A rodent breeding and ethology experiment device and a method for establishing a chronic social comparison stress model

Abstract

The present invention discloses a rodent breeding and ethology experiment device and a method for establishing a chronic social comparison stress model. The device includes a box body assembled by a bottom plate and side plates. The top of the box body is provided with a mesh box cover that can be opened. The box body is also provided with movable partitions for partitioning the internal space of the box body into several independent activity spaces. The partitions are one or several of transparent partitions, opaque partitions, and transparent or opaque partitions with through holes, and the size of the activity space can be adjusted according to the number and placement position of the partitions to control the activity area and communication degree of the rodents. The present invention can flexibly change the activity space according to the placement position of the movable partitions to form multiple activity spaces, explore the breeding and ethology characteristics of rodents from multiple angles, establish chronic social comparison stress models of different grades of rodents under different breeding environments, with low cost and the realization of diversified model establishment.

Description

Technical Field

[0001] The present invention relates to the technical field of experimental equipment, and particularly relates to a rodent breeding and ethology experiment device and a method for establishing a chronic social comparison stress model. Background Art

[0002] Social comparison refers to the process in which an individual compares his own environment, viewpoints, abilities, etc. with others. In the absence of an objective judgment standard, an individual will compare his own situation with others to obtain self-evaluation. Negative self-evaluation makes social comparison related to depression, and studies have shown that social comparison may induce depression. At present, most of the research on the brain neural mechanism of social comparison is based on humans. The social comparison behavior of non-human primates is less, and there is no research on social comparison of rodents. So far, there is no experimental modeling device and experimental paradigm for rodent social comparison stress at home and abroad.

[0003] The establishment and detection of animal behavior models are one of the important technologies in modern neuroscience and psychology research. By observing the social, decision-making, learning and other behaviors of animals, research on learning and memory, emotion and cognition can be carried out. The chronic social comparison stress model established through an animal breeding box can be used to explore the social comparison behavior of rodents and broaden the understanding of the social behavior of rodents. At the same time, animals that have undergone chronic social comparison show anxiety and / or depressive-like behaviors, providing a new way and means for further exploring the mechanisms of anxiety and depression. Summary of the Invention

[0004] In order to establish a chronic social comparison stress model for rodents, obtain the ethological characteristics of animals in different comparison environments, and provide a new means for exploring anxiety and depression, the present invention provides a rodent breeding and ethology experiment device and a method for establishing a chronic social comparison stress model.

[0005] The present invention adopts the following technical solutions:

[0006] On the one hand, the present invention provides a rodent breeding and ethology experiment device, including a box body assembled by a bottom plate and side plates. The top of the box body is provided with a mesh box cover that can be opened. The box body is also provided with a movable partition for separating the internal space of the box body into several movable spaces. The partition is one or several of a transparent partition, an opaque partition, and a transparent or opaque partition with through holes. And the size of the movable space can be adjusted according to the number and placement position of the movable partitions to control the activity area and communication degree of rodents.

[0007] Further, food trough openings and water bottle openings for hanging food are also provided on the side plates, and at least one of each of the food trough openings and at least one of the water bottle openings are formed on the side plates corresponding to each activity space in the box body.

[0008] Preferably, the through holes on the partition are one or a combination of regular graphic holes and / or irregular graphic holes; or a control door for controlling the opening and closing of the through holes is further provided on the partition.

[0009] More preferably, fixing holes for fixing the partition are further provided at two opposite positions of the side plates on both sides of the box body, and both ends of the partition are fixed to the corresponding side plates through connecting fasteners.

[0010] Preferably, two partitions are provided inside the box body to form a three-compartment social comparison experimental box.

[0011] Preferably, a box body activity space extends outward from both sides of the middle activity space of the box body, and each activity space in the box body is separated by the partition into a five-compartment social comparison experimental box with a cross shape.

[0012] On the other hand, the present invention also provides a method for establishing a chronic social comparison stress model, characterized in that,

[0013] Step 1, randomly match rodents and place them in pairs or groups in an undivided breeding experimental device for adaptive breeding to form an experimental group;

[0014] Step 2, conduct an animal social status dominance experiment test on the animals adapted to the breeding environment, and determine the animals with higher social status as social comparison stress-applying animals and the animals with lower social status as social comparison stress animals according to the test results;

[0015] Step 3, put the matched pairs or groups of animals into an experimental device with two or more independent spaces formed by a transparent partition with through holes, and give the social comparison stress-applying animals better breeding stimulation conditions than the social comparison stress animals to start the formal experiment;

[0016] Step 4, give continuous stimulation to the social comparison stress-applying animals and the social comparison stress animals for 24 hours respectively. When the given breeding stimulation conditions reach the set stimulation time, give the social comparison stress-applying animals and the social comparison stress animals breeding stimulation conditions different from the previous breeding stimulation conditions respectively;

[0017] Step 5, after the stress stimulation intervention for a set time, conduct a modeling effect test on the rodents in the box body to obtain the anxiety or depression-like behavior indexes of the tested rodents, and make an evaluation of the chronic social comparison of the rodents after statistical analysis.

