource and social contact separation to form resource and social contact differences, the scene that the pressure of the peers is generated due to non-uniform resource and social contact relationship distribution in human life is accurately simulated, and the scene is highly matched with the actual attack inducement of human anxiety disorder; the physiological process of pressure-induced anxiety of the peers can be reflected more truly, and a more reliable model is provided for research on pathogenesis of human anxiety disorder and drug evaluation. The method is easy to operate and non-invasive, interference of non-experimental factors on mouse behaviors can be reduced to the maximum extent, and reliability and repeatability of the model are guaranteed.

Description

Technical Field

[0001] This invention relates to the field of animal models, specifically to a method for constructing and applying an anxiety disorder model based on peer pressure stimulation. Background Technology

[0002] Anxiety disorders are among the most prevalent mental health problems worldwide, primarily characterized by anxiety, fear, and autonomic dysfunction. Their prevalence ranks first among various mental illnesses. Anxiety disorders not only severely damage an individual's mental health but also negatively impact physical health and social functioning. Currently, treatment for anxiety disorders mainly involves psychotherapy and medication, primarily antidepressants such as serotonin reuptake inhibitors (SRIs) and anti-retroviral drugs (ARBs). However, approximately 30% of patients do not respond to existing treatments (Garakani, A., et al.). Pharmacotherapy of Anxiety Disorders: Current and Emerging Treatment Options (Frontiers in Psychiatry, 2020. 11: p. 595584.) This greatly increases the disease burden of anxiety disorders on patients, families, and society. However, the pathogenesis of anxiety disorders is unclear, which greatly hinders the development of new anti-anxiety drugs and intervention strategies.

[0003] The pathogenesis of anxiety disorders is complex and not yet fully understood, with social environmental factors playing a significant role. In human society, individuals often face competition for resources (such as material and opportunity resources) and social relationships (such as emotional support and social recognition). This competition and the resulting peer pressure are a major trigger for anxiety. For example, in the workplace, competition for promotions and differences in resource allocation among colleagues; in the school environment, differences in social popularity and material conditions among classmates, can all cause psychological stress, which, over time, can easily develop into anxiety disorders.

[0004] Existing research on constructing mouse anxiety models mainly focuses on social isolation, chronic unpredictable mild stress, etc. (Zhao, H., et al., Recent advances in anxiety disorders: Focus on animal models and pathological mechanisms(Animal Model Exp Med, 2023. 6(6):p. 559-572.) Although these models can induce anxiety-like behaviors in mice to a certain extent, they have two major drawbacks: First, they are difficult to accurately simulate the peer pressure scenarios caused by resource and social competition in human life, and have a weak correlation with the actual causes of anxiety disorders in humans; Second, the efficiency of obtaining control and experimental groups is low, and it is usually necessary to set up different cages to raise control mice and experimental mice separately, which not only increases the breeding space and cost, but may also introduce additional interference variables due to the environmental differences of different cages, affecting the reliability of experimental results.

[0005] Therefore, there is an urgent need to develop a mouse anxiety disorder model that can more realistically simulate peer stress scenarios in humans and efficiently obtain control and experimental groups, so as to provide a more reliable experimental tool for the study of the mechanism of anxiety disorders and the development of new drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a mouse anxiety disorder model based on peer pressure stimulation, so as to overcome the shortcomings of existing mouse anxiety models that cannot simulate human peer pressure scenarios, have weak correlation with actual disease inducing factors, and have low efficiency in obtaining control and experimental groups.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for constructing an anxiety disorder model based on peer pressure stimuli, comprising the following steps: (1) Adaptation of experimental animals: Two male adult mice of the same age were housed together in the same cage for a week to allow them to adapt to the group living environment; (2) Introduce resources and social elements: Introduce marshmallows and female adult mice into the mouse cage and continue to raise them together for another week; (3) Establishing a peer pressure scenario: Two male mice in the cage were separated by a transparent perforated partition, so that the marshmallow and the female mouse were kept with only one of the male mice for a week. The male mice kept with the marshmallow and the female mouse showed anxiety-like phenotypes, and the anxiety disorder model based on peer pressure stimulation was obtained.

