A chronic restraint stress depression model based on improved restraint device and construction method and use thereof

Abstract

This invention belongs to the field of animal model technology for diseases, specifically relating to a chronic restraint stress depression model based on a modified restraint device, its construction method, and its applications. The construction method includes the following steps: using a modified ventilated restraint device to restrict the activity of experimental mice while avoiding injury; establishing standardized conditions for restraint of 4-8 hours daily for 4-8 weeks; subjecting the mice to chronic restraint stress; and combining behavioral evaluation and neurotransmitter detection to obtain a stable animal model with a depressive-like phenotype. This invention, by optimizing the restraint intensity, duration, and modeling cycle of the experimental mice, achieves stable and efficient induction of depressive-like behavioral phenotypes in animals, providing a standardized and reliable animal model scheme for evaluating antidepressant activity and studying related mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of animal model technology for diseases, specifically relating to a chronic restraint stress depression model based on a modified restraint device, its construction method, and its applications. Background Technology

[0002] Depression is a common mental illness with a complex pathogenesis. Currently, stress is widely considered one of the core triggers for depression. The clear link between stress and depression has led researchers to establish various stress-related animal models to explore the pathogenesis of depression and screen antidepressants. Animal models of depression mainly include social frustration, unpredictable chronic stress, learned helplessness, maternal deprivation, isolation stress, sleep deprivation, and forced swimming models. Among these, the chronic social frustration stress model, the chronic unpredictable stress model, the learned helplessness model, and bilateral olfactory bulbectomy are currently the most widely used models and have a strong correlation with antidepressant research.

[0003] Chronic social frustration stress is a widely used model in rodent depression research. Its core principle is to induce phenotypes similar to human depression in animals by simulating frustration and conflict in human social interactions. These phenotypes mainly include anhedonia, reduced social behavior, slowed weight gain, and increased anxiety. The specific procedure is as follows: Male C57BL / 6J mice are used as experimental subjects. For 10 consecutive days, they are exposed to different aggressive CD1 mice for 10 minutes daily. After the experimental mice exhibit obvious failure behaviors (such as curling up, avoidance, and lack of resistance), two mice are separated by a perforated partition and housed for 24 hours to allow the animals to experience continuous stress. After the final modeling, the C57BL / 6J mice are housed individually to complete the model construction. This model can simulate depressive-like behaviors, and its social frustration stress closely resembles the inducing factors of human depression, making it a commonly used animal model of depression.

[0004] Chronic mild stress and chronic unpredictable mild stress are based on the core principle that chronic, uncontrollable stress impairs the brain's reward system and are widely used to study the neuropathological mechanisms of depression. By applying long-term, low-intensity, unpredictable mild stimuli, depressive-like phenotypes are induced in animals. The specific procedure for chronic stress is as follows: animals are continuously exposed to a series of unpredictable mild stimuli, such as slight temperature drops, disrupted light-dark cycles, changes in cage companions, or damp bedding, while being randomly deprived of water or food daily. This long-term, chronic, low-intensity stress simulates depressive symptoms such as anhedonia induced by long-term life stress in humans. The modeling period typically lasts 3 weeks to 3 months. The advantage of this model is that it can reflect the clinical depressive phenotype of patients with depression. Disadvantages include: high modeling difficulty, time-consuming experimental procedures, slight variability in phenotypes, difficulty in cross-laboratory data replication and validation; some high-frequency experiments require large spaces, which can lead to lower experimental efficiency.

[0005] Learned helplessness is a classic animal model of depression. Its core principle is to induce a depressive-like state in animals by gradually eroding their will to escape through uncontrollable and unpredictable foot shocks. The behavioral and physiological changes closely resemble the clinical phenotypes of patients with depression. Helpless animals exhibit persistent changes such as weight loss, disrupted sleep patterns, abnormal hypothalamic-pituitary-adrenal (HPA) axis activity, and loss of hippocampal spinous synapses. The advantage of this model is its high similarity to clinically diagnosed depression, allowing it to be directly used to validate the pathophysiology of depression and the efficacy of antidepressants. Limitations include the relatively short duration of uncontrollably induced depressive-like symptoms and strain-specific sensitivity differences.