[0018] Further, in step 1, two rodents matching the social ranks of the two animals in step 2 are provided as a control group, and the two rodents in the control group are subjected to undifferentiated feeding stimulation conditions.

[0019] Further, in step 2, for the two rodents adapted to the environment of the feeding experiment device, an animal social status dominance experiment is used to determine the social rank. The specific method is as follows: a transparent pipe with a set length that allows a rodent to pass through is used, and a mark is made at the center position of the transparent pipe; the two rodents are simultaneously placed at both ends of the pipe and timed; within the set time, if one rodent is pushed out of the pipe by the other rodent, the pushed-out rodent is set as the social comparison stress-applying animal, and the other pushed-out rodent is set as the social comparison stressed animal; if neither of the two rodents exits the pipe within the set time, the rodent closer to the same-side pipe outlet is set as the social comparison stressed animal, and the other rodent is set as the social comparison stress-applying animal.

[0020] Further, in step 3, the matched paired or grouped rodents are placed in one of a two-chamber social comparison experiment chamber, a three-chamber social comparison experiment chamber, or a five-chamber social comparison experiment chamber, and differentiated feeding stimulation conditions are provided for the social comparison stress-applying animal and the social comparison stressed animal.

[0021] Further, in step 4, at least one good feeding stimulation condition is provided to the animal with a higher social rank in the experimental group every day, which is set as the social comparison stress-applying animal, and normal feeding conditions or at least one bad environmental stimulation condition is provided to the animal with a lower social rank in the experimental group every day, which is set as the social comparison stressed animal; regardless of the social rank of the animals in the control group, normal feeding conditions or at least one good stimulation condition or at least one bad stimulation condition is provided to the animals in the control group every day.

[0022] Further, in step 5, during the period of performing stress stimulation intervention, feces are collected from the rodents once a week, and the 24-hour sucrose preference is measured once. After the intervention, all animals are tested for three-chamber social interaction, open field, open field social interaction, elevated plus maze, 12-hour sucrose preference test, and forced swimming test to obtain their behavioral data for evaluating their depressive or anxious behaviors.

[0023] Further, the method further includes step 6 of taking samples for biochemical detection, which is used to detect the content of related proteins in the brain nuclei of the social comparison stressed animals and evaluate the changes in the levels of depression-related proteins in the rodents after chronic social comparison stress.

[0024] The technical solution of the present invention has the following advantages:

[0025] A. The present invention forms a spliced box body composed of a bottom plate, side plates and a box cover, and partitions that can form various communication levels and activity spaces are arranged in the box body. According to the experimental purpose, the interior of the box body is partitioned into multiple independent activity spaces. The two-box or three-box experimental boxes formed by splicing can stably establish a chronic social stress model of rodents such as rats for differential social comparison. After several days of chronic treatment, rats treated with different chronic environments show differences in anxiety or depressive behavioral indicators, which is of great significance for conducting behavioral feeding experiments on rodents.

[0026] B. The experimental box model established by the present invention can establish a stress model in the form of one-to-one or one-to-many, saving the amount of experimental rats, enhancing the uniformity of stress received among different experimental groups, expanding the number of samples in unit time or unit batch experiments, and reducing breeding and experimental costs.

[0027] C. The experimental device provided by the present invention can flexibly change the size of the activity space according to the placement position of the movable partition, and can form differences in each activity space. It not only realizes environmental changes in animal breeding conditions, but also provides comfort changes for animals due to changes in the size of the activity area, exploring the breeding and behavioral characteristics of rodents from multiple angles, and establishing a chronic social comparison stress model for different grades of rodents in different breeding environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for the specific embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 The front view of the two-box social comparison experimental box provided by the present invention;

[0030] Figure 2 is Figure 1 the top view shown;

[0031] Figure 3 The top view of the three-box social comparison experimental box provided by the present invention;

[0032] Figure 4 is Figure 3 the schematic diagram of the side plate structure in;

[0033] Figure 5 The schematic diagram of the partition structure;

[0034] Figure 6 The schematic diagram of the box cover structure;

[0035] Figure 7 Top view of the five-chambered social comparison experimental chamber provided by the present invention;

[0036] Figure 8 Schematic diagram of the experimental procedure provided in Example 1;

[0037] Figure 9 A - F in [figure] are schematic diagrams of the inspection effect data provided in Example 1.

