A method for simulating feed and modeling rodent anxiety

Abstract

The application relates to a method for simulating feed and modeling rodent anxiety, and belongs to the technical field of animal experiment models. The simulating feed is used for modeling the anxiety of rodent experimental animals, and comprises a shell, an end cover is screwed at one end of the shell, a plurality of through holes are arranged on the shell wall, real feed is arranged in the shell, the real feed cannot leak out through the through holes, but the smell of the real feed can be diffused through the through holes, the shell is hard, and the real feed cannot be broken by the experimental animals biting the shell, the shape of the shell is consistent with the shape of the feed eaten by the experimental animals in daily life, and the shell and the end cover are non-toxic and odorless. The method for modeling the anxiety comprises a modeling method and a research method. The application can build an effective experimental animal anxiety model through the simulating feed, and the accuracy and reliability of the model are verified through comparative experiments and drug efficacy experiments.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal experimental models, and particularly relates to a simulation feed and a method for modeling anxiety of rodents. BACKGROUND

[0002] Anxiety is a common chronic mental illness, which is generally caused by irrational emotions such as tension, depression, worry, restlessness and fear. It has attracted more and more researchers' attention due to its wide range of somatic symptoms, complex complications, high recurrence rate and significant gender difference.

[0003] The inducing factors of anxiety are related to psychology, society, physiology and other factors, and its pathogenesis is complex, which has not been elucidated. Studies have shown that it may be related to neurobiochemistry, neuroimmunity, neuroendocrine and other aspects. However, in the clinic, the anti-anxiety drugs are limited due to their many adverse reactions. Therefore, anxiety and new anti-anxiety drugs have become a hot spot in medical research.

[0004] Animal models are a simplified method for studying human diseases, and anxiety animal models can help researchers indirectly analyze the complex psychological activity of anxiety, which has important value in the discovery, improvement of anxiety drugs and the study of the neurobiological mechanism of anxiety diseases. The transformation research between diseases and animals and between animals and humans has always been a problem to be solved in the field of animal model research. In the past 20 years, in order to improve the poor clinical use effect and the large adverse reactions of existing anti-anxiety drugs, research on new anti-anxiety drugs has increased, but many compounds have shown inconsistent pharmacological effects with expectations in clinical and / or non-clinical trials, one of the key reasons for which is the lack of an experimental animal anxiety model that is universally applicable to different types of drugs.

[0005] Anxiety behavior experiment is a necessary means to study the mechanism of neurosychiatric diseases such as anxiety and to develop preventive measures. The anxiety behavior experiment method is mainly based on the conflict between the exploratory nature of rodents and the external environment, thereby causing anxiety, which is established when animals face inevitable or impending aversive stimuli. The commonly used anxiety behavior experiment method of rodents can be divided into two categories, unconditioned reflex and conditioned reflex. Unconditioned reflex includes exploratory behavior, social behavior and defensive behavior. The classic experimental methods include open field test (OFT), elevated plus maze (EPM), light-dark box (LDB) and novel environment food intake inhibition experiment, which are also the most commonly used anxiety behavior test methods. OFT, EPM and LDB are classic test methods for evaluating animal anxiety, which can reflect the changes of animal's own anxiety behavior during the test. Therefore, OFT, EPM and LDB can be used to monitor the changes of animal's anxiety behavior or evaluate the anti-anxiety effect of drugs, but they cannot be used to prepare experimental animal models of anxiety. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the application discloses a kind of simulation feed and the method for modeling research of rodent anxiety, the simulation feed can be used to prepare effective anxiety experimental animal model by experimental verification, the modeling research method discloses the method for modeling experimental animal anxiety model by simulation feed and the method for drug research based on experimental animal anxiety model.

[0007] In order to achieve the above object, the technical scheme of the present application is:

[0008] A kind of simulation feed, the simulation feed is used for the anxiety model modeling of rodent experimental animal, including shell, one end of the shell is screwed with end cap, shell wall is equipped with a plurality of through holes, the shell is equipped with real feed, real feed cannot leak out through the through hole, but the smell of real feed is emitted through the through hole, shell is hard, experimental animal cannot break real feed by tearing shell, the shape of the shell is consistent with the shape of the feed that experimental animal eats daily, and shell, end cap are all non-toxic, odorless.

[0009] Preferably, the shell is cylindrical, the shell wall is also equipped with a plurality of blind holes, the inner diameter of the through hole is 1mm.

[0010] Preferably, the shell and end cap are both made of ABS engineering plastic material, the diameter of the shell is 12mm, the length is 10-30mm, the outer surface of the shell is also doped with yellow dye to make the shell closer to the color of real feed.

[0011] A kind of experimental animal anxiety modeling research method by simulation feed, including modeling method and research method;

[0012] The modeling method includes: by time limit feeding, let rodent experimental animal develop the habit of fixed point fast diet, then in time limit feeding, under the condition that rodent experimental animal does not obtain sufficient diet, food is deprived, rodent experimental animal is hungry and cannot eat, and then produces anxiety emotion;By fixed time and fixed quantity diet, limit the food intake of rodent experimental animal, rodent experimental animal is hungry and cannot eat, and then produces anxiety emotion;On the basis of the above, real feed used daily is mixed with simulation feed and fed, since simulation feed appearance is close to real feed, and can emit real feed smell, rodent experimental animal cannot distinguish real and false feed, produces uncertainty in the process of eating, produces anxiety emotion by continuous stimulation;Finally, by: hungry and cannot eat + eat and cannot get + get and cannot be full + uncertainty, rodent experimental animal produces anxiety emotion;

[0013] The research method includes:

[0014] A. In the process of modeling by simulation feed, the anxiety behavior changes of rodent experimental animals are monitored by one or more combinations of OFT, EPM and LDB experiments until the modeling is successful;

[0015] B. The method of modeling by simulation feed is compared with the chronic restraint stress-induced anxiety modeling method in two stages of 7 days and 14 days; in the process, the anxiety behavior changes of rodent experimental animals are monitored by one or more combinations of OFT, EPM and LDB experiments;

[0016] C. The anxiety model of rodent animals modeled by simulation feed is intervened by drugs, the anxiety behavior changes of rodent experimental animals are monitored by one or more combinations of OFT, EPM and LDB experiments, and the therapeutic effect of drugs on the anxiety model of experimental animals is evaluated.