[0008] Specifically, step (1) is the littermate adaptation stage (S1). Male adult mice of the same age are selected as littermates and placed in the same standard cage for a week. The purpose of this stage is to allow the mice to adapt to group life, establish stable peer relationships, and lay the foundation for the subsequent introduction of competitive factors. During the rearing period, sufficient food and water are ensured, the light cycle is strictly controlled, and the environment is quiet and undisturbed.

[0009] Preferably, the same-age male adult mice are C57 mice, and the initial feeding age is 8 weeks.

[0010] As a preferred option, the rearing conditions are: temperature 22-25℃, humidity 45-55%, providing sufficient standard mouse feed and sterile water, controlling the light cycle to 12 h light / 12 h dark (e.g., 07:00-19:00 light), while keeping the environment quiet and free from additional stimuli.

[0011] As a preferred method, during the feeding period, the mice were stroked along their backs with their fingertips for 1-2 minutes at a fixed time each day to reduce the mice's stress response to the experimenters.

[0012] Step (2) is the introduction of resource and social elements (S2). After completing the S1 rearing, commercially available edible marshmallows (as a representative of material resources) and age-matched female mice (as representatives of social resources) are introduced into the cages. All mice continue to be reared together for one week. This stage allows the mice to become familiar with the living environment containing resource and social elements, so that subsequent differences in the allocation of resources and social elements can more significantly generate peer pressure. The same environmental conditions as in S1 are maintained during the rearing period.

[0013] Preferably, the female mouse is of the same breed and age as the adult male mouse. Specifically, the female mouse is an age-matched female C57 mouse.

[0014] As a preferred option, marshmallows are continuously supplied during the feeding period, with each supply consisting of 4-5 g of marshmallows.

[0015] Step (3) is the peer pressure scenario establishment stage (S3). Two male mice in the cage are separated using a transparent perforated partition. At the same time, the marshmallow and female mice are kept with only one male mouse, forming a contrasting scenario of "resource-social exclusivity" (experimental group) and "resource-social deprivation" (control group). This is continued for one week. During the rearing period, the same environmental conditions as in S1 are maintained to ensure the stability of the mice's physiological needs and to avoid other factors interfering with the induction of peer pressure.

[0016] In this invention, a transparent perforated partition effectively separates two male mice without affecting sensory input. Preferably, the transparent perforated partition completely separates the two male mice on both sides of the cage, with the space on both sides being the same size; the height of the partition is the same as the height of the cage, the thickness is 2 mm, and the perforation diameter is 1 cm.

[0017] After the peer pressure stimulus ended, the anxiety phenotypes of mice in the experimental and control groups were verified. As an optimal candidate, the anxiety-like phenotypes of two male mice were assessed using the open field test and the elevated cross maze test.

[0018] The study found that mice in the experimental group, which received exclusive access to resources and social elements, exhibited significantly higher anxiety-like phenotypes than the control group, thus successfully establishing a model of anxiety disorder based on peer pressure. The control group showed no significant phenotypic differences compared to conventionally fed mice that did not receive the necessary resources and social elements.

[0019] In this invention, by simulating resource and social competition scenarios and using the same rat cage to separate the control group and experimental group simultaneously, the efficiency of experimental group acquisition is greatly improved while ensuring that the model closely resembles the actual human pathogenesis scenario. This provides a more efficient and reliable experimental model for the study of the mechanism of anxiety disorders and drug development.