[0038] The markings shown in the figure are as follows:

[0039] a - box body; 1 - bottom plate; 2 - side plates, 21 - food trough opening, 22 - water bottle mouth opening, 23 - fixing hole, 24 - hanging hole; 3 - box cover, 31 - hinge, 32 - hook; 4 - partition, 41 - through hole; 5 - connecting fastener. Detailed implementation manners

[0040] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0041] As Figure 1 、 Figure 2 and Figure 3 shown, the present invention provides a rodent breeding and ethology experiment device, including a box body a assembled by a bottom plate 1 and side plates 2. A mesh box cover 3 that can be opened is provided at the top of the box body a. An activity partition 4 is further provided inside the box body a for separating the internal space of the box body a into several independent activity spaces. The partition 4 is one or several of a transparent partition, an opaque partition, and a transparent or opaque partition with through holes 41, and the size of the activity space can be adjusted according to the number and placement position of the activity partitions 4 to control the activity area and communication degree of the rodents.

[0042] Food trough openings 21 for hanging food and water bottle mouth openings 22 are further provided on the side plates 2, and at least one food trough opening 21 and at least one water bottle mouth opening 22 are formed on the side plates 2 corresponding to each activity space inside the box body a. These food trough openings 21 and water bottle mouth openings 22 are used to provide food and water for the rodents. As Figure 5 shown, the through holes 41 on the partition 4 are one or several combinations of circular, square, oblong, and strip-shaped. Of course, through holes 41 with small sizes can be provided, and through holes 41 with large sizes can also be provided on the partition. Of course, through holes 41 with mixed large and small sizes can also be provided on the same partition 4. Of course, it is not limited toFigure 5 The structure of the through holes 41 in the partition board 4 provided therein. Through holes with larger sizes can improve the communication between animals in adjacent activity spaces more effectively than those with smaller sizes.

[0043] As Figure 1 and Figure 4 shown, fixing holes 23 for fixing the partition board 4 are also provided at two opposite positions on the two side plates 2 on both sides of the box body a. The two ends of the partition board 4 are fixed to the corresponding side plates 2 through connecting fasteners 5; in combination with Figure 1 and Figure 2 shown, two screw holes are respectively provided on the upper parts of the two side plates 2. The gap formed by the two screw holes is larger than the thickness of the partition board 4. When the fastening connectors 5 are installed on the two screw holes on both sides, the gap formed by the two fastening connectors 5 is slightly larger than the thickness of the partition board, which is convenient for disassembling and assembling the partition board 4.

[0044] As Figure 2 shown, one partition board is arranged inside the box body. The internal space of the box body is divided into two by the partition board, forming a two - chamber social comparison experimental box. That is, the rectangular side plates and the two end side plates are stapled with an air gun to form the side walls of the box body that are closed on all four sides. Note that the short - side side plates are pressed on the outer vertical surface of the long - side side plates. Then the side walls of the box body are stapled and fixed to the bottom plate. The box cover 3 is made of two pieces of wire mesh. One side of the box cover 3 is fixed to the upper edge position of one side plate 2 by a hinge 31, and the other side is fixed to the hanging hole 24 on the upper part of the opposite side plate 2 through a hook 32. This box body a can be used to raise paired groups of animals. By replacing the middle partition board 4, the different social communication degrees of the two groups of animals can be controlled: if an opaque partition board without holes is inserted, the requirements for raising animals separately can be met; if a transparent partition board with holes is inserted, visual and olfactory communication of the animals can be ensured. On this basis, different living environments in the units on both sides of the partition board can be designed according to the experiment to establish a paired chronic social comparison stress animal model.

[0045] As Figure 3 and Figure 4 shown, two partition boards 4 are arranged inside the box body a, forming a three - chamber social comparison experimental box. One of the two side plates 2 here can be the Figure 4 shown structure, while the opposite side plate of it and the two opposite side plates in the other direction are both of opaque plate structures. This box body a is a three - chamber social comparison experimental box. This box body can be used to raise three animals / group with different environmental treatments. By replacing the middle partition board, the different social communication degrees of the three groups of animals can be controlled. Removing the partition board can combine the internal space of the breeding cage into one, increasing the animal breeding space.

[0046] As Figure 7As shown in the figure, it is also possible to extend both sides of the movable space in the middle of the box outward to form a movable space for the box, and the various movable spaces in the box are separated by partitions into a five-box social comparison experimental box with a cross shape, which can be used for social comparison modeling experiments of one-to-many for rodents. It uses the social comparison stressor animals in the central area and cooperates with multiple peripherally experimental activity spaces that can be plugged and assembled externally, enabling the stressor animals to be utilized efficiently by multiple times, reducing the cost of experimental animals, and ensuring that the stress intensity received by each experimental animal is consistent.

[0047] In this box, social comparison stressor animals can be raised in the box located in the center, and social comparison stressed animals can be raised in other boxes. Or social comparison stressed animals can be raised in the box located in the center, and social comparison stressor animals can be raised in other boxes to increase the stress intensity. By replacing the intermediate partition, the different social communication degrees of the three groups of animals can be controlled.

[0048] The design of inserting partitions in the box of the present invention makes the space use flexible and convenient. The detachable design greatly improves the utilization rate of the experimental space and the breeding space. After being disassembled, it can be flatly stored and transported, reducing the waste of space of experimental equipment.

