A Method for Constructing an Animal Model of Recurrent Depression and Its Application

Abstract

This invention belongs to the field of animal model construction technology. To address the technical problem that classic CUMS (Chronic Unpredictable Stress Medication) models cannot stably simulate the critical process of "relapse after clinical cure," this invention provides a method for constructing a relapsing depression animal model and its application. The model uses chronic unpredictable stress CUMS to simulate depressive triggers, combined with paroxetine administration intervention, withdrawal treatment, and mild CUMS relapse induction to construct a relapsing depression animal model. The constructed animal model not only reflects the two pathological stages of depressive onset and relapse, but also accurately simulates the complete clinical course of "chronic stress sensitization - drug relief - withdrawal relapse." The model has a high success rate, good stability, and strong reproducibility. Furthermore, a multi-dimensional evaluation system of behavioral, physiological, and biological indicators has been established, making it suitable for evaluating the efficacy of antidepressant relapse drugs and studying relapse mechanisms. It can provide a reliable experimental model and scientific basis for research on the mechanism of depression relapse and the development of related new drugs.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing a relapsing depression animal model and its application. Background Technology

[0002] Depression is a chronic, relapsing mental disorder characterized by persistent low mood and loss of interest and pleasure. It is often accompanied by varying degrees of cognitive impairment, which has a long-tail effect. Currently, depression is one of the most prevalent mental illnesses globally, and the most common type of illness among the more than one billion people worldwide suffering from mental disorders. It has a wide reach and a high degree of social harm.

[0003] High relapse rates are a core challenge in the clinical diagnosis and treatment of depression, and a key reason why the disease is difficult to cure and why the social burden of illness remains high. Epidemiological data show that the risk of relapse in depression increases progressively with the number of episodes: after symptom relief with standardized treatment, the short-term relapse rate is approximately 50% for first-time patients; after a second episode, the relapse risk rises to 70%; and for patients with three or more episodes, the relapse risk can reach over 80%. More than 80% of relapses occur within the high-risk window of six months after symptom relief. Repeated fluctuations in the condition cause patients to experience persistent low mood, decreased energy, cognitive abnormalities, and physical discomfort, easily leading to negative and hopeless emotions, and significantly increasing the risk of disability and suicide. Relapses not only significantly increase the difficulty of clinical treatment and the economic burden on patients' families, but also continuously consume public medical resources. Clinical relapse has a characteristic course: after long-term maintenance medication achieves symptom relief, once medication is discontinued, the body's stress sensitivity increases significantly, and relapse can be induced even by mild environmental stressors, with relapse symptoms often more severe than the first episode. This typical clinical relapse characteristic is a core technical shortcoming that current animal models generally cannot accurately replicate.

[0004] Constructing animal models of relapsing depression that closely resemble the clinical course is fundamental for developing new drugs to prevent and treat relapsed depression and for evaluating the efficacy of preventative anti-relapse drugs. The classic chronic unpredictable mild stress (CUMS) model can only induce a single primary depressive episode through chronic stress, without drug intervention or withdrawal. It is only suitable for acute depression research and cannot simulate the core clinical course of "drug maintenance remission - relapse after drug withdrawal," thus it cannot be used for evaluating the efficacy of anti-relapse and preventative drugs. Conventional relapse models often rely on secondary high-intensity stress to induce relapse, deviating from the actual clinical relapse mechanism. They generally suffer from long modeling periods, unstable relapse patterns, significant individual variability, and insensitivity to preventative drugs, resulting in low reproducibility and reliability in drug evaluation. In comparison, the withdrawal-mediated relapse depression model constructed in this invention accurately replicates the clinical process of "chronic stress sensitization - drug maintenance therapy - withdrawal sensitization - subthreshold mild stress triggering relapse". Moreover, the relapse symptoms are more severe than the actual course of the initial disease. It effectively makes up for the shortcomings of the classic CUMS model, which has no relapse course and the low fit of the mechanism of conventional relapse models. The model has a stable phenotype and is suitable for screening preventive anti-relapse drugs. It can provide reliable experimental support for the study of the mechanism of depression relapse and the development of new anti-relapse drugs.