[0020] Another object of the present invention is to provide an anxiety disorder model prepared by the above-described construction method and the uses of the model, the uses of which include using the model to study the pathogenesis of peer pressure-induced anxiety disorders or to screen or identify drugs that have therapeutic effects on anxiety disorders.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The scenario simulation is closer to human reality: This invention accurately simulates the scenario of peer pressure caused by the uneven distribution of resources (such as material resources) and social relationships (such as opposite-sex social opportunities) in human life through the steps of "fitting in the same nest - introducing resources and social interaction - separation to form resource and social differences". It is highly consistent with the actual causes of human anxiety disorders and can more realistically reflect the physiological process of anxiety induced by peer pressure, providing a more reliable model for studying the pathogenesis of human anxiety disorders.

[0022] (2) High efficiency in obtaining control and experimental groups: The present invention innovatively adopts the same cage partition design, so that two male mice from the same litter can simultaneously become the experimental group with "exclusive resources and social interaction" and the control group with "lack of resources and social interaction" under the same initial environment and the same feeding conditions. There is no need to set up a separate control cage, which not only saves feeding space and cost, but also eliminates the additional variables caused by the differences in the environment of different cages (such as temperature, light uniformity, noise interference) to the greatest extent, significantly improving the efficiency of obtaining experimental groups and the reliability of results. It is especially suitable for batch experimental scenarios that require the simultaneous construction of a large number of control and experimental groups.

[0023] (3) Simple and non-invasive operation: The model construction process of this invention does not require complex surgery or high-intensity stress treatment. It can be completed simply by adjusting the feeding environment and separating the mice with partitions. The operation steps are simple and easy to perform, causing little physiological damage to the mice. It can minimize the interference of non-experimental factors on the behavior of the mice and ensure the reliability and repeatability of the model. Attached Figure Description

[0024] Figure 1This is a schematic diagram of the model construction process of the present invention, showing the cage layout and mouse status in three stages: S1 (livestock mice are raised together), S2 (introducing marshmallows and female mice are raised together), and S3 (separated by partitions, marshmallows and female mice are raised together with one of the male mice); in addition, to reflect whether the anxiety level of the control group is normal, a group of mice in the "no-operation group" was set up.

[0025] Figure 2 This is a comparison chart of open field test results. The horizontal axis represents the three groups of mice (experimental group: resource-social exclusivity group, control group: resource-social deficiency group, and no-operation group: social group), and the vertical axis represents the time spent in the central region (seconds). It clearly shows that the experimental group mice spent significantly less time in the central region than the control group, while the control group and the no-operation group had similar times. ** indicates... p <0.01, ns indicates no significant difference.

[0026] Figure 3 This is a comparison chart of the results of the elevated cross maze test. The vertical axis represents the dwell time on the open arm (seconds), and the horizontal axis represents the three groups of mice. The experimental group mice showed a shorter dwell time on the open arm, while the control group and the no-operation group had similar times. *** indicates... p <0.001, ns indicates no significant difference.

[0027] Figure 4 The results of the elevated cross maze test after injecting an anxiolytic drug into a mouse model of anxiety disorder are shown. The vertical axis represents the open arm dwell time (seconds), and the horizontal axis represents the saline-treated control group and the fluoxetine-treated group. The fluoxetine-treated group had a significantly longer open arm dwell time than the saline-treated group. ** indicates that the open arm dwell time was significantly longer in the fluoxetine-treated group. p <0.01. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] Example 1: Construction of an Anxiety Disorder Model 1. Preparation and Adaptation of Laboratory Animals Twenty-four 8-week-old male C57BL / 6J mice were purchased from a reputable laboratory animal supplier and housed in a standard animal laboratory (temperature 22-25℃, humidity 45-55%). The mice were randomly divided into 12 groups of two mice each (library mice) and placed in 12 standard cages (each cage measuring 30×18×15 cm). 3 ), and carry out S1 stage feeding (1 week).

[0031] Gently stroke the mice at a fixed time each day (e.g., 9 a.m.) (wear gloves and stroke the mouse’s back with your fingertips for 1-2 minutes) to reduce the mice’s stress response to the experimenters and ensure stable behavior in subsequent experiments.

