Construction method and application of lonely mouse model

Abstract

The invention relates to a construction method and application of a loneliness mouse model, and the method comprises the steps: placing an experimental mouse in an isolation region in a transparent acrylic isolation cover through an environment in which physical isolation and social deprivation stimulation coexist, forming a visually accessible but physically isolated social environment with five normal living mice, and continuously inducing chronic loneliness for 4 weeks. The effectiveness of the model is verified through a multi-dimensional evaluation system, including behavioristics (open field, elevated maze and three-box experiment), neuroendocrine (serum noradrenaline and angiotensin II), immune inflammatory factors (IL-1beta, IL-6, TNF-alpha and CCL5) and nerve activation (blue spot, almond nucleus, lateral nucleus septum, medial prefrontal cortex and center joint dorsal nucleus C-Fos staining) indexes. According to the intervention method, dynamic environment abundance and social stimulation are adopted, running wheels are arranged, a modular abundance device is arranged, and a social environment of new mice is introduced, so that pathological phenotypes caused by loneliness are reversed. And a systematic experiment tool is provided for exploring association of aloneness and diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of experimental animal model construction, and in particular to a loneliness mouse model construction method and application thereof. Background Art

[0002] Loneliness is a subjective and painful emotional experience caused by the inability of the quantity or quality of social connections to meet the needs of the individual, which will have a negative impact on the individual's physical and mental health. With the rapid changes in society and the accelerated pace of modern life, loneliness has become one of the main issues affecting human health and has attracted much attention in the research field. Studies have shown that loneliness increases the risk of various diseases in individuals, or has a potential causal relationship with some diseases. However, there is still a lack of in-depth basic research on the pathophysiological mechanism between loneliness and various diseases. The main reason is the lack of an effective and comprehensive animal model to simulate the characteristics of loneliness.

[0003] However, most existing mouse models focus on the single characteristics of social isolation or social stress, lack the simulation of the complex social interactions of loneliness, and fail to reproduce the context of human loneliness. In addition, social isolation models generally rely on a single behavioral indicator to evaluate the impact of social isolation on mice, and lack a comprehensive reflection of the impact of loneliness on multiple systems such as physiology, immunity, and neuroendocrine.

[0004] Therefore, developing a new loneliness mouse model that can simulate the physiological, psychological, and emotional changes of loneliness in multiple dimensions and explore the pathophysiological mechanisms linking loneliness to disease is an important issue that needs to be addressed urgently. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides a method for constructing a loneliness mouse model and application thereof.

[0006] In order to solve the above technical problems, the present invention is implemented by the following technical solutions: In the first aspect, a method for constructing a loneliness mouse model comprises the following steps:

[0007] S1: The experimental mice were placed in an independent isolation area with a transparent acrylic isolation cover, which was set in an environmental area accommodating 5 normal mice to form a visually accessible but physically isolated social environment;

[0008] S2: Maintain the isolation state for 4 weeks, during which the isolation structure is maintained while the environment is cleaned every day;

[0009] S3: Verify the effectiveness of the model through the following multi-dimensional evaluation system:

[0010] s31: The open field test and elevated maze test were used to detect changes in depression and anxiety-like behaviors, and the three-box experiment was used to detect the degree of social willingness of mice.

[0011] s32: Measuring the levels of serum norepinephrine and angiotensin II by ELISA

[0012] s33: Quantitative analysis of the activation degree of neurons in the locus coeruleus (LC), basolateral amygdala (BLA), lateral septal nucleus (LSV), medial prefrontal cortex (mPFC), and dorsal raphe nucleus (DRN) by immunofluorescence C-Fos staining

[0013] In this aspect, existing animal models are difficult to accurately simulate the physiological-psychological comprehensive pathological state caused by long-term social isolation in humans, especially lacking a systematic modeling method that can simultaneously reflect the changes at the neuroendocrine, immune, and behavioral levels.

[0014] This application establishes a mouse model that can simulate chronic loneliness by constructing an environment where physical isolation and social stimulation coexist and combining a multi-dimensional evaluation system. The specific steps are as follows:

[0015] Step S1: Environment construction

[0016] Place the experimental mice alone in an independent isolation area inside a transparent acrylic isolation cover. The isolation cover is equipped with an array of ventilation holes with a diameter of 1 cm to ensure air circulation while blocking physical contact. The isolation area is placed in a square environmental area containing 5 normal gregarious mice. The two areas are adjacent to form a social environment where visual contact is possible but physical contact is not. This design simulates the lonely state of "social visibility but inaccessibility" in humans and induces chronic social deprivation stress in the experimental mice.