[0005] In summary, developing an animal model that can accurately replicate the pathological characteristics of clinical depression relapse and has a stable course is of great theoretical research value and practical application prospects for elucidating the mechanism of depression relapse. There is still an urgent need for breakthroughs in standardized and clinically relevant modeling techniques in this field. Summary of the Invention

[0006] This invention aims to address the technical shortcomings of the classic CUMS model in simulating clinical relapse induced by drug maintenance remission and withdrawal stress, and provides a method for constructing a withdrawal-mediated relapsing-rhinoceros depression rat model and its application.

[0007] To achieve the above objectives, the technical solution adopted in this invention is as follows: a method for constructing a relapsing depression animal model, which uses chronic unpredictable stress (CUMS) to simulate depressive triggers, and combines paroxetine administration intervention, drug withdrawal treatment and mild CUMS relapse induction to construct a relapsing depression animal model.

[0008] Specifically, the following steps are included: (1) Adaptation period feeding: Healthy male rats were selected and subjected to an adaptation period of 1 week. The feeding environment conditions were: temperature 22±2℃, relative humidity 50±5%, circadian rhythm of 12h light / 12h darkness, and free access to food and water to ensure that the rats adapted to the feeding environment and eliminate the interference of environmental stress on subsequent experiments. (2) Modeling, grouping, and drug intervention: After 7 days of acclimatization, rats were randomly divided into 4 groups according to body weight to ensure a balanced weight distribution among the groups. From the second week, the control group rats maintained normal feeding conditions and were given the same volume of pure water by gavage as the drug treatment group daily; the model rats were given chronic unpredictable stress (CUMS) stimulation daily on the basis of normal feeding; the drug treatment group was given paroxetine 4.2 mg / kg by gavage daily on the basis of modeling, and chronic unpredictable stress (CUMS) was applied 1 hour after administration to construct the animal model of depression; the 1-hour interval between administration and stress could eliminate the interference of the drug sedation effect on the animal behavioral test results, and this stage was continuously treated for 4 weeks. After the treatment period, rats that were qualified for modeling were selected according to their body weight and sucrose preference rate. (3) Withdrawal recovery: Starting from week 6, based on the behavioral data results, the rats in the drug-treated group who were successfully modeled by combining the sugar water preference rate and body weight were divided into the continuous drug-treated group (PX-C) and the acute withdrawal group (PX-A). Except for the continuous drug-treated group, which continued to be drug-treated, the other groups were not drug-treated or stimulated. This withdrawal recovery phase lasted for 1-2 weeks.

[0009] (4) Relapse induction: After the drug withdrawal and recovery in the 7th-8th week, mild CUMS stimulation was continued for rats in all groups except the control group. This stage lasted for 2-4 weeks, and finally the constructed relapse-type depression animal model was verified. The CUMS stimulation uses a random combination of various stress methods, with one method administered daily to avoid using the same stress method for two consecutive days. The stress methods include: fasting for 24 hours, water restriction for 24 hours, day-night reversal for 24 hours, swimming in 4℃ cold water for 5 minutes, tail clamping for 2 minutes, foot electric shock (voltage 1-2mA, duration 1-2s, interval 10s, total 10 times), restraint for 3 hours, heat stimulation (45℃, 10 minutes), and ultrasound stimulation (60dB, Class A noise exposure for 3 hours).

[0010] The behavioral tests described in step (3) include the sucrose preference test, elevated maze, open field test, and forced swimming test. The behavioral tests are conducted in accordance with the "Guidelines for Experimental Animals Mouse and Rat Emotional Behavior Tests" and are recorded after each test cycle. The rat's weight is also recorded weekly. The indicators for verifying the constructed recurrent depression animal model in step (4) include: behavioral data; physiological status evaluation indicator body weight; biological indicator detection: HPLC detection of serum 5-hydroxytryptamine content, HE staining and Nissl staining to observe the morphology of hippocampal cells and neurons and intestinal tissue.

[0011] In step (2), the CUMS modeling is completed in the 5th week. The modeling success is determined by the sucrose preference rate and the change in body weight. If the sucrose preference rate is less than 70% and the body weight shows a significant decreasing trend, the animal model of depression is considered to be successfully established.

