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).

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 &#177; 20 g), were acquired and housed in a controlled environment with a temperature of 22&#176;C &#177; 2&#176;C and humidity of 55% &#177; 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
<ol>
	<li>Grouping
	<ol>
		<li>Divide the 24 rats randomly into 2 groups: the control (CON) group, which will not be exposed to isolation or stress, and the model (MOD) group. Each group contains 12 rats.</li>
		<li>House the rats in standard cages measuring 55 cm x 40 cm x 20 cm, with 6 rats per cage. Retain the cage assignment throughout the acclimatization period unless otherwise indicated.</li>
		<li>Fill each rearing cage with fresh bedding and replace it twice a week.</li>
		<li>Conduct a 1-week acclimatization period. Allow the rats unrestricted access to food and water, except during CUMS stressor application. Maintain a constant environment with a temperature of 22&#176;C &#177; 2&#176;C, humidity of 55% &#177; 15%, and a 12 h/12 h light/dark cycle from 08:00 to 20:00, unless otherwise stated.</li>
		<li>Prior to initiating the experiment, handle the rats daily to acclimate them to the researcher and to minimize additional stress during the experimental phase.</li>
	</ol>
	</li>
	<li>Isolation with chronic unpredictable mild stress
	<ol>
		<li>Concurrently place the MOD and CON groups in separate rooms. House the MOD group rats individually, while keeping the CON group rats together. Maintain all other conditions constant.</li>
		<li>Implement a 28-day stressor regimen25. To prevent habituation and to ensure stressor unpredictability, administer one random stressor per day, avoiding the use of the same stressor on consecutive days.</li>
		<li>Randomly apply one of the following nine stressors26,27 on different days: 24 h water deprivation, 24 h food deprivation, wet padding, cage incline, inversion of the light/dark cycle, cold exposure at 4 &#176;C, heat exposure at 45 &#176;C, 1 min tail clamping, or 15 min shaking at 160 rpm. The specific design is outlined in Table 1.</li>
		<li>During the application of the stressor, restrict access to food and water to the MOD group until the stress was over, except during the inversion of the light/dark cycle. The CON group was not required to restrict water and diet.</li>
	</ol>
	</li>
	<li>Stress methods
	<ol>
		<li>Initiate the experiment by applying depression stimuli in conjunction with 28-day isolation to all rats, excluding the control group. House these rats in individual cages. Refer to Table 2 for conditions related to depression stimuli.</li>
		<li>To perform the tail clamp method, secure the tail of a rat from the MOD group by clamping the tail with a standard paper clip at a distance of 1-2 cm from the root of the rat&#39;s tail. Measure the clamping time for a duration of 1 min (n = 12).</li>
		<li>In the water deprivation method, withhold water from a MOD group rat by removing its water&#160;bottle and record for 24 h. 
		NOTE: The time of the start of the water deprivation was recorded so that the end time could be accurately calculated; the behavior of the rats during this period was observed, including activity, appetite and mental state.</li>
		<li>In the food deprivation method, withhold food from a MOD group rat and record for 24 h. 
		NOTE: Record the time when food deprivation begins so that the end time can be accurately calculated; ensure that the rats are adequately hydrated during this period; and observe the behavior of the rats during this period, including activity and mental status, etc.</li>
		<li>For cold stimulation at 4 &#176;C, place a rat from the MOD group in a bucket of cold water and record for 5 min. Ensure that the water temperature remains consistent throughout the test. At the conclusion of the experiment, dry the rat with a blower and return it to its original cage. 
		NOTE: Maintain the cold water temperature at 4 &#176;C using a thermometer and ice cubes to regulate the water temperature when it rises. The water should be changed regularly to ensure that the water quality is clean and the temperature is constant. When swimming, all limbs and trunks of the rats should be immersed in cold water except the head. The depth of the water must be greater than the length of the rat&#39;s body to prevent the rat from jumping out of the water due to contact with the bottom of the bucket.</li>
		<li>To administer 45 &#176;C heat stress, place a MOD group rat in an incubator and record for 5 min, ensuring that the temperature remains stable throughout the test.</li>
		<li>In the inversion of the light-dark cycle, envelop the cage in a black cloth for 1 h&#160;to simulate daytime darkness. Subsequently, illuminate the cage for 12 h during the night to mimic daylight. Record the rat&#39;s&#160;behavior, food and water intake, and sleep patterns for 24 h.</li>
		<li>For the wet padding experiment, introduce 200 mL of water into a cage containing 100 g of padding. House a MOD group rat in the wet cage and record the behavior of the rats under wet bedding stress, including activity, appetite, water intake, etc. Observe whether there are any abnormal behaviors or uncomfortable reactions, such as the skin and hair condition of the rats, which may be caused by the wet padding, and record them in time for subsequent analysis for 24 h. Following the test, dry the rat with a blower and return it to a cage with fresh wood shavings.</li>
		<li>In the cage incline method, situate a MOD group rat in a cage tilted at a 45&#176; angle against a wall and record for 24 h. Use the cage frame structure to adjust the angle and secure the cage in place. 