[0032] During the rearing period, provide sufficient standard mouse feed and sterile water, and strictly control the light cycle to 12 h / 12 ​​h (07:00-19:00 light).

[0033] 2. Introduce resources and social elements for raising animals. After completing the S1 stage of rearing, proceed to the S2 stage (1 week). Place one commercially available edible marshmallow (approximately 5g, placed next to the food bowl in the corner of the cage) and one 8-week-old female C57BL / 6J mouse in each cage and continue rearing.

[0034] Observe the mice's eating, drinking and interaction every day to ensure that the marshmallows are not consumed quickly (if the marshmallows are completely consumed within 24 hours, replenish them with the same weight of marshmallows in time), and record the interaction frequency between the female mice and the two male mice to ensure the stable presence of social elements.

[0035] 3. Create a peer pressure environment and raise [the animal / animal]. After phase S2, phase S3 (1 week) begins. A transparent perforated partition (the partition height should match the cage height, 2 mm thick, with 1 cm perforations, and made of acrylic) is inserted in the center of each cage, completely separating the two male mice to opposite sides of the cage, ensuring that the space on each side is the same size (30×9×15 cm). 3 Furthermore, mice can achieve sensory communication through perforations in the septum.

[0036] The positions of the marshmallow and female mice were adjusted so that they were only in the space occupied by one of the male mice on one side (defined as the experimental group: resource-social exclusivity group), while the space occupied by the male mice on the other side was empty of the marshmallow and female mice (defined as the control group: resource-social deprivation group). The same environmental conditions as S1 were maintained during the rearing period.

[0037] Male mice that were not subjected to steps 2 and 3 above and were continuously fed for 3 weeks in stage S1 were defined as the non-operation group.

[0038] Test Example 1: Open Field Test Mice from the experimental group, control group, and unmanaged group after the S3 phase in Example 1 were subjected to open field testing to detect anxiety-like phenotypes.

[0039] The core principle of the open field test is to assess the anxiety level of animals by observing their behavior in response to the conflict between their innate anxiety and exploratory instincts in unfamiliar open environments. The experimental setup is a square open field box with sides of 50 cm and a height of 30 cm. The bottom of the box is divided into 25 equally sized smaller squares, with the central nine squares forming the central area. The open field test method is as follows: Before testing, the open area box was wiped and disinfected with 75% ethanol and allowed to air dry before the experiment. Mice were removed from their cages and gently placed in the center of the open area box. The Anymaze animal behavior analysis system was immediately started, and the mouse's movement trajectory was continuously recorded for 10 minutes. The statistical indicator was the time spent in the central area. After the test, the mice were returned to their original cages, and the open area box was wiped with ethanol to remove residual odors before the next mouse was tested.

[0040] Statistical results are as follows Figure 2 As shown, the experimental group mice spent significantly less time in the central region than the control group ( p <0.05). In the open field test, the shorter the time mice spent in the central region, the higher their anxiety level. These results indicate that the experimental group mice had a higher level of anxiety.

[0041] Test Example 2: Elevated Cross Maze Test Mice from the experimental group, control group, and unmanaged group after the S3 phase in Example 1 were subjected to the elevated cross maze test to detect anxiety-like phenotypes.

[0042] The elevated cross maze utilizes the exploratory nature of animals towards novel environments and the contradictory conflict between their fear and anxiety about being suspended in an open arm to examine their anxiety state. The elevated cross maze setup consists of a central intersection area (5×5 cm). 2 ), 2 open arms (30×5 cm) 2 ) and 2 closed arms (30×5×15 cm) 3 It consists of [parts], and is erected as a whole at a height of 50 cm above the ground. The test method for the elevated cross maze is as follows: Before testing, wipe the surface of the device with 75% ethanol and allow it to dry before starting the experiment. During the test, place the mouse in the central junction area with its head facing the open arm, activate the Anymaze animal behavior analysis system, and record its behavior over 5 minutes. The statistical index is the time spent in the open arm. After the test, clean the device and proceed to the next mouse.