[0012] Step (4) The relapse induction phase lasts for 2 weeks, with daily mild CUMS stimulation, including ultrasound stimulation, thermal stimulation and restraint; The successful establishment of the recurrent depression animal model was achieved when, based on behavioral data and tissue sample analysis, the experimental animals exhibited three of the following primary and secondary symptoms: The main symptoms are: (1) a significant decrease in the preference for sugary drinks; (2) a significant decrease in weight; (3) a decrease in serum 5-hydroxytryptamine levels; (4) a decrease in the density of hippocampal tissue and a reduction in the number of neurons; (5) atrophy and a decrease in the number of colonic crypts, a decrease in the number of goblet cells, and significant damage to the intestinal mucosal barrier. The secondary symptoms are: (6) in the open field test, the time to escape the central grid is significantly increased, the number of times to stand upright is significantly reduced, and the number of grids traversed is reduced; (7) in the elevated maze test, the time to stay in the closed arm is significantly increased.

[0013] The rats were male Sprague Dawley rats (SPF grade, 8 weeks old), weighing 180-200 g.

[0014] The present invention also provides an animal model of relapsing depression constructed using the method described above.

[0015] The present invention also provides the application of the aforementioned relapsing depression animal model in screening anti-relapsing depression drugs.

[0016] The relapse depression model constructed using SD rats in this invention is simple to operate, has a high success rate, stable pathological changes, and strong reproducibility.

[0017] This invention provides a method for constructing and evaluating an animal model of depression relapse by simulating the core physiological course of "treatment-relief-relapse" in clinical depression relapse. The model group and experimental group rats are analyzed for behavioral data and histopathological changes to evaluate whether a relapse has occurred.

[0018] This invention establishes a multi-dimensional evaluation system based on behavioral and histopathological findings. By comparing key behavioral indicators such as sucrose preference rate, dynamic weight change, and open field activity in rats from the control group and model group, and combining this with histopathological observations of hippocampal HE and Nissl staining, a comprehensive evaluation of the depressive relapse model is formed. Statistical analysis using Graphpad Prism 10.1.2 software showed significant differences between the model group and the control group in terms of decreased sucrose preference rate, slower weight gain, and reduced open field activity. These behavioral changes reflect typical characteristics of anhedonia and behavioral inhibition in a state of depressive relapse. Simultaneously, histopathological results from the hippocampus further revealed corresponding neurological damage, thus jointly verifying the reliability of this depressive relapse model.

[0019] This invention constructs a criterion for determining depressive relapse, based on a comprehensive index of sucrose preference and body weight data. This criterion standardizes behavioral data from multiple periods (depressive phase, withdrawal phase, and relapse phase), scores them according to preset biological thresholds, and finally combines the two indicators for comprehensive evaluation to objectively and consistently define the "relapse" state of an animal. Definition: After experiencing a withdrawal phase, if an animal, under artificially applied subthreshold stress and natural observation, experiences a decrease in its "relapse phase" comprehensive score (Rscore) relative to its "remission phase" comprehensive score (Wscore) exceeding a preset threshold (a decrease ≥30%), it is determined that the animal has experienced "behavioral relapse of depression."

[0020] Compared with existing technologies, this invention not only provides a scheme for constructing a drug withdrawal-mediated relapse-recurrent depression animal composite model that simulates the real clinical course, but also achieves multiple technological breakthroughs from two dimensions: model construction and evaluation system. First, the modeling process completely replicates the complete clinical course of "chronic stress sensitization - drug intervention relief - drug withdrawal stress sensitization - subthreshold stimulation inducing relapse", making up for the shortcomings of traditional CUMS models that can only induce a single depressive episode, conventional relapse models that deviate from the clinical pathogenesis logic, and unstable model phenotypes. The model success rate can reach 62.5%, the modeling cycle is controllable, individual differences are small, and reproducibility is excellent. Second, it abandons the limitations of existing technologies that rely solely on a single behavioral indicator to judge the model evaluation, and builds a model that integrates behavioral indicators, physiological indicators, serum neurotransmitters, and brain tissue. The pathology-intestinal tissue pathology multidimensional integrated evaluation system simultaneously covers central hippocampal nerve damage and colonic intestinal mucosal barrier lesions, realizing the correlation observation of pathological damage at the brain and intestinal ends. It intuitively reveals the characteristics of the imbalance of bidirectional regulation of the brain-gut axis based on multi-level indicators. It can systematically quantify the differences in pathological damage among groups from multiple levels, including macroscopic behavioral phenotypes, physiological homeostasis, peripheral neurotransmitters, central neuron morphology, and intestinal mucosal barrier. The evaluation dimensions are comprehensive and the results are objective and reliable. Thirdly, it innovatively constructs a quantitative judgment standard for depression relapse based on the weighted calculation of sucrose preference rate and body weight. By setting a comprehensive score reduction of ≥30% as the judgment threshold, it realizes the objective quantitative differentiation of relapse status in animals, getting rid of the problems of strong subjectivity and inconsistent judgment standards in traditional manual qualitative interpretation.

