This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.
Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.
Major depressive disorder (MDD) is a prevalent mental illness, ranking as the third leading cause of disability globally and affecting over 300 million people1,2,3. Notably, it is estimated that at least half of the affected individuals do not receive adequate treatment4. Given this gap, animal models serve as a crucial tool for investigating the etiology of depression. To date, more than 20 different animal models for depression exist5. Among these, the chronic unpredictable mild stress (CUMS) model, refined by Paul Winer in 1987, is the most frequently utilized6. The CUMS model operates on the premise that exposing rodents to a diverse range of socio-environmental stressors leads to symptoms akin to anxiety, tension, and depression. The methodology involves exposing animals to various mild stressors over several weeks, culminating in a range of behavioral alterations, including anhedonia and depressive-like behaviors7,8. These changes are accompanied by shifts in endocrine and neurotransmitter profiles, such as a reduction in 5-HT9,10. These outcomes closely mirror those observed in humans diagnosed with MDD, thereby validating the model’s utility. The CUMS model is particularly valued for its effectiveness in evaluating antidepressants, manifesting high levels of surface, structural, and predictive validity11,12. Unlike other models, CUMS is sensitive to the effects of chronic administration of monoaminergic antidepressants. For instance, selective serotonin reuptake inhibitors (SSRIs) such as citalopram, paroxetine, and fluoxetine have been shown to prevent and reverse anhedonia under conditions of chronic stress12,13. Additionally, new rapid-acting antidepressants, such as ketamine, have also demonstrated efficacy in this model14,15. In contrast, other tests like the forced swimming test (FST) and tail suspension test (TST) are less reliable for modeling long-term behavioral changes, often reflecting adaptations to acute stress rather than enduring symptoms of depression16. These characteristics underscore the robust validity of the CUMS model in depression research. One of the most salient features of the CUMS model, recognized for its high reliability in classical studies, is anhedonia-the incapacity to experience pleasure or interest in everyday activities17,18. This phenomenon is commonly evaluated using sucrose preference tests, and many antidepressants have been shown to reverse reduced sucrose consumption. Several other metrics are also commonly employed in the CUMS literature, including the open field test (OFT), which assesses voluntary motor behavior, exploratory tendencies, and tension, thereby gauging the severity of depression19. Other tests such as the elevated plus maze (EPM) evaluate anxiety-like behaviors, the Morris water maze test (MWM) examines cognitive functioning20, and the FST assesses susceptibility to negative emotions and behavioral despair20. Furthermore, the majority of stressors that impact humans are social in nature. Individuals with suboptimal social relationships, characterized by limited social activities, networks, and support, are at a heightened risk for various diseases21,22. This is also relevant in the case of rodents, who are social animals living in groups. For example, rats that are housed in isolation exhibit characteristics of what is termed isolation syndrome, which induces social stress and expedites the onset of depression23.
Mongolian medicine, a significant branch of Chinese medicine, posits that the onset of disease is a complex interplay between intrinsic and external factors. These external factors, referred to as the four auxiliary conditions, encompass climate change, diet, lifestyle, and sudden events such as infections, startling incidents, and psychological disorders. The disease process is conceptualized as an ongoing interaction between three elements-referred to as the three types of homors-and the Seven Bodily Constituents in concert with the four auxiliary conditions24. Mongolian medicine holds that the human body functions as an integrated entity, maintained by a relative balance among the three homors. A disruption in this balance is considered to be a precursor to illness24. Given the pivotal role of animal experimentation in bridging traditional and modern medicine, it is crucial to develop animal models that are relevant for research in the field of Mongolian medicine. Accordingly, we employed a 28-day isolation methodology coupled with CUMS to simulate these physiological and psychological stressors. We selected nine specific unpredictable stressors and sought to underpin this modeling method through the Three homors theory of Mongolian medicine. Establishing a robust animal model is fundamental for advancing basic research in Mongolian medicine and will contribute significantly to its foundational studies.
The experimental protocols received approval from the Ethics of Animal Experiment Care Committee of Inner Mongolia Medical University (YKD202301172) and adhered to the National Institutes of Health guidelines for animal care and ethics. The license number for our animal center is NO.110324230102364187. Twenty-four male Sprague-Dawley (SD) rats, each 8 weeks old (200 g ± 20 g), were acquired and housed in a controlled environment with a temperature of 22°C ± 2°C and humidity of 55% ± 15%. Feed the rats with a rodent maintenance-feed diet and pure water with corn cobs for bedding. The rats were subjected to a 12 h/12 h light/dark cycle for 1 week prior to experimentation.