[0049] The present invention also provides a method for establishing a chronic social comparison stress model, including the following steps:

[0050]

S001

[0051]

S002

[0052] The present invention preferably uses an animal social status dominance experiment to determine the social hierarchy for two rodents adapted to the breeding and experimental device environment, and uses the rodent with a higher social hierarchy after the hierarchy test as the social comparison stressor animal, and uses the other rodent as the social comparison stressed animal, and gives the social comparison stressor animal better breeding stimulation conditions than the social comparison stressed animal. Similarly, for the animals in the control group, the above-mentioned animal social status dominance experiment method is also used to determine the two animals with high and low social hierarchies, and the rodent with a higher social hierarchy is used as the social comparison stressor animal, and the other animal is used as the social comparison stressed animal. However, the two animals in the control group receive non-differentiated breeding stimulation conditions, that is, the same breeding conditions, usually using the normal breeding method.

[0053]

S003

[0054]

S004

[0055] Provide at least one good feeding stimulation condition for the socially comparing stressor animals in the experimental group every day, provide normal feeding conditions for the socially comparing stress animals in the experimental group or at least one bad environmental stimulation condition every day; provide normal feeding conditions for the socially comparing stressor animals and the socially comparing stress animals in the control group or at least one good stimulation condition or bad stimulation condition every day.

[0056] It mainly includes three environmental conditions. The general environment is the ordinary feeding environment condition, which only includes bedding, ordinary feed and water. The good stimulation condition is the rich feeding environment condition. The rich feeding environment condition can include high-protein food, nest box, toys, drill barrels, mazes, running wheels, supporting boards, inclined ladders, female partners, etc.; the bad feeding stimulation condition can include water deprivation, food deprivation, wet bedding, stroboscopic lights, continuous light, 42 °C oven heat stress, empty bottle stimulation (pour out the drinking water), narrow and crowded space, inclined bottom plate, etc. According to the above environmental stimulation needs, prepare stimuli such as dried mealworms, condensed milk, female animals, partition boards for restricting activities and crowded other male animal companions.

[0057]

S005

[0058]

S006

[0059] Take rats as an example to establish a social comparison animal model. The specific experimental method is:

[0060] Prepare about 250 g of adult male SD rats and pair-feed them in a common breeding cage, two rats per cage. Set the breeding cage to have day and night reversed from the outside world, with a temperature of 23 - 25 degrees Celsius, and let the rats adapt to the breeding environment for at least 7 days. During the adaptation period, the experimenter needs to hold the rats for at least 3 days, at least 10 minutes per day, to let them get used to the experimenter. When the rats weigh about 300 g, conduct a social hierarchy test on the rats using the animal social status dominance experiment. Two days before the social hierarchy test, each rat needs to be allowed to adapt to the hierarchy test pipeline twice. The method for adapting to the hierarchy test pipeline is to prepare a transparent acrylic tube with a length of 1.5 m and an inner diameter of 60 cm, put the rat into one end of the tube, and if it comes out smoothly from the other end, it is considered a successful adaptation once.

[0061] Weigh the rats pair-fed in the common breeding cage and then conduct the animal social status dominance experiment test. The specific method is as follows: Make a mark at the center position of the pipeline, put the two rats into the pipeline from both ends at the same time and start timing. If one rat pushes the other rat out of the pipeline within two minutes, the rat that pushes out is judged to have a higher rank, and the rat that is pushed out has a lower rank; if the two minutes have not ended, then judge that the rat closer to the same-side pipeline exit has a lower rank.

[0062] Before formally conducting chronic social comparison stress intervention, the corresponding number of experimental devices (i.e., experimental boxes) and corresponding environmental stimuli should also be prepared in advance. Chronic social comparison stress intervention mainly separates two rats of different social hierarchies that have lived together for a long time through experimental boxes with perforated partitions of different specifications, but the two can communicate through the perforated transparent partition, and can hear, see, and smell each other. The experimenter creates different living environments artificially to prompt the rats to make social comparisons, thus generating a psychological stress of a social comparison nature. Chronic social comparison stress lasts for at least 14 days, with at least one good / poor environmental stimulus per day, and the stimulation duration is 24 hours.

[0063] Taking one stimulus per day and intervening at 10 am as an example. As shown in the following table, there are a total of seven stimuli, which are processed in a cycle.

[0064]

[0065] After being tested in the animal social status dominance experiment, pairs of rats were transferred to a two-chamber chronic social comparison experimental chamber for feeding. Among them, the rats with a higher social rank were placed on the side with better environmental conditions, and the rats with a lower rank were placed on the side with poorer environmental conditions. For example, in the two-chamber setting of an enriched feeding environment and a normal feeding environment, the rats with a higher rank were placed in the space of the enriched environment; in the two-chamber experimental chamber of a normal feeding environment and a poor feeding environment, the rats with a higher rank were placed in the space of the normal feeding environment, and so on. One or several environmental feeding stimulation conditions were changed between the enriched feeding environment and the poor feeding environment every day for stimulation, and this environmental stimulation was maintained for 24 hours.