[0021] In summary, this invention provides a standardized, quantifiable, and clinically relevant animal modeling and evaluation system for relapsed depression. The model is highly stable, has high specificity in pathological features, and provides intuitive and clear observation indicators. It can be accurately used for efficacy evaluation of candidate antidepressant drugs for relapse, analysis of the molecular mechanisms of depression relapse, and screening and development of novel anti-relapse drugs. It has extremely high basic scientific research value and promising prospects for new drug translation and application. Attached Figure Description

[0022] Figure 1 The changes in body weight of rats in each group at different time points; Figure 2 The weight gain of each group of rats at different time points; Figure 3 The sugar water preference rate of each group of rats at different time points; Figure 4 The escape time of the central grid for each group of rats at different time points; Figure 5 The number of times each group of rats stood upright at different time points; Figure 6 The number of grids traversed by each group of rats at different time points; Figure 7 Images of elevated cross maze trajectories for each group of rats at different time points; Figure 8 Pathological morphological changes in hippocampal tissue of rats in each group (HE staining, ×10); Figure 9 Nissl body changes in hippocampal tissue of rats in each group (Nissl staining, ×10). Figure 10Pathological morphological changes in colon tissue of rats in each group (HE staining, ×20); Figure 11 Serum 5-HT levels at different time points for each group of rats. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0025] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0027] A method for constructing an animal model of relapsing depression is proposed, which uses chronic unpredictable stress (CUMS) to simulate depressive triggers, and combines paroxetine administration intervention, withdrawal treatment and mild CUMS relapse induction to construct a relapsing depression animal model.

[0028] Specifically, the steps include the following: (1) Adaptation period feeding: Healthy male rats were selected and subjected to an adaptation period of 1 week. The feeding environment conditions were: temperature 22 ± 2℃, relative humidity 50 ± 5%, circadian rhythm of 12 h light / 12 h dark, and free access to food and water, in order to ensure that the rats adapted to the feeding environment and eliminate the interference of environmental stress on subsequent experiments. (2) Grouping treatment: After the adaptive feeding period, the rats were randomly divided into a blank group (NC), a model group (MS), and a paroxetine administration group (PX) by weight stratification. The weight ratio of rats in each group was consistent to ensure the balance of grouping. (3) Modeling and drug intervention in weeks 2-5: Starting from week 2, the control group rats maintained normal feeding conditions and were given the same volume of pure water as the model rats by gavage every day; the model rats were given chronic unpredictable stress (CUMS) stimulation every day on the basis of normal feeding; the drug treatment group was given paroxetine every day on the basis of modeling, and the drug treatment group was given paroxetine by gavage to achieve simultaneous modeling and drug treatment. This stage lasted for 4 weeks. Withdrawal recovery period (weeks 6-7): Starting from week 6, based on behavioral data and the combined assessment of sucrose preference rate and body weight, the rats in the drug-treated group that were successfully modeled were divided into a continuous drug-treated group (PX-C) and an acute withdrawal group (PX-A). Except for the continuous drug-treated group, which continued to receive drug treatment, the other groups did not receive any drug treatment or stimulation. This withdrawal recovery period lasted for 1-2 weeks.

[0029] Relapse induction period (weeks 8-9): After the drug withdrawal recovery period, the model group and the drug withdrawal group were given mild CUMS stimulation, while the control group was fed normally. This relapse induction period lasted for 3-4 weeks. The entire modeling cycle lasted about 9 weeks, and finally a relapse-type depression animal model was obtained.

[0030] After the model was established, behavioral tests were performed on each group of animals. Finally, brain tissue and serum samples were collected from the animals. The changes in pathological sites of brain tissue and the changes in 5-hydroxytryptamine content were observed by HE staining and HPLC.