1. Establishment of CUMS rat model
2. Behavioral tests
3. Statistical analysis
Results from the behavioral tests in the CUMS-induced rat depression model
To corroborate the efficacy of the CUMS procedure for inducing depression-like behaviors, a manipulation check was performed. Male Sprague-Dawley (SD) rats were randomly allocated to either the MOD or CON group for a period of 4 weeks, as outlined in step 1.2.3. Subsequently, the rats were sacrificed, and their hippocampi were completely dissected for assessment of 5-HT, a neurotransmitter strongly associated with depression pathophysiology10, using enzyme-linked immunosorbent assay (ELISA)28.
Before CUMS induction, no significant differences were observed among the tested groups in the OFT score, sucrose preference, or MWM analysis.
After establishing the CUMS model, a one-way analysis of variance (ANOVA) revealed a significant impact of CUMS on rat anxiety behaviors. Specifically, in the OFT, significant differences were observed between the groups. The MOD group exhibited lower vertical and horizontal scores compared to the CON group (**P < 0.01; Figure 1A,B,E), suggesting that the CUMS procedure was effective in inducing anxiety.
Subsequently, the sucrose preference test (SPT) was administered over 5 days, as described in step 2.4, to evaluate whether the rats showed signs of anhedonia post-CUMS exposure. Results indicated no significant differences in sucrose preference percentage between the groups on day 0. However, one-way ANOVA revealed a significant difference in sucrose intake between the groups on day 28. Specifically, the sucrose preference percentage in the MOD group was lower than that in the CON group (**P < 0.01; Figure 1C), confirming the effectiveness of the CUMS protocol in inducing anhedonia.
A one-way ANOVA revealed that CUMS significantly impacted the spatial memory and learning abilities of rats, as assessed by the MWM test. The latency for the MOD group to locate the platform was notably longer than that for the CON group (**P < 0.01). In terms of crossing times, the MOD group crossed fewer times than the CON group (**P < 0.01; Figure 1D,F). These findings support the efficacy of the CUMS protocol in inducing memory impairment in rats.
Results of hippocampal 5-HT levels in the CUMS-induced rat depression model
One-way ANOVA on hippocampal 5-HT levels indicated a significant difference among the groups (**P < 0.01). The MOD group exhibited reduced hippocampal 5-HT levels when compared to the CON group (Figure 2). These results suggest that the CUMS protocol effectively decreased hippocampal 5-HT levels, a phenomenon commonly observed in human depression29.
Figure 1: Effects of the CUMS-induced rat depression model. (A) Open field test (OFT) vertical scores. (B) Open field test horizontal scores. (C) Sucrose consumption levels (%) on days 0 and 28. CUMS-induced anhedonia and reduced sucrose preference in rats. (D) Latency time and zone crossing numbers in the MWM test. Results are presented as the mean ± SE (n = 12 rats per group). **P < 0.01 indicates significant changes in the model (MOD) group compared to the control (CON) group calculated using one-way analysis of variance (ANOVA). (E) OFT tracks for rats in each group. (F) MWM tracks for each group of rats. Please click here to view a larger version of this figure.
Figure 2: Effect on brain 5-HT levels in CUMS-induced depression model in rats. Rats subjected to 4 weeks of CUMS demonstrated a significant reduction in hippocampal 5-HT levels compared to the control (CON) group. Results are presented as the mean ± SE (n = 12 rats per group). **P < 0.01 indicates significant changes in the model (MOD) group compared to the CON group calculated using one-way analysis of variance (ANOVA). Please click here to view a larger version of this figure.
Stressors | Days |
Tail clamp 1 min | 1, 7, 20 |
Water deprivation 24 h | 3, 18, 28 |
Food deprivation 24 h | 2, 16, 25 |
Swimming in 4 °C cold water 5 min | 4, 11, 19, 23 |
45 °C heat stress 5 min | 5, 14, 17, 27 |
Inversion of light dark cycle | 6, 12, 22 |
Wet padding | 9, 15, 26 |
Cage incline 24 h | 10, 21 |
High-speed shaking 15 min | 8, 13, 24 |
Table 1: Chronic unpredictable mild stress (CUMS) schedule. Stressors and execution dates for the induction of CUMS in rat subjects.
