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 it 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'll 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. 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. After dividing the rats into a control or a CON, and model or MOD group, house them in the 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 5HT levels compared to the CON group.