[0006] Bilateral olfactory bulbectomy: In rodents, the olfactory system is an important component of the limbic system, closely linked to brain regions related to emotion regulation such as the amygdala and hippocampus, directly influencing animal behavior and emotion regulation. Bilateral olfactory bulbectomy involves surgically removing both olfactory bulbs in rodents, disrupting their olfactory system and related emotion regulation pathways, thereby inducing depression-related phenotypes. The core characteristics of this model are a series of behavioral changes in rodents, including hyperactivity, abnormal alterations in social behavior, increased nocturnal activity, learning and memory deficits, and altered taste aversion. It stably simulates depression-related phenotypes, is relatively simple to perform, and is suitable for rapid screening of depression-related phenotypes. While this model can simulate depression-related phenotypes, it cannot elucidate the etiological mechanisms or corresponding pathological causes.

[0007] Currently, the existing traditional methods for constructing animal models of depression caused by chronic restraint stress are among the most commonly used classic methods. These methods typically involve using centrifuge tubes, restraint cages, or self-made restraint devices to continuously restrict the activity of experimental animals. By applying restraint stress over a long period, animals exhibit depressive-like behavioral phenotypes such as despair and anhedonia. This approach is widely used in antidepressant drug screening and research on the pathogenesis of depression. Common modeling parameters for existing chronic restraint stress (CRS) depression models are shown in Table 1. Restraint duration typically ranges from 2 to 6 hours per day, and the modeling period is generally concentrated in the range of 2 to 4 weeks. Forced swimming tests and sucrose preference tests are used as the main behavioral evaluation indicators.

[0008] Table 1. Summary of modeling parameters for the chronic restraint stress (CRS) depression model.

[0009] However, existing traditional models of chronic restraint stress-induced depression have the following significant shortcomings in practical applications: 1. Inconsistent modeling intensity and cycle, resulting in poor model stability: Existing technologies set the restraint duration, daily stress intensity, and total modeling cycle arbitrarily, lacking systematic comparison and optimization. This leads to large differences in the success rate of modeling in different laboratories and different batches of animals. The depressive-like behavioral phenotype is unstable and has poor repeatability, making it difficult to meet the requirements of standardized pharmacological experiments.

[0010] 2. Short-term modeling is difficult to induce stable and repeatable depressive-like behavior: The conventional short-term restraint stress of about 2 weeks usually only puts the animals into the stress adaptation stage. The behavioral indicators (such as the time of forced swimming without moving) are not significantly different from the control group. The model phenotype is atypical, and false negative results are easy to occur, which affects the reliability of the experiment.

[0011] 3. Low-intensity stress cannot effectively induce significant depressive-like behavior: Existing techniques often use low-intensity restraint schemes, which are simple to operate, but are insufficient to induce stable and obvious depressive-like behavior, resulting in weak model phenotypes that are difficult to use for drug efficacy evaluation or mechanism research.

[0012] 4. Lack of research on the synergistic effect of modeling period and intensity: Existing publicly available technologies mostly focus on restraint time or modeling period alone, without systematically comparing the modeling effects under different combinations of intensity and period, making it impossible to determine the optimal modeling conditions, resulting in low modeling efficiency, blindly extending the period, and increasing experimental costs and animal consumption.

[0013] In summary, existing depression models suffer from problems such as inconsistent modeling cycles, ambiguous restraint intensity settings, unstable induction of depressive-like behavioral phenotypes, and poor reproducibility. These issues result in low model construction success rates, long experimental cycles, and significant differences in behavioral results, making it difficult to meet the needs of antidepressant drug screening and depression mechanism research for stable and reliable animal models. Therefore, there is an urgent need to provide a method for constructing a stable, reproducible, and clearly defined method for chronic restraint stress depression models with specific intensity and cycle. Summary of the Invention

[0014] To address the shortcomings of the existing technologies, this invention provides a chronic restraint stress depression model based on a modified restraint device, along with its construction method and applications. By optimizing the restraint strength, duration, and modeling cycle of experimental mice, it achieves stable and efficient induction of depressive-like behavioral phenotypes in animals, providing a standardized and reliable animal model scheme for evaluating antidepressant activity and studying related mechanisms.