		NOTE: Calculate the time from start to finish and observe the behavior of the rats during the period of tilting the cage, including activity, appetite, and mental state, and ensure that the angle of the tilted cage is set correctly and remains stable to ensure the accuracy and reproducibility of the experiment.</li>
		<li>For high-speed shaking, place a MOD group rat in a mechanical shaker set to 160 rpm and record the rat for 15 min. Behavioral testing methods are subsequently utilized to evaluate the successful establishment of the model.</li>
		<li>After applying stressors, relocate the MOD group cages from the CUMS room back to the housing room. During the 4-week stress exposure period, maintain the CON group in their home cages situated in the housing room.</li>
	</ol>
	</li>
	<li>Precautions during experiments
	<ol>
		<li>Transfer cages of the MOD group back to the general housing room after applying the stressor in the CUMS room.</li>
		<li>Animal monitoring during CUMS modeling
		<ol>
			<li>During tail clamping, the animal is likely to struggle due to the induced stimulation. Throughout this period, continuously monitor the clamp. If it becomes dislodged, pause the timer, reapply the clamp, and then resume the timer for 1 min.</li>
			<li>Do not impose&#160;water deprivation and wet bedding stressors simultaneously. 
			NOTE: Avoiding the simultaneous imposition of wet padding and water deprivation helps maintain experimental integrity, reduces confounding variables, and promotes animal welfare.</li>
			<li>The animal&#39;s body temperature and ambient room temperature may raise the water temperature during cold water swimming. Therefore, adjust by adding ice water or ice cubes to maintain a constant water temperature.</li>
			<li>Observe the rats at 30 min intervals during the application of stressors, except during diurnal reversal. Pay special attention to signs of unusual distress, such as shivering, lethargy, or lack of movement. If such symptoms are observed-particularly potential hypothermia during 4 &#176;C cold water swimming and wet bedding-immediately remove the rat from the stressor. 
			NOTE: Remove the experimental animals from the study when they have&#160;health problems, such as infection, severe trauma, aggressive behaviours, abnormal mobility, etc.,The conditions for removing the animals from the study typically revolve around protecting their health and safety while ensuring the integrity of experimental outcomes.</li>
			<li>Conduct daily inspections for wounds or other physical or behavioral abnormalities in each rat. If any abnormalities are observed, consult the laboratory veterinarian to determine whether the rat should be excluded from the experiment.</li>
			<li>Weigh each rat every 3 days. If an animal loses more than 20% of its pre-feeding basal body weight, it must be excluded from the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Behavioral tests
<ol>
	<li>To begin, administer the depression stimuli in conjunction with isolation for 28 days to all rats, excluding the control group. House the rats in individual cages. Refer to Table 2 for specifics on the depression stimuli conditions.</li>
	<li>For the open field test, partition a black box into 25 square sections of equal area. Install a video tracking analysis system in the box. Place the rat in the central square and monitor its horizontal and vertical activities for a duration of 5 min. 
	NOTE: The dimensions of the box are 500 mm x 500 mm x 300 mm. Activity data will be gathered using the video tracking system to assess anxiety-related behaviors in the rodents when exposed to a novel environment.</li>
	<li>Subsequently, tally the number of squares traversed by the rat, using all paws, to quantify horizontal activity. Count instances of standing and grooming as indicators of vertical activity. After each test, sanitize the box using 75% alcohol to eliminate any residual rat odors for subsequent tests.</li>
	<li>Next, anhedonia is assessed through the sucrose preference test. Place two bottles on the cage lid: bottle A contains pure water, while bottle B contains 1% sucrose solution. Allow ad libitum access to both solutions for the rat. Weigh the bottles before and after consumption to calculate the 60 min sucrose preference rates on days 0, 7, 14, 21, and 28. The formula is as follows: 
	Sucrose consumption = <img alt="Equation 1" src="/files/ftp_upload/65889/65889eq01.jpg" style="margin: auto;" /> &#215; 100%</li>
	<li>To measure spatial memory and learning abilities, employ the Morris water maze test. Divide the pool into four quadrants, numbering them from one to four. Place a submerged resting platform 1 cm below the water surface in the third quadrant.</li>
	<li>Introduce milk to the pool to increase water opacity and maintain a water temperature of approximately 23 &#176;C throughout the experimental procedure.</li>
	<li>Place each rat in various quadrants of the maze, allowing them 120 s to locate the hidden platform. The rats must rely on spatial memory and learning skills to remember the platform&#39;s location. Once they learn the platform&#39;s location, they can swim directly to it. Record the latency time using the Morris water maze video tracking system.</li>
	<li>Position the rat at a fixed location in the pool. If the subject fails to locate the hidden platform within 120 s, record the latency as 120 s.</li>
	<li>Finally, remove the hidden platform, place the rat back into the water, and record the number of zone-crossings over a 120 s period.</li>
</ol>
3. Statistical analysis
<ol>
	<li>To assess significant differences in biochemical parameters, employ one-way analysis of variance (ANOVA) followed by Duncan&#39;s post hoc test. Present the data as mean &#177; standard error (SE) and regard a p-value of less than 0.05 as statistically significant.</li>
</ol>

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

The authors have no conflicts of interest to disclose.