[0066] After completing the stress stimulation intervention, it is necessary to test the modeling effect. After several days of stress intervention, a behavioral test was conducted on the rats in a cycle every day to detect the anxiety or depression-like behavior indicators of the rats. The experimental methods included: behavioral experimental results such as the three-chamber social test, open field test, open field social test, locomotor activity test, elevated plus maze, forced swim test, and sucrose preference test, and samples were taken to detect the content of related proteins in the brain nuclei.

[0067] Three-chamber social test: Transport the experimental chamber for chronic social comparison stress and the rats in it to the test room to adapt for at least one hour. Then place the rats in the three-chamber social comparison experimental chamber to adapt to the environment for 10 minutes. After a 5-minute interval, the first-stage test was carried out. Place the cage containing the strange companion 1 and an empty cage (or a cage containing a toy rat) on both side boxes, open the middle door, and let the test rat shuttle freely. Record for 10 minutes and count the proportion of the communication time of the test rat with the strange companion 1. Under normal circumstances, the rat should sniff the companion for much more time than the empty cage, otherwise it represents a decrease in social interest. After a 5-minute interval, the second-stage test was carried out. Replace the empty cage with a cage containing another strange companion 2 (a stranger companion) and place it on the other side. Open the middle door and let the test rat shuttle freely. Record for 10 minutes and count the proportion of the sniffing time of the test rat for the already familiar strange companion 1 and the stranger companion 2. Under normal circumstances, the rat should like to communicate with the stranger companion 2 more. Otherwise, it may represent an abnormality in the social novelty index of the rat, that is, an abnormality in social recognition memory or social ability. During the test interval, the three-chamber device was cleaned with 75% alcohol. After the test, the rats were sent back to the breeding room.

[0068] Open field test: Transport the experimental chamber for chronic social comparison stress and the rats in it to the test room to adapt for at least one hour. Then place the test rat in the center of a 1 m × 1 m open field device and let it move freely. Record the behavior for 10 minutes and count the proportion of the movement time of the rat in the central area of the open field. If the movement time in the central area decreases compared with the control group, it represents that the rat has anxiety-like behavior. After the test, the open field box was cleaned with 75% alcohol, and then the test rat was sent back to the breeding room.

[0069] Open field social test: One day before the test, the rats housed in the experimental cages for chronic social comparison stress and their age-matched unfamiliar rats (weight difference ±10 g) for social matching were transported to the test room and allowed to acclimate for at least one hour. Then each rat was placed in the center of a 1 m × 1 m open field apparatus and allowed to move freely for 10 minutes. After each rat was acclimated, the open field cage was cleaned with 75% alcohol, and then the animals were returned to the breeding room. On the day of the test, the above rats were also transported to the test room and allowed to acclimate for at least one hour. The rats for social matching were marked in advance. During the test, the experimental rats and the rats for social matching were placed together in the center of the open field apparatus, and their behaviors were recorded for 10 minutes. The proportions of the rats' social behaviors (such as sniffing, approaching, following, licking, and contacting unfamiliar rats) and non-social anxiety behaviors (such as walking, running, and standing upright in the open field unrelated to companions) were statistically analyzed, which represented the social interaction ability index. After the test, the open field cage was cleaned with 75% alcohol, and then the animals were returned to the breeding room.

[0070] Locomotor activity test: The experimental cages for chronic social comparison stress and the rats in them were transported to the test room and allowed to acclimate for at least one hour. Then the test rats were placed in the center of a 50 cm × 50 cm behavior cage and allowed to move freely. Their behaviors were recorded for 30 minutes, and the locomotor distance of the rats was statistically analyzed. After the test, the behavior cage was cleaned with 75% alcohol, and then the test rats were returned to the breeding room.

[0071] Elevated plus maze test: The experimental cages for chronic social comparison stress and the rats in them were transported to the test room and allowed to acclimate for at least one hour. Then the test rats were placed in the central area of the elevated plus maze with their heads uniformly facing the open arms and allowed to move freely. Their behaviors were recorded for 5 minutes. The time and number of times the rats entered the open arms were statistically analyzed. If it was lower than that of the control group, it represented an increase in the anxiety level. The statistical significance of the relevant indicators of the closed arms was the opposite. After the test, the behavior cage was cleaned with 75% alcohol, and then the test rats were returned to the breeding room.