[0043] Statistical results are as follows Figure 3 As shown, the experimental group mice spent a significantly shorter time in the open arm than the control group ( p<0.05). In the elevated cross maze test, the shorter the time the mice stayed in the open arm, the more obvious the anxiety-like phenotype of the mice. The above results further verified the anxiety-like phenotype of the experimental group mice.

[0044] Application Example 1 The anxiety disorder model mice constructed in Example 1 were divided into two groups. The mice were acutely injected with saline and the classic anti-anxiety drug fluoxetine (15 mg / kg). Half an hour later, the mice underwent the elevated cross maze test, which was performed in the same way as in Example 2, to detect the anxiety-like phenotype.

[0045] Statistical results are as follows Figure 4 As shown, the time spent in the open arm was significantly longer in the fluoxetine-treated group than in the saline-treated group (p < 0.05). These results confirm that fluoxetine alleviated the anxiety levels in the experimental group mice.

[0046] In summary, the core operation of the above embodiments revolves around "efficient grouping by separating mice in the same cage." There are no complex or invasive procedures. Researchers can simultaneously construct control and experimental groups using healthy C57 mice by following the above steps, significantly improving model preparation efficiency and achieving a 100% success rate in constructing anxiety disorder models. Furthermore, because the initial state and breeding environment of the two groups of mice in the same cage are completely consistent, experimental errors are significantly reduced, and the model reliability is higher.

[0047] Therefore, the mouse model constructed in this invention can be mass-produced in the industry and applied to the study of the mechanism of peer pressure-induced anxiety disorder and the development of anti-anxiety drugs.

[0048] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for constructing an anxiety disorder model based on peer pressure stimuli, characterized in that, Includes the following steps: (1) Adaptation of experimental animals: Two male adult mice of the same age were housed together in the same cage for a week to allow them to adapt to the group living environment; (2) Introduce resources and social elements: Introduce marshmallows and female adult mice into the mouse cage and continue to raise them together for another week; (3) Establishing a peer pressure scenario: Two male mice in the cage were separated by a transparent perforated partition, so that the marshmallow and the female mouse were kept with only one of the male mice for a week. The male mice kept with the marshmallow and the female mouse showed anxiety-like phenotypes, and the anxiety disorder model based on peer pressure stimulation was obtained.

2. The construction method as described in claim 1, characterized in that, The same-age male adult mice were C57 mice, and the initial age of the mice was 8 weeks.

3. The construction method as described in claim 1 or 2, characterized in that, The female mice were of the same breed and age as the adult male mice.

4. The construction method as described in claim 1, characterized in that, In steps (1)-(3), the feeding conditions are: temperature 22-25℃, humidity 45-55%, providing sufficient standard mouse feed and sterile water, controlling the light cycle to 12 h of light / 12 h of darkness, and keeping the environment quiet and free from additional stimuli.

5. The construction method as described in claim 1, characterized in that, In step (1), during the feeding period, the mice were stroked along their backs with their fingertips for 1-2 minutes at a fixed time each day to reduce the stress response of the mice to the experimenters.

6. The construction method as described in claim 1, characterized in that, In steps (2) and (3), marshmallows are continuously supplied during the feeding period, with each supply consisting of 4-5 g of marshmallows.

7. The construction method as described in claim 1, characterized in that, In step (3), the transparent perforated partition completely separates the two male rats on both sides of the cage, with the space on both sides being the same size; the height of the partition is the same as the height of the cage, the thickness is 2 mm, and the aperture is 1 cm.

8. The construction method as described in claim 1, characterized in that, In step (3), after the feeding was completed, the anxiety-like phenotypes of the two male rats were assessed using the open field test and the elevated cross maze test.

9. The use of an anxiety disorder model prepared by the construction method according to any one of claims 1-8, characterized in that, include: The model can be used to study the pathogenesis of peer pressure-induced anxiety disorders or to screen or identify drugs that have therapeutic effects on anxiety disorders.

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