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 l...

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.

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chronic unpredictable mild stress in rats based on the mongolian medicine

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.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

The scope of our research is Mongolian medical research. In this study, we are investigating the factors contributing to depression in Mongolian medicine, with a particular emphasis on the interaction between internal factors, such as wind, biofilm, and external factors. The problem of conforming to the lack of traditional Mongolian medical models was solved.This protocol addresses the establishment of an animal model based on Mongolian medical theory and then provides some suggestions for future research in Mongolian medicine. In a depressive disorder study, establishing an animal model is crucial for understanding new drugs'pathological mechanisms and effects. Compared with other techniques, this protocol is more in line with the pathogenesis and treatment principles of depression in traditional Mongolian medicine, which will help future research on the treatment of depression in Mongolian medicine.The protocol can demonstrate the relationship between external and internal factors interacting to produce disease and is in accordance with the theoretical system of Mongolian medicine. It will advance the basic research of traditional medicine on depression and provide some recommendations that will help subsequent researchers to research, develop, and utilize traditional medicines.

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...

Watch this Scientific Journal Video about Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine at JoVE.com

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 ...

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Stress Induction Methods for Establishing the Chronic Unpredictable Mild Stress Rat Model

After dividing the rats into a control, or con, and model, or mod, group, house them in individual cages. For the tail-clamp method, secure the tail of a rat and measure the clamping time for one minute. In the water deprivation method, remove the feeding bottle from the cage and record the rat for 24 hours.For the food deprivation method, remove the food from the cage and record the rat for 24 hours. For cold stimulation at four degrees Celsius, place a rat in a bucket of cold water and monitor its activity for five minutes. Then dry the rat with a blower and return it to its original cage.To administer 45 degrees Celsius heat stress, place the rat in an oven and monitor for five minutes. For the inversion of the light-dark cycle, wrap the cage in a black cloth for one hour to stimulate daytime darkness. Subsequently illuminate the cage for 12 hours at night to mimic daylight and record the data for 24 hours.For the wet padding experiment, pour 200 milliliters of water into a cage containing 100 grams of padding. Place the rat in the wet cage and monitor for 24 hours. After drying the rat as demonstrated earlier, return it to a cage containing fresh wood shavings.In the cage incline method, place the rat in a cage angled at 45 degrees against a wall and monitor it for 24 hours. For high speed shaking, place the rat in the mechanical shakers set at 160 RPM, and record data for 15 minutes. After applying stressors, transfer the mod group cage from the CUMS room to the housing room.

Behavioral Tests for Assessing Depression-Related Behaviors in Rats

After dividing the rats into a control, or a CON, and model, or MOD group, house them in individual cages. For the open field test, partition a black box into 25 square sections of equal area. Install a video tracking analysis system in the box.Place the rat in the central square and monitor its horizontal and vertical activities for five minutes. After the test, sanitize the box with 75%alcohol to remove any remaining rat odors. To assess anhedonia through the sucrose preference test, weigh bottle A containing water and bottle B containing 1%sucrose.Place both bottles on the cage lid and allow ad libitum access to both solutions for the rat. After consumption, weigh the bottles, and, using the given formula, calculate the 60 minute sucrose preference rates on given days. To perform the Morris water maze test, divide the pool into four quadrants, and number them from one to four.In the third quadrant, place a resting platform one centimeter below the water surface. Add milk to the pool to increase water opacity and maintain a water temperature of approximately 23 degrees Celsius throughout the experiment. Place the rat in one quadrant of the maze, and using the Morris Water Maze video tracking system, record the latency time to identify the platform for 120 seconds.Finally, remove the hidden platform from the pool. Then, place the rat into the water and record the number of zone crossings for 120 seconds. In the open field test, the MOD group exhibited lower vertical and horizontal scores compared to the CON group, indicating successful induction of anxiety.After 28 days, the MOD group showed a lower sucrose preference percentage than the CON group. The Morris water maze test revealed that the MOD group had a longer latency to locate the platform and crossed fewer times compared to the CON group. Further, the MOD group exhibited reduced hippocampal 5-HT levels compared to the CON group.

528 Views.  Inner Mongolia Medical University.  This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

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