[0072] Sucrose preference test: At 10:00 am on the day of the locomotor activity test, each rat was acclimated to two bottles of 1% sucrose solution for 24 hours. At 10:00 am on the day of the elevated plus maze test, one of the two bottles of 1% sucrose solution was replaced with ordinary water and the rats were acclimated for 24 hours. At 12 hours, the positions of the sucrose solution and water were swapped. On the morning of the day after the elevated plus maze test, all rats were deprived of sucrose solution, water, and food and fasted and water-deprived for 24 hours. At 10:00 am on the second day after the elevated plus maze test, each rat underwent the sucrose preference test. One bottle of 1% sucrose solution and water were weighed separately and placed at the drinking port without placing food. At 6 hours, the positions of the water and sucrose solution were swapped, and they were weighed after 12 hours of testing. The percentage of sucrose consumption in the total water consumption was statistically analyzed. If the proportion of sucrose consumption decreased, it represented that the rats had depressive-like emotions.

[0073] Forced swimming test: On the second day of the sucrose preference test, forced swimming adaptation was carried out. The chronic social comparison stress experimental box and the rats in it were transported to the test room for at least one hour of adaptation. Then, the test rats were placed in a forced swimming bucket with a height of 50 cm, a diameter of 20 cm, and a water level of 30 cm for 15 minutes of adaptation. The water temperature was 23 - 25 degrees. The forced swimming test was carried out at the same time on the next day after adaptation. The chronic social comparison stress experimental box and the rats in it were transported to the test room for at least one hour of adaptation. Then, the rats were placed in the forced swimming bucket under the same conditions as above, and the test was carried out for 6 minutes and video-recorded. After counting the video, the stationary time of the rats within the last 5 minutes and its proportion were statistically analyzed. An increase in the stationary time or percentage indicates an increase in despair behavior; the active swimming index can also be statistically analyzed, that is, the time of swimming upward or swimming around. If the active swimming time is lower than that of the control group, it can be used as an indicator of depressive-like behavior, indicating depressive-like behavior.

[0074] Sample collection and detection: The test rats were anesthetized with chloral hydrate, the chest was opened to expose the apex of the heart, and the blood of the rats was collected with a disposable vacuum blood collection tube. Then, the rats were immediately decapitated to remove the brain, which was embedded and frozen in a -80-degree refrigerator. Alternatively, after anesthetizing the rats, perfusion with normal saline and 4% tissue fixative can be carried out. The above behavioral tests were subjected to video analysis and data statistics according to the required indicators. Sampling can be subjected to biochemical detection according to needs to further evaluate the changes in the levels of depression-related proteins in rodents after chronic social comparison stress.

[0075] Example 1

[0076] This example is a method for establishing an animal model of anxiety- and depression-like rats provided in accordance with the present invention, and an animal model of anxiety- and depression-like rats induced by social comparison stress is established. The flow chart is as Figure 8 shown. First, the rats were randomly paired in a combined animal breeding cage in pairs to adapt to the environment of the combined breeding cage. After the adaptation period, the paired rats were subjected to an animal social status dominance experiment to determine the social rank, and the breeding environment of the rats was determined according to the test results of the social rank. The two rats were respectively bred on both sides of a two-chamber social comparison experimental box, separated by a transparent perforated acrylic board in the middle. The experimental group was set as the rats with a high social rank receiving a treatment of a good condition stimulus every day, designated as the social comparison pressure application animal group, and the stimulus was changed at 10:00 every morning for a total of 31 days. The rats with a lower social rank matched with them received no additional treatment except for normal breeding conditions, designated as the social comparison stress animal group (stress group). The control group was set as the rats with a high social rank and the rats with a low social rank matched with them only receiving normal breeding conditions. Feces were collected from the rats once a week during the modeling period, and the sucrose preference for 24 hours was measured once. On the 23rd day of modeling, all animals were tested for three-chamber social interaction, open field, open field social interaction, elevated plus maze, 12-hour sucrose preference test, and forced swimming test.

[0077] All behavioral test results were statistically analyzed using unpaired t-tests. * represents p < 0.05; ** represents p < 0.01. The sample sizes of the control group and the social comparison stress group (stress group) were 12 and 10 rats, respectively. The results of the three-chamber social interaction test ( Figure 9 -A) showed that there were no significant statistical differences between the two groups in terms of social interest ( Figure 9 -A left) and social novelty recognition ( Figure 9 -A right) (p = 0.854 and p = 0.910, respectively). In the forced swimming test ( Figure 9 -B), the immobility time of the social comparison stress group was significantly increased compared to the control group (p = 0.047), indicating that after chronic social comparison stress, rats in the social comparison stress group exhibited depressive-like behaviors. The results of the open field social interaction test ( Figure 9 -C) showed that compared to the control group, the percentage of social time of the social comparison stress group decreased, while the percentage of non-social time increased, with the significance levels of the two indicators being p = 0.055 and p = 0.092, respectively. In the sucrose preference test ( Figure 9 -D), there was a marginal significance between the two groups in terms of sucrose consumption (p = 0.099), indicating that after chronic social comparison stress, rats in the stress group tended to exhibit depressive-like behaviors. In the elevated plus maze test ( Figure 9 -E), there was no significant statistical difference between the two groups in the percentage of time spent in the open arms ( Figure 9 -E left) (p = 0.499), but the results of the number of entries into the closed arms ( Figure 9 -E right) showed that compared to the control group, the number of entries into the closed arms of the social comparison stress group was significantly increased (p = 0.005), indicating that rats in the social comparison stress group exhibited anxiety-like behaviors. In the open field test ( Figure 9 -F), compared to the control group, the residence time of the social comparison stress group in the central area was significantly decreased (p = 0.009), indicating that rats in the social comparison stress group exhibited anxiety-like behaviors.