[0015] The specific technical solution is as follows: The first objective of this invention is to provide a method for constructing a chronic restraint stress depression model based on a modified restraint device, comprising the following steps: using a modified ventilated restraint device to restrict the activity of experimental mice while avoiding injury, defining standardized conditions of restraint for 4-8 hours per day and continuous modeling for 4-8 weeks, performing chronic restraint stress treatment, and combining behavioral evaluation and neurotransmitter detection to obtain an animal model with a stable depression-like phenotype.

[0016] Furthermore, the improved ventilated restraint device is a modified 50 mL centrifuge tube with ventilation holes, a restraint cylinder, a restraint box, or other movement restriction device with a ventilation structure.

[0017] Furthermore, the experimental mice are C57BL / 6 mice, ICR mice, BALB / c mice, or rats.

[0018] Furthermore, the construction method also includes: supplementing the chronic restraint stress treatment with mild unpredictable stress.

[0019] Furthermore, the mild unpredictable stress refers to at least one of the low-intensity stimuli such as damp bedding, reversed day and night cycles, and tail clamping.

[0020] Furthermore, the behavioral evaluation uses the immobility time in the last 4 minutes of the forced swimming experiment as the core evaluation indicator.

[0021] Furthermore, the behavioral evaluation also includes at least one of the following: sucrose preference test, open field test, and elevated cross maze.

[0022] Furthermore, the neurotransmitter detection uses the effects of different restraint stress intensities on the mouse monoamine neurotransmitter system as a neurobiochemical indicator.

[0023] The second objective of this invention is to provide a chronic restraint stress depression model based on a modified restraint device, obtained using the construction method described above.

[0024] The third objective of this invention is to provide the use of the chronic restraint stress depression model described above in the screening, efficacy evaluation and mechanism research of antidepressants.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The construction method of the present invention involves subjecting experimental mice to chronic restraint stress treatment for a specific duration and period, combined with behavioral evaluation and neurotransmitter detection, to obtain reasonable modeling conditions and optimal modeling conditions that can stably induce depressive-like behavior, thereby obtaining an animal model with a stable depressive-like phenotype. (2) The model constructed in this invention has the characteristics of short modeling cycle, significant phenotype, strong repeatability and high animal safety, and can be used for antidepressant drug screening, efficacy evaluation and mechanism research. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the construction and experimental process of the chronic restraint stress depression model in Embodiment 1 of the present invention.

[0027] Figure 2 This is a diagram showing the results of chronic restraint stress of different durations (intensities) and cycles stably inducing depressive-like behavior in mice in Example 2 of the present invention. Among them, (A) immobility time in the forced swimming test (FST) after 2 weeks of chronic restraint stress (CRS) treatment; (B) immobility time in the forced swimming test (FST) after 4 weeks of chronic restraint stress (CRS) treatment; (C) immobility time in the forced swimming test (FST) after 8 weeks of chronic restraint stress (CRS) treatment; data are expressed as mean ± standard error (Mean ± SEM), and the sample sizes of experimental animals were: control group n=10, CRS 4 h group n=10, and CRS 8 h group n=9; CRS: chronic restraint stress; one-way ANOVA was used for inter-group comparisons. Compared with the control group, * P <0.05, *** P <0.001.

[0028] Figure 3 This is a graph showing the results of high-intensity chronic restraint stress significantly reducing the level of peripheral monoamine neurotransmitters in mice in Example 3 of the present invention; Among them, (A) plasma 5-hydroxytryptamine (5-HT) concentration; (B) plasma 5-hydroxyindoleacetic acid (5-HIAA) concentration; (C) plasma norepinephrine (NE) concentration; (D) plasma tryptophan (Trp) concentration; (E) plasma 5-hydroxytryptamine (5-HTP) concentration; data are expressed as mean ± standard error (Mean ± SEM), and the sample sizes of experimental animals were: control group n=10, CRS 4 h group n=9, CRS 8 h group n=9; CRS: chronic restraint stress; one-way ANOVA was used for inter-group comparisons, compared with the control group, * P <0.05, *** P <0.001. Detailed Implementation

[0029] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0030] Example 1: Chronic restraint stress treatment (modeling) Thirty 8-week-old male C57BL / 6J mice were randomly divided into a blank control group, a 4 h / d restraint group, and an 8 h / d restraint group, with n=10 in each group. A modified 50 mL centrifuge tube with ventilation holes was used as the restraint device, which restricted the mice's free movement and prevented physical compression and injury. Mice in the model group were restrained at fixed times each day. Mice in the blank control group were fasted and deprived of water under the same environment, but were not restrained. Different daily restraint durations (4 h / d and 8 h / d) and different modeling periods (2 weeks, 4 weeks, and 8 weeks) were designed for the experiment.