[0031] Based on the results of behavioral data and tissue sample analysis, the rat model of relapsing depression was successfully established when experimental animals exhibited three of the following major and minor symptoms. The main symptoms are: (1) a significant decrease in sugar water preference rate; (2) a significant decrease in weight; (3) a decrease in serum 5-hydroxytryptamine content; (4) a decrease in hippocampal tissue compactness and a decrease in the number of neurons; (5) atrophy and a decrease in the number of colonic crypts, a decrease in the number of goblet cells, and significant damage to the intestinal mucosal barrier. The secondary symptoms were: (6) a significant increase in the time to escape the central grid, a significant decrease in the number of upright positions, and a decrease in the number of grids traversed during the open field test. The specific results are as follows: Figure 4 , Figure 5 , Figure 6 As shown; (7) The dwell time of the closed arm in the elevated maze test increased significantly, and the specific results are as follows: Figure 7 As shown.

[0032] Statistical analysis of biomarker levels showed that if the serum 5-hydroxytryptamine level in model rats showed a decreasing trend, vacuolar structures appeared in the hippocampus, and the number of neurons was significantly reduced. Specific changes included... Figure 11 As shown, the results are as follows: The 5-HT levels in the NC group remained high at different time points, with no significant difference among the three groups; however, the MS group during the depressive phase showed a highly significant difference compared to the NC group. P <0.0001, confirming successful establishment of the depression model and the presence of significant 5-HT dysfunction; the difference in the withdrawal period compared with the NC group was extremely significant ( P <0.0001, the content dropped to its lowest point; the difference during the relapse period was significant compared with the NC group ( P The concentration was <0.01%, and the content slightly increased, but it was still significantly lower than the normal level.

[0033] Although the 5-HT levels in the PX-A group were higher during the depressive phase, there was no significant difference compared to the MS group; during the relapse phase, the 5-HT levels in the PX-A group were significantly lower than those in the MS group. The PX-C group may be due to the combined effect of central 5-HT autoreceptor-mediated inhibition of synthesis and peripheral 5-HT transporter (SERT) blockade after paroxetine administration, leading to impaired platelet uptake.

[0034] HE staining results of hippocampal tissue are as follows: Figure 8 As shown, the results indicated that in the control group, the hippocampal tissue structure of rats was compact, the neurons were intact and full with irregular polygonal outlines, the cell nuclei were deeply stained, and no vacuolar changes were observed in the tissue. In the model group, vacuolar structures of varying sizes were visible, and the number of neurons was significantly reduced. After drug intervention, the hippocampal tissue state of the PX-C group was improved to varying degrees compared with the model group, with better tissue compactness, fewer vacuolar structures, slightly more regular neuronal outlines, and a recovery in the number of neurons. After drug withdrawal, the PX-A group showed a large number of vacuolar structures of varying sizes compared with the control group, and the number of neurons was significantly reduced. Moreover, compared with the model group, the number of vacuolar structures further increased, and the number of neurons was relatively reduced.

[0035] HE staining results of colon tissue are as follows Figure 10 As shown, the results indicated that the colonic mucosa of rats in the blank group was intact, with regular villi and crypts, continuous and intact epithelium, abundant goblet cells, and no congestion, edema, or mucosal damage. In the model group, villi were atrophied and broken, crypts were sparse, epithelium was shed over a large area, mucosal congestion and edema were accompanied by extensive inflammatory infiltration, goblet cells were reduced, and the intestinal mucosal barrier was damaged. After continuous drug intervention, the colonic mucosal damage in the PX-C group was significantly repaired compared to the model group, the structure of villi and crypts was improved, epithelial shedding and inflammatory infiltration were reduced, and goblet cells increased. After drug withdrawal and relapse, the colonic mucosa in the PX-A group was eroded and defective, villi and crypts were extensively destroyed, inflammatory cells infiltrated extensively in the lamina propria, goblet cells were depleted, and the mucosal damage was more severe than in the model group.

[0036] Nissl staining results as follows Figure 9As shown, the results indicated that in the control group, the hippocampal tissue of rats had regular morphology, dense arrangement, and abundant Nissl bodies in the cytoplasm. In the model group, the hippocampal tissue structure was damaged, neurons were disorganized, cytoplasm was fragmented, and Nissl bodies were absent. Compared with the model group, the PX-C group showed improved hippocampal tissue density and a significant recovery in the number of Nissl bodies; compared with the control group, the PX-A group showed scattered neuronal arrangement, extensive cytoplasmic fragmentation, and severe Nissl body absence, and compared with the model group, the degree of neuronal disorganization was more severe and the cytoplasmic damage was more significant.