Stressors | Four Auxiliary Conditions | Three Homors change |
Tail clamp 1 min | Sudden factors | Wind↑ Bile↑ |
Water deprivation 24 h | Diet | Wind↑ |
Food deprivation 24 h | Diet | Wind↑ |
Swimming in 4 °C cold water 5min | Living | Phlegm↑ |
45 °C heat stress 5min | Living, Climate change | Bile↑ |
Inversion of light dark cycle | Living | Wind↑ |
Wet padding | Living | Phlegm↑ |
Cage incline 24 h | Living | Wind↑ |
High-speed shaking 15min | Sudden factors | Wind↑ |
Table 2. Nine stressors with the four auxiliary conditions and the theory of three homors in Mongolian medicine. The intrinsic relationship between the nine stressors, the theory of three homors, and four auxiliary conditions in Mongolian medicine.
Depression is a mental disorder characterized by symptoms such as low mood, lack of pleasure, and reduced energy30. In the field of depression research, the establishment of a reliable animal model is crucial for advancing therapeutic interventions. Among various animal models, the CUMS model is particularly noteworthy for its high reliability, validity, and its congruence with the characteristics of human depression31. It is well-suited to mimic the cumulative effects of low-level stressors over an extended period across various settings. In this study, we established a CUMS-induced depression model in rats, employing a range of stressors outlined in step 1.2.3 and Table 2. Several aspects of the stress protocol are critical for the model’s success. Firstly, rats in all MOD groups must be housed individually, unlike those in the CON group. Secondly, the CUMS stress regimen should be carefully planned to maintain unpredictability. Specifically, rats should be subjected to a randomized selection of seven out of the nine possible stressors on a weekly basis. The variability and unpredictability of these stressors are essential for the model’s efficacy32,33. Lastly, repeated use of the same stressor should be minimized to prevent the rats from adapting, which could compromise the integrity of the experimental outcomes. Moreover, the stressor settings were designed to be adjustable throughout the course of the study.
The CUMS model offers several advantages as a depression model. It closely aligns with the pathogenesis of human depression and accurately represents most clinical symptoms and underlying mechanisms34. One of its key strengths is that it uses anhedonia, or the lack of pleasure, as a quantifiable indicator6,7. The model also accounts for the various factors that contribute to the onset of depression35,36. Importantly, the CUMS model is designed for long-term application, spanning several months. This duration is consistent with the needs of a depression model and allows for the assessment of drugs in a chronic dosing regimen, as well as the identification of compounds with rapid mechanisms of action36. Due to its comprehensive nature, the CUMS model has become widely accepted in the scientific literature.
Despite these advantages, the CUMS model is not without limitations5,37. One of the primary drawbacks is its resource-intensive nature, requiring significant investments in environment, manpower, and time, which consequently results in low reproducibility38. There are also inconsistencies in the literature regarding the selection and intensity of stressors, as well as the sequence in which they are applied38. Such inconsistencies make it challenging to replicate the model across different laboratories. Furthermore, the literature lacks a detailed time schedule for applying stressors, adding another layer of ambiguity39,40. Another issue arises from the variation in combination methods employed across different studies; these often lack explicit rationale, creating difficulties in model replication. Subject animals are highly sensitive to minor alterations in the stressor design, which also contributes to low reproducibility40. Moreover, using a limited number of stressors can lead to habituation among the subject animals, making the stressor design predictable. Finally, animals from different laboratories show varied responses to stressors, contributing to the poor reproducibility across studies. In the existing literature, Wistar Kyoto (WKY) rats have been identified as particularly susceptible to a depression-like phenotype following exposure to CUMS25,41. This suggests that inter-strain variability can significantly affect susceptibility to stress-induced behavioral changes. Similarly, the SPT, a key metric often used for assessing the efficacy of depression models, presents its own set of challenges. Specifically, individual rats show variability in their consumption of a 1% sucrose solution20,42. Under stress conditions, both stress-prone and stress-resistant animals exhibit alterations in their consumption of sweet solutions. Consequently, it becomes challenging to ascertain the proportion of animals within a given batch that are genuinely stress-resistant or belong to the opposite category. This variability may result in some animals displaying SPT outcomes that do not accurately reflect their disease state.