[0078] Example 2

[0079] This embodiment is a method for establishing an animal model of anxiety- and depression-like rats according to the present invention, which can enhance the stress level of social comparison stress and induce an animal model of anxiety- and depression-like rats. The experimental equipment uses a three-chamber social comparison experimental chamber. First, the rats are randomly grouped and raised in groups of three. After the animals adapt to the breeding environment, they are randomly divided into groups. The social hierarchy of the rats is detected according to the animal social status dominance experiment, and the breeding positions and breeding environments of the rats are determined according to the test results of the social hierarchy. For example, the rats with the lowest social hierarchy are set as the social comparison stress group and raised in the middle chamber of the three-chamber social comparison experimental chamber, with a normal breeding environment; the other two rats with higher social hierarchies are respectively raised in two independent spaces on both sides of the three-chamber social comparison chamber, with a better breeding environment, and each independent space is separated by a transparent perforated acrylic board. Another example is to set the rats with the highest social hierarchy as the social comparison stress application group and raise them in the middle chamber of the three-chamber social comparison experimental chamber, with a good breeding environment; the other two rats with lower social hierarchies are respectively raised in two independent spaces on both sides of the three-chamber social comparison chamber, with a normal breeding environment, and each independent space is separated by a transparent perforated acrylic board. The experimental group is set as the rats with a high social hierarchy to receive the treatment of two good stimulation conditions every day (the social comparison stress application animal group), and the stimulation is changed at 10:00 every morning for a total of several days. The rats with a lower social hierarchy (the social comparison stress animal group) matched with them have no additional treatment except for normal breeding conditions. The control group is set as the rats with a high social hierarchy and the rats with a low social hierarchy both only receive normal breeding conditions. After the stress is over, all animals are tested for three-chamber social interaction, open field, open field social interaction, locomotor activity, elevated plus maze, sucrose preference test, and forced swimming test to obtain relevant behavioral index data, and subsequent analysis of the depression or anxiety level is carried out. Of course, the present invention can also set more control groups. For example, the rats with a high social hierarchy and the rats with a low social hierarchy in the control group both receive the breeding conditions of two good stimulations every day, which will not be elaborated here.

[0080] Furthermore, to enhance the level of social comparison stress, it can be extended to a five-chamber social comparison experimental chamber. Rats are randomly grouped and raised in groups of five. After the animals adapt to the breeding environment, they are randomly divided into groups. According to the animal social status dominance experiment, the social hierarchy of the rats is detected, and the breeding positions and environments of the rats are determined according to the test results of the social hierarchy. For example, the rats with the lowest social hierarchy are set as the social comparison stress group and are raised in the central chamber of the five-chamber social comparison experimental chamber, with a normal breeding environment; the other four rats with higher social hierarchies are respectively raised in four independent spaces on the periphery of the five-chamber social comparison chamber, with a good breeding environment, and each independent space is separated by a transparent perforated acrylic board. Another example is that the rats with the highest social hierarchy are set as the social comparison stress application group and are raised in the middle chamber of the five-chamber social comparison experimental chamber, with a good breeding environment; the other four rats with lower social hierarchies are respectively raised in four independent spaces on the periphery of the five-chamber social comparison chamber, with a normal breeding environment, and each independent space is separated by a transparent perforated acrylic board.

[0081] Example 3

[0082] This example is a multi-chamber social comparison stress chamber provided according to the present invention, which can be flexibly applied to the preparation of chronic social comparison stress animal models under various social comparison stress experimental conditions. For example, a three-chamber social comparison experimental chamber is used to establish social comparison stress rats with different pressure-bearing levels. First, rats are randomly grouped and raised in groups of three; after the animals adapt to the breeding environment, they are randomly divided into groups. According to the animal social status dominance experiment, the social hierarchy of the rats is detected, and the breeding positions and environments of the rats are determined according to the test results of the social hierarchy; the rats with the middle social hierarchy are set as the social comparison normal group and are raised in the middle chamber of the three-chamber social comparison experimental chamber, with a normal breeding environment; the rats with the highest and lowest social hierarchies are respectively raised in two independent spaces on both sides of the three-chamber social comparison chamber, with the best and worst breeding environments, and each independent space is separated by a transparent perforated acrylic board. The experimental group is set such that the rats with a high social hierarchy receive the treatment of two good stimulation conditions every day, and the stimulation is changed at 10:00 every morning for a total of several days, and it is set as the social comparison stress application animal group; the rats with the middle social hierarchy receive no additional treatment except normal breeding conditions and are set as the social comparison normal animal group; the rats with the lowest social hierarchy receive the treatment of two harsh stimulation conditions every day, and the stimulation is changed at 10:00 every morning for a total of several days, and it is set as the social comparison stress animal group. The control group is set such that regardless of the social hierarchy, the rats only receive normal breeding conditions. After the stress is over, all animals are tested for three-chamber social interaction, open field, open field social interaction, locomotor activity, elevated plus maze, sucrose preference test, and forced swim test.