[0037] This indicates that the animal model of relapsed depression has been successfully established and the model is stable.

[0038] The rats were male Sprague Dawley rats, weighing 180-200 g, and the acclimatization period was 7 days.

[0039] Blank group (n=10); Model group (n=10); Drug treatment group (n=30).

[0040] The model was created using the CUMS modeling method, with one type of stress administered daily to avoid using the same stress method for two consecutive days. The stress methods included: fasting for 24 hours, water restriction for 24 hours, day-night reversal for 24 hours, swimming in 4°C cold water for 5 minutes, tail clamping for 2 minutes, foot shock (voltage 1-2 mA, duration 1-2 s, interval 10 s, for a total of 10 times), restraint for 3 hours, heat stimulation (45°C, 10 minutes), and ultrasound stimulation (60 dB, Class A noise exposure for 3 hours), totaling 9 types of stimulation.

[0041] Paroxetine should be dissolved in pure water before administration and should be prepared and used immediately. The dosage is 4.2 mg / kg / day. CUMS stimulation should be performed one hour after administration to avoid interference with animal behavior due to drug sedation.

[0042] Paroxetine is one of the representative drugs of highly selective serotonin reuptake inhibitors (SSRIs). It has irreplaceable value in the treatment of diseases such as depression and anxiety disorders. It is a first-line drug recommended by domestic and international guidelines for the diagnosis and treatment of depression. It should be prepared with pure water and used fresh daily.

[0043] Validation metrics for the construction method include: (a) behavioral evaluation: sucrose preference test; elevated maze; open field test; (b) Physiological status assessment: body weight; (c) Detection of biological indicators: The content of 5-hydroxytryptamine in serum was detected by HPLC, and the morphology of hippocampal cells and neurons was observed by HE staining and Nissl staining. At the same time, the morphology of colon tissue was observed by HE staining.

[0044] Standardized scoring for individual indicators: Sugar water preference rate: A classic core indicator of depression, reflecting the degree of anhedonia. Sugar water preference rate percentage (SPP) (calibrated with the mean of the healthy control group at 100%): In the formula: X is the original value of sucrose preference for a single experimental rat; The average sucrose preference rate of all animals in the healthy control group during the same period; Sugar water preference score: Threshold Explanation: A score of 75% is set as the cutoff for complete recovery. This threshold is based on empirical data from previous literature (Berrio JP, Kalliokoski O. Rethinking data treatment: The sucrose preference threshold for anhedonia in stress-induced rat models of depression. J Neurosci Methods. 2023 Jul 15;395:109910. DOI: 10.1016 / j.jneumeth.2023.109910), showing that sucrose preference rates in depressive-like animal models are typically significantly lower than in control groups (approximately a 25%-40% decrease). A score below 75% is defined as 0.

[0045] Weight change: Degree of restoration of metabolism and physiological homeostasis. Body weight percentage (BW) (calibrated to 100% of the mean of the healthy control group at the same time): In the formula: X represents the body weight of the experimental group. Weight of the healthy control group during the same period; weight change score: Threshold explanation: 10% is set as an acceptable range of normal physiological fluctuations (Luo J, Tang C, Chen X, et al. Impacts of Aerobic Exercise on Depression-Like Behaviors in Chronic Unpredictable Mild Stress Mice and Related Factors in the AMPK / PGC-1α Pathway. Int J Environ Res Public Health. 2020 Mar 19;17(6):2042. DOI: 10.3390 / ijerph17062042). A return to body weight within 100% ± 10% of the control group is considered a basic restoration of physiological homeostasis. A score decreasing to 0 is applied to any weight exceeding 10%.

[0046] Overall Score: A comprehensive assessment of the animal's performance at a specific stage (e.g., the depressive phase D).score Remission period W score Relapse period R score The overall condition of the patient is assessed by weighting and integrating the two scores mentioned above. Since craving for sucrose is a classic core indicator of depression, the weight for craving for sucrose is defined as 0.6, and weight change as 0.4. The calculation formula is as follows: The score ranges from 0 to 1, with a score closer to 1 indicating that the behavior and physiological phenotype during that period are closer to the level of a healthy control.