[0031] Example 2: Behavioral Evaluation (Forced Swimming Experiment) Reference Figure 1 After each modeling time point, the forced swimming test (FST) was used for behavioral evaluation. The immobility time in the last 4 minutes was recorded as the core indicator for judging the depressive-like behavior of mice, so as to obtain reasonable and optimal modeling conditions that can stably induce the depressive-like phenotype.

[0032] The forced swimming test is primarily used to assess hopeless behavior in laboratory animals and is a behavioral indicator for measuring antidepressant-like effects in depression models. At the start of the experiment, a single animal is placed in a transparent acrylic cylinder (16 cm in diameter, 20 cm deep, water temperature 25 ± 1 °C). The animal's movement in the water is recorded via video for 6 minutes. The water is replaced with clean water after each animal's session. The immobility time of the animal in the last 4 minutes is analyzed.

[0033] The results of chronic restraint stress of different durations (intensities) and cycles stably inducing depressive-like behavior in mice are shown in Table 2 and [Table data missing]. Figure 2 .

[0034] Table 2. Chronic restraint stress of different durations (intensities) and cycles can stably induce depressive-like behavior in mice.

[0035] Note: Data are expressed as mean ± SEM; the sample sizes of experimental animals were: control group n=10, CRS 4 h group n=10, CRS 8 h group n=9; FST immobility time unit: seconds (s); one-way ANOVA was used for statistical analysis; compared with the control group, * P <0.05, *** P <0.001.

[0036] From Table 2 and Figure 2 The results from the study A showed that, at 2 weeks of modeling, compared with the blank control group, the immobility time of mice restrained for 4 h and 8 h daily during FST showed an increasing trend, but there was no statistically significant difference. The results indicate that short-term CRS stimulation is insufficient to induce stable and clearly defined depressive-like hopelessness behavior in mice, suggesting that within this time window, mice may still be in the stress adaptation phase, and their behavioral phenotypic changes are not yet typical.

[0037] From Table 2 and Figure 2 The results from the study showed that when the modeling period was extended to 4 weeks, the behavioral effects induced by CRS began to appear significantly. Compared with the blank control group, the immobility time of mice in the 8-hour daily restraint group during FST significantly increased from 169.95±9.85 s to 199.77±5.87 s. P =0.021), indicating that mice exhibited significant depressive-like behavior under this modeling condition. In contrast, the immobility time in the 4-hour daily restraint group showed an increasing trend, but there was no statistically significant difference, suggesting that low-intensity chronic restraint stress was insufficient to induce depressive-like behavioral phenotypes in mice, while higher-intensity stress could induce behavioral abnormalities earlier.

[0038] From Table 2 and Figure 2 The results from the study showed that when the model was prolonged to 8 weeks, the behavioral abnormalities induced by chronic restraint stress were more severe, and the intensity effect was more pronounced. Compared with the blank control group, the immobility time (FST) of mice in the 4 h and 8 h daily restraint groups was significantly increased, rising from 184.93±6.32 s to 201.30±2.46 s and 217.64±5.66 s, respectively. P=0.0003). Among them, the immobility time increased more significantly in the 8h group, indicating that the depressive-like behavior of the model animals was more stable with the extension of stress treatment time and intensity.

[0039] Example 3: Neurotransmitter Detection (Neurobiochemical Indicators) The detection of neurobiochemical indicators further evaluated the effects of different stress intensities on the monoamine neurotransmitter system in mice. At week 8 of modeling, this study quantitatively analyzed the levels of monoamine neurotransmitters, metabolites, and their precursors in the plasma of mice in each group. The results are shown in Table 3 and [Table data missing]. Figure 3 .

[0040] Table 3. High-intensity chronic restraint stress significantly reduced peripheral monoamine neurotransmitter levels in mice.