[0083] The experimental box model established by the present invention can establish a stress model in the form of one-to-one or one-to-many, saving the usage of experimental mice, enhancing the uniformity of stress suffered among different experimental groups, expanding the number of samples in unit time or unit batch of experiments, and reducing the breeding and experimental costs.

[0084] Obviously, the above-mentioned embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for establishing a chronic social comparison stress model, characterized in that, The method includes the following steps: Step 1: Randomly match the rodents and group them in an undivided breeding experimental device for adaptive breeding to form an experimental group. Step 2: Conduct an animal social status dominance experiment test on the animals that have adapted to the breeding environment. Determine the animals with a higher social rank as the animals subjected to social comparison stress, and the animals with a lower social rank as the animals subjected to social comparison stress. In Step 2, to determine the social rank of two rodents that have adapted to the breeding experimental device environment using an animal social status dominance experiment, the specific method is as follows: Use a transparent pipe with a set length that allows one rodent to pass through, and make a mark at the center position of the transparent pipe. Put the two rodents into the pipe from both ends simultaneously and start timing. Within the set time, if one rodent is pushed out of the pipe by the other rodent, set the pushed-out rodent as the animal subjected to social comparison stress, and the other pushed-out rodent as the animal subjected to social comparison stress. If neither of the two rodents exits the pipe within the set time, set the rodent closer to the same-side pipe exit as the animal subjected to social comparison stress, and the other rodent as the animal subjected to social comparison stress. Step 3: Put the grouped animals after matching into an experimental device formed by multiple independent spaces separated by a transparent partition with through holes, and provide better breeding stimulation conditions for the animals subjected to social comparison stress than for the animals subjected to social comparison stress, and start the formal experiment. Step 4: Apply continuous stimulation for 24 hours to the animals subjected to social comparison stress and the animals subjected to social comparison stress respectively. When the provided breeding stimulation conditions reach the set stimulation time, provide breeding stimulation conditions different from the previous breeding stimulation conditions for the animals subjected to social comparison stress and the animals subjected to social comparison stress respectively. Step 5: After the stress stimulation intervention for the set time, conduct a modeling effect test on the rodents to obtain the anxiety or depression-like behavior indicators of the tested rodents, and make an evaluation of the chronic social comparison of the rodents after statistical analysis.

2. The method for establishing a chronic social comparison stress model according to claim 1, characterized in that In Step 1, two rodents matched with the social ranks of the two animals in Step 2 are also set as a control group, and the two rodents in the control group receive undifferentiated breeding stimulation conditions.

3. The method for establishing a chronic social comparison stress model according to claim 1, characterized in that, In Step 3, put the grouped and matched rodents into one of a two-chamber social comparison experimental box, a three-chamber social comparison experimental box, or a five-chamber social comparison experimental box, and provide differentiated breeding stimulation conditions for the animals subjected to social comparison stress and the animals subjected to social comparison stress.

4. The method for establishing a chronic social comparison stress model according to claim 3, characterized in that, In Step 4, provide at least one good breeding stimulation condition for the animals with a higher social rank in the experimental group every day, set as the animals subjected to social comparison stress, provide normal breeding conditions or at least one bad environment stimulation condition every day for the animals with a lower social rank in the experimental group, set as the animals subjected to social comparison stress. Regardless of the social rank of the animals in the control group, provide normal breeding conditions or at least one good stimulation condition or at least one bad stimulation condition every day for the animals in the control group.

5. The method for establishing a chronic social comparison stress model according to claim 1, characterized in that, In step 5, during the period of performing stress stimulation intervention, feces of the rodents are collected once a week, and the 24-hour sucrose preference is measured once. After the intervention, all animals are tested for three-chamber social interaction, open field, open field social interaction, elevated plus maze, 12-hour sucrose preference test, and forced swimming test to obtain their behavioral data for evaluating their depressive or anxious behaviors.

6. The method for establishing a chronic social comparison stress model according to claim 1, wherein The method further includes step 6 of sampling for biochemical detection, which is used to detect the content of related proteins in the brain nuclei of the animals subjected to social comparison stress and evaluate the changes in the levels of depression-related proteins in the rodents after chronic social comparison stress.

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