[0047] Overall Composite Index and Relapse Determination: To quantify the dynamic trajectory of animals throughout the experimental process and ultimately determine relapse, the following definition is used: After experiencing a remission period (withdrawal period), if an animal is subjected to artificially applied subthreshold stress and natural observation, its "relapse period" (Rrelapse) is determined. score The difference between its own "remission period" and its own "remission period" (W) score The decrease exceeds a pre-set threshold (decrease ≥ 30%), that is: The animal is then considered to have "relapsed depressive behavior".

[0048] Body weight changes of rats in each group at different time points are as follows: Figure 1 As shown, weight gain at different times is as follows: Figure 2 As shown, the preference rate for sugary drinks at different times is as follows: Figure 3 As shown in the figure; the specific experimental data for each group are shown in Table 1.

[0049] Table 1: Statistical Results of Experimental Data CUMS modeling was performed, and the success of the model was determined by the sucrose preference rate and weight change after week 5. A sucrose preference rate below 70% and significant weight loss were considered indicative of successful modeling. After a 4-week modeling period, a withdrawal recovery period was initiated. During this period, the model group received continuous stimulation to maintain a state of chronic depression, while the drug-treated group received no stimulation and was fed normally.

[0050] Before entering the withdrawal recovery period, rats that successfully developed the paroxetine model were screened from the treatment groups and randomly divided into two groups, receiving different treatments: The continuous paroxetine administration group (PX-C) received paroxetine 4.2 mg / kg / day as normal. The acute paroxetine withdrawal group (PX-A) stopped administration after entering the withdrawal recovery period, without any additional stimulation.

[0051] Rats that successfully developed the paroxetine model were selected from the drug-treated groups and randomly divided into two groups to ensure randomness and balance. Each group had an equal number of rats, a body weight difference of ≤10%, and similar levels of model development. The groups were: a continuous paroxetine administration group (n=10) and an acute paroxetine withdrawal group (n=10).

[0052] After entering the relapse induction period, mild CUMS stimulation, including ultrasound stimulation, thermal stimulation, and restraint, was administered daily to the model group, continuous drug administration group, and acute withdrawal group. The relapse induction period lasted for two weeks. After the relapse induction was completed, weight changes and behavioral data were recorded for each group.

[0053] The constructed model of depressive relapse was validated by analyzing behavioral changes and pathological characteristics in rats from the blank control group and the experimental group. Anhedonia is a crucial indicator of depressive relapse. In translational medicine, the sucrose preference test serves as a vital bridge connecting clinical symptoms and animal models. Clinically, the Snyder-Hamilton Anhedonia Scale is used to assess anhedonia, while sucrose preference is used in animal models to simulate it. In preclinical animal research on depression, the sucrose preference test is considered the "gold standard" in behavioral testing due to its direct, objective, and quantitative measurement of the core symptom of anhedonia, its sound neurobiological basis, and its sensitivity to pharmacodynamic evaluation. Definition: After a withdrawal period, if an animal, under artificially applied subthreshold stress and natural observation, experiences a decrease in its "relapse period" composite score (Rscore) relative to its "remission period" composite score (Wscore) exceeding a pre-set threshold (a decrease ≥30%), it is considered to have experienced "behavioral relapse of depression." The results showed that the relapsed depression animal model constructed using the method described in this invention not only reflects the two pathological stages of depression onset and relapse, but also accurately simulates the complete clinical course characteristics of "chronic stress sensitization-drug relief-drug withdrawal relapse". The model has a high success rate, good stability and strong reproducibility. Furthermore, a multi-dimensional evaluation system of behavioral, physiological and biological indicators has been established, which is suitable for the efficacy evaluation of antidepressant relapse drugs and the study of relapse mechanism. It can provide a reliable experimental model and scientific basis for the study of depression relapse mechanism and the development of related new drugs.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an animal model of relapsing depression, characterized in that: A relapsing-remitting animal model of depression was constructed by simulating chronic unpredictable stress (CUMS) as a trigger for depression, combined with paroxetine administration intervention, withdrawal treatment, and mild CUMS relapse induction.