[0041] Note: Data are expressed as mean ± standard error (Mean ± SEM). The sample sizes of experimental animals were: control group n=10, CRS 4 h group n=9, and CRS 8 h group n=9; CRS: chronic restraint stress; 5-HT: serotonin; 5-HIAA: 5-hydroxyindoleacetic acid; NE: norepinephrine; Trp: tryptophan; 5-HTP: 5-hydroxytryptophan; One-way ANOVA was used for inter-group comparisons. Compared with the control group, * P <0.05, *** P <0.001.

[0042] From Table 3 and Figure 3 As shown in A and B, prolonged high-intensity restraint stress significantly disrupted the homeostasis of the peripheral 5-HT system. Compared with the control group, the plasma 5-HT level in mice in the CRS 8 h group was significantly decreased (Control: 408.79±60.57 ng / mL vs. CRS 8 h: 132.02±46.77 ng / mL). P =0.018), while the level of its main metabolite 5-HIAA also showed a highly significant decrease (Control: 40.18±2.23 ng / mL vs. CRS 8 h: 24.75±1.18 ng / mL, P =0.0002). There was no statistically significant difference in 5-HT and 5-HIAA levels between the CRS 4 h group and the blank control group.

[0043] From Table 3 and Figure 3The CE (corticotropic enzyme) analysis revealed a significant alteration in the level of norepinephrine, a key neurotransmitter in the sympathetic nervous system. Compared to the control group, the plasma norepinephrine level in mice in the CRS 8 h group was significantly decreased (Control: 322.20±28.09 ng / mL vs. CRS 8 h: 194.84±7.01 ng / mL). P =0.0008), while the NE level in the CRS 4 h group showed a decreasing trend, but the difference was not statistically significant. In addition, the 5-HT synthesis precursors tryptophan (Trp) and 5-hydroxytryptophan (5-HTP) were detected simultaneously in the experiment. Furthermore, there were no statistically significant differences in the levels of Trp and 5-HTP in the plasma of mice in each group, suggesting that the decrease in peripheral 5-HT in the CRS 8 h group may be related to excessive neurotransmitter consumption or changes in metabolic enzyme activity.

[0044] In summary, the results indicate that chronic restraint stress of 4 hours daily did not significantly alter the peripheral monoamine neurotransmitter system; however, high-intensity stress of 8 hours daily for 8 weeks induced significant depletion of peripheral 5-HT, 5-HIAA, and NE, leading to neurobiochemical disorders. The peripheral plasma biochemical phenotype in mice was consistent with their depressive-like behavioral manifestations.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a chronic restraint stress depression model based on a modified restraint device, characterized in that, Includes the following steps: A modified ventilated restraint device was used to restrict the activity of experimental mice while avoiding injury. Standardized conditions of restraint for 4-8 hours per day and continuous modeling for 4-8 weeks were established. Chronic restraint stress treatment was carried out, and combined with behavioral evaluation and neurotransmitter detection, a stable animal model with a depressive-like phenotype was obtained.

2. The construction method according to claim 1, characterized in that, The behavioral evaluation uses the immobility time in the last 4 minutes of the forced swimming experiment as the core evaluation indicator.

3. The construction method of claim 2, wherein, The behavioral evaluation also includes at least one of the following: sucrose preference test, open field test, and elevated cross maze.

4. The construction method of claim 1, wherein, The neurotransmitter detection used the effects of different restraint stress intensities on the mouse monoamine neurotransmitter system as a neurobiochemical indicator.

5. The construction method of claim 1, wherein, The improved ventilated restraint device is a modified 50 mL centrifuge tube with ventilation holes, a restraint cylinder, a restraint box, or other restrictive movement device with a ventilation structure.

6. The construction method of claim 1, wherein, The experimental mice were C57BL / 6 mice, ICR mice, BALB / c mice, or rats.

7. The construction method of claim 1, wherein, Also includes: In addition to chronic restraint stress treatment, unpredictable stress is also introduced.

8. The construction method of claim 7, wherein, The unpredictable stress is at least one of the following: damp bedding, reversed day and night cycle, and tail clamping.

9. A chronic restraint stress depression model based on a modified restraint device, characterized in that, Obtained by the construction method described in any one of claims 1-8.

10. The use of the chronic restraint stress depression model as described in claim 9 in the screening, efficacy evaluation and mechanism research of antidepressants.

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