[0017] Step S2: Model induction

[0018] Maintain the above isolation state for 4 weeks. During daily environmental cleaning, keep the isolation structure intact to avoid short-term contact between the experimental mice and the mice in the non-isolation area. This stage induces physiological and behavioral abnormalities related to long-term loneliness in the experimental mice.

[0019] Step S3: Model verification

[0020] Verify the effectiveness of the model through the following multi-dimensional evaluation system:

[0021] s31: Behavioral detection

[0022] Use the open field test (detecting spontaneous activity and exploratory behavior) and the elevated plus maze test (evaluating anxiety level) to analyze the changes in anxiety-like behaviors of the experimental mice.

[0023] s32: Neuroendocrine indicators

[0024] The levels of norepinephrine (NE) (reflecting sympathetic nerve activation) and angiotensin II (AngII) (indicating cardiovascular stress response) in serum were measured by enzyme-linked immunosorbent assay (ELISA).

[0025] s33: Neural activation analysis

[0026] Immunofluorescence C-Fos staining was performed on brain tissues, and the degree of neuronal activation in the locus coeruleus, amygdala, lateral septal nucleus, medial prefrontal cortex, and dorsal raphe nucleus regions was quantitatively analyzed to characterize the abnormal regulation of the neural circuit related to loneliness.

[0027] Technical effect: Through the standardized isolation environment and multi-dimensional verification system, this method realizes for the first time the synchronous characterization of sympathetic hyperactivity, elevated inflammatory factors, anxiety behavior, and activation of specific brain regions in the loneliness mouse model, providing a highly simulated animal model for studying loneliness-related diseases.

[0028] In a specific embodiment of the first aspect, the transparent acrylic isolation cover is provided with a ventilation hole array with a diameter of 1 cm. The isolation area is adjacent to the environmental area, and the environmental area uses a square breeding cage with a diameter of 50 cm.

[0029] In the second aspect, an intervention method for a loneliness mouse model is characterized by including: after completing the model construction described in claim 1, transferring the experimental mice to a gregarious environment equipped with a running wheel, multi-layer platform, and replaceable environmental enrichment device (including a tree hole module, building block module, and tunnel module), and maintaining an intervention cycle of 2 weeks.

[0030] In this aspect, the existing intervention means lack the simulation of the dynamics of "social support" and are difficult to effectively reverse the multi-system pathological changes caused by long-term loneliness.

[0031] After the model construction of this application is completed, intervention is carried out through dynamic environmental enrichment and social stimulation, specifically including:

[0032] Transfer the experimental mice to a gregarious environment, equipped with a running wheel (promoting exercise), multi-layer platform (increasing spatial complexity), and replaceable environmental enrichment device, including a tree hole module (providing a hidden space), building block module (stimulating exploratory behavior), and tunnel module (promoting social interaction).

[0033] Maintain an intervention cycle of 2 weeks, and randomly change the module combination of the enrichment device every day to avoid the attenuation of the stimulation effect caused by environmental adaptation.

[0034] Introduce new same-age mice (supplement 1-2 mice per week) to activate the social drive of the experimental mice through continuous social stimulation.

[0035] This method significantly improves the anxiety behavior, inflammation level, and neuroendocrine indicators of model mice through the synergistic effect of the dynamic environment and social stimuli, providing a standardized protocol for studying the social support intervention mechanism.

[0036] In a specific implementation of the second aspect, the environmental enrichment device randomly changes the module combination daily and adds newly introduced mice of the same age for social stimulation.

[0037] In the third aspect, an application of a loneliness mouse model in the study of disease mechanisms is provided for:

[0038] Studying the activation mechanism of the sympathetic - adrenal medulla system in chronic stress - related cardiovascular diseases; using this model to reveal the pathological mechanisms such as reduced heart rate variability and abnormal vascular tone caused by the over - activation of the sympathetic - adrenal medulla system (SAM) due to loneliness. Quantitatively evaluate sympathetic nerve activity through heart rate variability spectral analysis (detecting the low - frequency / high - frequency power ratio).