To enhance the reliability of the CUMS model, we propose several methodological refinements. First, it is crucial to segregate animals in the CON group from those in the MOD group to mitigate the impact of olfactory, visual, and auditory stimuli, which can induce stress in the animals43,44. Second, to minimize variability, it is advisable to designate one or two experienced laboratory personnel to conduct all experiments. This approach serves to reduce errors attributable to individual factors. In addition, prior research has identified stress-resilient test animals13; hence, it would be prudent to screen for such groups either prior to formal experimentation or post hoc to better interpret the results. Regarding the SPT test, which is a reward-based behavioral assessment that gauges intrinsic anhedonia, it is essential to determine the optimal sucrose concentration before initiating the CUMS protocol. Preliminarily establishing preference based on sucrose concentration can markedly enhance the test’s discriminative sensitivity20. However, it should be noted that rats subjected to 24 h food and water deprivation may indiscriminately consume any available liquid due to extreme thirst35,45. To obtain a more accurate measure of sucrose preference, we recommend assessing consumption during the nocturnal phase of the circadian cycle, when rodents are most active. Stressors applied during the diurnal (inactive) phase could result in chronic sleep deprivation, thereby introducing an unintended stressor46,47. Finally, it is imperative to meticulously control for the influence of manageable factors described above to ensure that the data collected are as reliable as possible.
The current Western clinical treatment regimens for depression often come with pronounced toxic side effects and a high likelihood of relapse upon cessation of medication. Investigating treatment approaches rooted in traditional medicine and delving into the pathogenesis of depression through the lens of Mongolian medicine theory could offer innovative insights into the disease’s etiology. In this context, the development of animal models of depression that align with traditional medicine principles stands as a pivotal step in Mongolian medicine research. The selection of the nine stressors in the solitary-culture CUMS modeling method is particularly noteworthy; it is informed by Mongolian medicine’s theory of the three homors and the inherent relationship between the four auxiliary conditions and the three homors. This approach exhibits a high degree of compatibility with the unique tenets of Mongolian medicine. Phenotypic analyses of depression, in conjunction with external factors, reveal significant correlations with the Mongolian medical concepts of Wind and Phlegm (see Table 2). Specifically, wind-related dysfunctions manifest clinically as symptoms such as loss of energy, insomnia, forgetfulness, fatigue, and pain. Dysfunctions related to Phlegm, on the other hand, result in clinical manifestations like unresponsiveness, blurred consciousness, pessimism, and negativity. These symptoms align closely with the core symptoms of MDD48,49. Therefore, the aforementioned CUMS model serves as a pathological model that effectively simulates the complex interactions among external conditions and the balance of the three types of homors, as posited by Mongolian medicine theory, thereby contributing to the onset of disease.
The strengths of this model are multifaceted. Firstly, it adheres to contemporary medical etiological theories, satisfying the criteria set for animal models within the realm of modern medicine. Secondly, it incorporates principles from traditional Mongolian medicine to replicate specific symptoms of human diseases in animals, thus embodying characteristics unique to Mongolian medical practices. Given its alignment with both modern and traditional medical theories, this model serves as an effective platform for foundational research in Mongolian medicine. Therefore, the integration of diseases and symptoms within this animal model represents a confluence of research in Mongolian and modern medicine. While the model has its limitations, its ongoing refinement will significantly contribute to the future of basic research in Mongolian medicine.
Animal models have become indispensable in the exploration of depression due to the ethical and practical challenges associated with direct human brain study. Despite their inherent limitations in fully replicating the complexities of depression, the evolution and validation of these models remain a continual process. In this study, we have combined principles from both Mongolian and modern medicine to develop an animal model of depression. This collaborative approach offers valuable insights for constructing a more comprehensive animal model that encompasses both traditional Mongolian and modern medical theories.
The authors have nothing to disclose.
We are grateful for the instrumentation and laboratory provided byextend our gratitude to the Mongolian medical faculty of the Inner Mongolian Medical University, China.This study was supported by, for providing the necessary instrumentation and laboratory facilities. This study received financial support from the National Natural Sciences Foundation of China (Grant No. 81760762) and), the Inner Mongolia Medical University Project of China (Grant No. YKD2022MS074), and the Scientific Research Project of Higher Education in Inner Mongolia, China (Grant No. NJZY22661) and the Open Fund Project of Key Laboratory of Chinese and Mongolian Medicine in Inner Mongolia Autonomous Region,China (Grant No. MYX2023-K07).
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Rat 5-HT ELISA Kit | Lian Ke bio Co., Ltd,China | 96T/48T | |
SPF grade Sprague Dawley (SD) rats | SPF (Beijing) Biotechnology Co | SCXK(JING)2019-0010 | |
Sprague Dawley rats | Beijing Biotechnology Co., Ltd, China | SCXK (JING) 2019-0010 | |
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