2. The construction method according to claim 1, characterized in that: Specifically, the following steps are included: (1) Adaptation period feeding: Healthy male rats were selected and subjected to an adaptation period of 1 week. The feeding environment conditions were: temperature 22±2℃, relative humidity 50±5%, circadian rhythm of 12h light / 12h darkness, and free access to food and water to ensure that the rats adapted to the feeding environment and eliminate the interference of environmental stress on subsequent experiments. (2) Modeling, grouping and drug administration intervention: After 7 days of adaptive feeding, rats were randomly divided into 4 groups according to body weight to ensure a balanced weight distribution in each group. From the second week, the control group rats maintained normal feeding conditions and were given the same volume of pure water as the drug administration group by gavage every day. The model rats were given chronic unpredictable stress (CUMS) stimulation every day on the basis of normal feeding. The drug administration group was given paroxetine 4.2 mg / kg by gavage every day on the basis of modeling. One hour after administration, chronic unpredictable stress was applied to construct the animal model of depression. The 1-hour interval between administration and stress can eliminate the interference of drug sedation effect on the animal behavior test results. This stage was continuously treated for 4 weeks. After the treatment period, qualified rats were selected based on rat body weight and sugar water preference rate. (3) Withdrawal recovery: Starting from week 6, based on the behavioral data results, the rats in the drug-treated group who were successfully modeled by combining the sugar water preference rate and body weight were divided into the continuous drug-treated group PX-C and the acute withdrawal group PX-A. Except for the continuous drug-treated group, which continued to be drug-treated, the other groups were not drug-treated or stimulated. This withdrawal recovery phase lasted for 1-2 weeks. (4) Relapse induction: After the recovery period of drug withdrawal in week 8-9, rats in the acute drug withdrawal group were subjected to mild CUMS stimulation for 2-4 weeks to verify the constructed animal model of relapse-type depression. The CUMS stimulation employed a random combination of various stress methods, with one method administered daily to avoid using the same stress method for two consecutive days. The stress methods included: fasting for 24 hours; water restriction for 24 hours; circadian rhythm reversal for 24 hours; swimming in 4°C cold water for 5 minutes; tail clamping for 2 minutes; foot shock: voltage 1-2mA, duration 1-2 seconds, interval 10 seconds, for a total of 10 times; restraint for 3 hours; heat stimulation at 45°C for 10 minutes; 60dB ultrasound stimulation; and Class A noise exposure for 3 hours.

3. The construction method according to claim 2, characterized in that: The behavioral data mentioned in step (3) include the sucrose preference test; the elevated maze; the open field test; and the forced swimming test. The indicators for verifying the constructed recurrent depression animal model in step (4) include: behavioral data; physiological status evaluation indicator body weight; biological indicator detection: HPLC detection of serum 5-hydroxytryptamine content, HE staining and Nissl staining to observe the morphology of hippocampal cells and colon tissue.

4. The construction method according to claim 2, characterized in that: In step (2), the CUMS modeling is completed in the 5th week. The modeling success is determined by the sucrose preference rate and the change in body weight. If the sucrose preference rate is less than 70% and the body weight shows a significant decreasing trend, the animal model of depression is successfully established.

5. The construction method according to claim 2, characterized in that: Step (4) The relapse induction phase lasts for 2 weeks, with mild CUMS stimulation administered daily, including ultrasound stimulation, thermal stimulation and restraint. The stimulation conditions are as follows: 60dB ultrasound stimulation, Class A noise exposure for 3 hours; 45℃ thermal stimulation for 10 minutes; restraint for 3 hours. The successful establishment of the recurrent depression animal model was achieved when, based on behavioral data and tissue sample analysis, the experimental animals exhibited three of the following primary and secondary symptoms: The main symptoms are: (1) a significant decrease in sugar water preference rate; (2) a significant decrease in weight; (3) a decrease in serum 5-hydroxytryptamine content; (4) a decrease in hippocampal tissue compactness and a decrease in the number of neurons; (5) atrophy and a decrease in the number of colonic crypts, a decrease in the number of goblet cells, and significant damage to the intestinal mucosal barrier. The secondary symptoms are: (6) in the open field test, the time to escape the central grid is significantly increased, the number of times to stand upright is significantly reduced, and the number of grids traversed is reduced; (7) in the elevated maze test, the time to stay in the closed arm is significantly increased.

6. The construction method according to claim 2, characterized in that: The rats were male Sprague Dawley rats, weighing 180-200g.

7. An animal model of relapsing depression constructed using the method described in any one of claims 1-6.

8. The application of the relapsing depression animal model described in claim 7 in screening anti-relapsing depression drugs.

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