[0039] Exploring the role of abnormal neuro - immune regulation in the pathogenesis of metabolic syndrome; analyzing the macrophage polarization state (M1 / M2 type ratio) in the adipose tissue of model mice and combining with the serum inflammatory factor level to clarify the action path of abnormal neuro - immune regulation in promoting insulin resistance and lipid metabolism disorders.

[0040] Evaluating the correlation between social behavior disorders and hypothalamic - pituitary - adrenal axis dysfunction; by detecting the expression level of glucocorticoid receptor (GR) in the hippocampal region, evaluating the regulatory mechanism of hypothalamic - pituitary - adrenal axis (HPA axis) dysfunction on social behavior withdrawal and cognitive function decline.

[0041] The beneficial effects of the present invention are as follows:

[0042] 1. By providing an effective animal model of mouse loneliness, the present invention overcomes the deficiencies of existing models in simulating the characteristics of loneliness. This model can comprehensively reflect the characteristics of human loneliness by simulating the situations that generate loneliness. Through this model, the effects of loneliness on multiple systems such as physiology, immunity, and neuroendocrine can be studied more deeply, and the pathophysiological mechanisms between loneliness and various diseases can be explored, providing a more reliable and accurate experimental tool for the study of loneliness and the mechanism research of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a schematic structural diagram of the isolation device of the loneliness mouse model of the present invention.

[0044] Figure 2 is a schematic structural diagram of the rich social environment stimulation device of the present invention.

[0045] Figure 3It is a graph for verifying the anxiety behavior characterization and environmental enrichment intervention effect of the open field experiment based on the visual social deprivation model of the present invention.

[0046] Figure 4 It is a graph for verifying the anxiety behavior characterization and dynamic enrichment intervention effect of the elevated plus maze based on the visual social deprivation model of the present invention.

[0047] Figure 5 It is a graph for verifying the behavior characterization and dynamic enrichment intervention effect of the three-chamber experiment based on the visual social deprivation model of the present invention.

[0048] Figure 6 It is a graph for verifying the activation characterization of the sympathetic-adrenal medullary system and RAAS system induced by the visual social deprivation model of the present invention and the effect of dynamic environmental enrichment intervention.

[0049] Figure 7 It is a graph for verifying the activation characterization of systemic inflammation induced by the visual social deprivation model of the present invention and the reversal of neuroimmune regulation by dynamic environmental enrichment intervention.

[0050] Figure 8 It is a graph for verifying the specific activation characterization of neurons in the locus coeruleus, amygdala, lateral septal nucleus, medial prefrontal cortex, and dorsal raphe nucleus induced by the visual social deprivation model of the present invention and the regulation of neural activity by dynamic environmental enrichment intervention. Detailed implementation manners

[0051] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0052] Please refer to Figures 1 to 8 A method for constructing a loneliness mouse model and its application as shown.

[0053] Example 1: Construction and verification of a loneliness mouse model

[0054] In combination with Figure 1 The isolation device structure shown, the specific implementation steps are as follows:

[0055] Construction of the isolation environment

[0056] An isolation cover (size 15×10×10 cm) is prepared using transparent acrylic material, and an array of ventilation holes with a diameter of 1 cm (hole spacing 5 cm) is set on the surface to ensure an air exchange rate > 15 L / min.

[0057] Place the isolation cage in the center of a square group cage with a side length of 50 cm (accommodating 5 C57BL / 6J homologous mice), with a distance of 10 cm between the isolation area and the group area.

[0058] Model induction

[0059] Place the experimental group mice (n = 20) individually in the isolation cage for 4 weeks. During daily cleaning, use special clamping tools to maintain the integrity of the isolation structure and avoid physical contact.

[0060] The control group (n = 20) was placed in the same environment but without the restriction of the isolation cage.

[0061] Multi-dimensional verification

[0062] Behavioral verification: In the open field experiment, the exploration time of the model group in the central area was significantly lower than that of the control group (p < 0.001). The proportion of time spent in the open arms of the elevated plus maze decreased significantly (p < 0.01), and the frequency of the number of contacts with strange mice in the three-chamber experiment decreased significantly (p < 0.05).

[0063] Neuroendocrine detection: The serum norepinephrine (NE) level in the model group was significantly higher than that in the control group (p < 0.001), and the concentration of angiotensin II (AngII) increased significantly (p < 0.001).

[0064] Please refer to Figure 8 , Neuroactivation analysis: The density of c-Fos positive neurons in the locus coeruleus, amygdala, lateral septal nucleus, medial prefrontal cortex, and dorsal raphe nucleus brain regions was significantly increased compared with the control group (p < 0.001), and the volume of the activated area showed an expanding trend.

[0065] Conclusion: In this example, a loneliness mouse model with anxiety behavior, sympathetic hyperactivity ( Figure 7 A-B) and specific brain region activation characteristics was successfully constructed.

[0066] Implementation of the dynamic environmental enrichment intervention method

[0067] Combined with Figure 2 The rich environmental device shown, the specific implementation steps are as follows:

[0068] Intervention environment construction

[0069] Use a multi-layer cage with a diameter of 50 cm (height 60 cm), configured with:

[0070] Rotating running wheel (rotational speed adjustable 10 - 30 rpm)

[0071] Modular enrichment device (randomly combine tree hole, tunnel, and building block modules daily)

[0072] Interaction platform (set contact sensors to record social behavior)

[0073] Intervention program

[0074] Transfer the mice in the model group to the intervention environment, and replace the enrichment module combination at 09:00 and 15:00 every day.

[0075] Introduce 2 unfamiliar mice of the same age every week (the cumulative social stimulation index reaches 85±5).

[0076] Verification of intervention effect

[0077] Please refer to Figure 5 As shown: Behavioral recovery: The social contact frequency in the intervention group was increased by 2.5-3.5 times compared with the model group (p<0.001), and recovered to the normal social behavior level.

[0078] Physiological reversal:

[0079] The serum IL-6 concentration decreased significantly compared with the model period (p<0.001);

[0080] The norepinephrine (NE) level recovered to within ±15% of the baseline value (p<0.001);

[0081] Please refer to Figure 8 As shown, neural remodeling: The density of c-Fos positive neurons in the locus coeruleus, amygdala, lateral septal nucleus, medial prefrontal cortex, and dorsal raphe nucleus decreased significantly compared with the model group (p<0.001), and the distribution characteristics of the activated areas approached normal.

[0082] Conclusion: The dynamic enrichment intervention significantly reverses the multi-system pathological phenotypes caused by loneliness through modular environmental stimulation and social contact activation, verifying the technical effect.

[0083] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a mouse model of loneliness, characterized in that, It includes the following steps: S1: Place the experimental mice in an independent isolation area equipped with a transparent acrylic isolation cover. The isolation area is set in an environmental area accommodating 5 normal mice, forming a socially accessible but physically isolated social environment; S2: Maintain the isolation state for 4 weeks, and keep the isolation structure during daily environmental cleaning; S3: Verify the effectiveness of the model through the following multi-dimensional evaluation system: S31: The open field test and elevated plus maze test are used to detect changes in depressive and anxiety-like behaviors, and the three-chamber test is used to detect the degree of social willingness of mice; S32: The ELISA method is used to measure the levels of serum norepinephrine and angiotensin II; S33: Immunofluorescence C-Fos staining is used for quantitative analysis of the activation degrees of neurons in the locus coeruleus (LC), basolateral amygdala (BLA), lateral septal nucleus (LSV), medial prefrontal cortex (mPFC), and dorsal raphe nucleus (DRN).

2. The method for constructing a loneliness mouse model according to claim 1, characterized in that: The transparent acrylic isolation cover is provided with an array of ventilation holes with a diameter of 1 cm. The isolation area is adjacent to the environmental area, and the environmental area uses a square breeding cage with a side length of 50 cm.

3. An intervention method for a loneliness mouse model, characterized in that, It includes: After completing the model construction described in claim 1, transfer the experimental mice to a social environment equipped with a running wheel, multi-layer platform, and replaceable environmental enrichment device (including a tree hole module, building block module, and tunnel module), and maintain an intervention cycle of 2 weeks.

4. The intervention method for a loneliness mouse model according to claim 3, wherein: The module combinations of the environmental enrichment device are randomly replaced daily, and newly introduced same-age mice are added for social stimulation.

5. Use of a loneliness mouse model in the study of disease mechanisms, characterized in that, For: Studying the activation mechanism of the sympathetic-adrenal medullary system in chronic stress-related cardiovascular diseases; Exploring the role of abnormal neuroimmune regulation in the pathogenesis of metabolic syndrome; Evaluating the correlation between social behavior disorders and hypothalamic-pituitary-adrenal axis dysfunction.

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