[0017] The method of simulation feed and rodent anxiety modeling research has the following beneficial effects:

[0018] 1. The simulation feed provided by the application can be used in the modeling environment of rodent experimental animals, and experimental verification shows that the simulation feed can produce an effective anxiety model of rodent experimental animals. The anxiety model of rodent experimental animals prepared by simulation feed stress can more accurately simulate the comprehensive reaction changes of the psychological state of rodent experimental animals. In the modeling process, the anxiety behavior changes of animals are monitored by OFT, EPM and LDB, and the chronic restraint stress-induced anxiety modeling method is compared in two stages of 7 days and 14 days. Finally, drug intervention is performed to evaluate the therapeutic effect of drugs on the anxiety model. The anxiety model is fully verified and necessary verification by the above method, which proves the accuracy and reliability of the anxiety model of experimental animals prepared by simulation feed stress stimulation. The experimental method of the application is simple, the experimental cost is low, a few experimental personnel are needed, the modeling success rate is high, and it is worth popularizing and further developing.

[0019] 2. After the modeling by simulation feed is successful, the anxiety model of rodent experimental animals shows a sustained anxiety state in the anxiety behavior test, the number of times and the distance of rodent experimental animals entering the central area of the open field are significantly reduced, the number of explorations and the residence time of rodent experimental animals in the open arms of the elevated plus maze are significantly reduced, and the residence time and the number of entries in the light-dark box experiment are significantly reduced. It is proved that the simulation feed stress successfully induces the anxiety symptoms of rodent experimental animals, and the anxiety model of rodent experimental animals is successfully prepared. At the same time, the experiment shows that diazepam can effectively relieve the anxiety response of rodent experimental animals after simulation feed stress, which further proves the effectiveness of the anxiety model of rodent experimental animals induced by simulation feed stress, and suggests that the model can be used for the feasibility of exploring the efficacy and mechanism of anti-anxiety drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The structural diagram of the simulation feed of the present application;

[0021] Figures 2-9 In order: total distance of open field experiment after 7 days of simulation feed adaptation, center area distance of open field experiment, time in center area of open field experiment, number of central area of open field experiment, open arm entering time percentage of elevated plus maze experiment, open arm entering number percentage of elevated plus maze experiment, light area entering time of light-dark box experiment, light area entering number of light-dark box experiment;

[0022] Figures 10-17 In order: total distance of open field experiment after 7 days of simulation feed modeling, center area distance of open field experiment, time in center area of open field experiment, number of central area of open field experiment, open arm entering time percentage of elevated plus maze experiment, open arm entering number percentage of elevated plus maze experiment, light area entering time of light-dark box experiment, light area entering number of light-dark box experiment;

[0023] Figures 18-25 In order: total distance of open field experiment after 14 days of simulation feed modeling, center area distance of open field experiment, time in center area of open field experiment, number of central area of open field experiment, open arm entering time percentage of elevated plus maze experiment, open arm entering number percentage of elevated plus maze experiment, light area entering time of light-dark box experiment, light area entering number of light-dark box experiment;

[0024] Figures 26-33 In order: total distance of open field experiment after 7 days of drug intervention gavage, center area distance of open field experiment, time in center area of open field experiment, number of central area of open field experiment, open arm entering time percentage of elevated plus maze experiment, open arm entering number percentage of elevated plus maze experiment, light area entering time of light-dark box experiment, light area entering number of light-dark box experiment;

[0025] 1, shell; 2, through hole; 3, end cover; 4, blind hole;

[0026] The English part of the figure is translated as follows:

[0027] Total distance (Total distance), center area distance (Center area distance), time in center area (Time in center area), number of central area (Number of central area), open arm entering time percentage (OT%), open arm entering number percentage (OE%), light distance (Light distance), light entering number (Number of light), time in light (Time in light). Detailed Implementation

[0028] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0029] Example 1

[0030] A type of simulated feed, such as Figure 1 As shown, the simulated feed is used to create an anxiety model in rodent laboratory animals. It includes a shell 1, with an end cap 3 screwed to one end of the shell. Several through holes 2 are provided on the shell wall. Real feed is placed inside the shell. The real feed cannot leak out through the through holes, but the odor of the real feed can be released through the through holes 2. The shell 1 is hard, and the experimental animals cannot break the real feed by biting the shell. The shape of the shell 1 is consistent with the shape of the feed that the experimental animals usually eat. Moreover, the shell and the end cap are non-toxic and odorless.

[0031] In this embodiment, the shell and end caps are non-toxic, odorless, and tear-resistant, preventing rodents from breaking the shell with their teeth or squeezing it, causing the real feed inside to leak out through the opening. In other words, the shell prevents the animal from experiencing the excitement of tasting food through forceful biting. The animal, despite constantly biting, cannot obtain food, leading to anxiety. Secondly, the simulated feed of this invention is mixed with the animal's daily feed. Because its appearance is identical to the animal's daily feed and it emits the odor of real feed through the opening, the animal cannot distinguish between the real and fake feed, creating significant uncertainty during feeding and further increasing anxiety. Since the feeding is continuous, the continuous stimulation from the simulated feed can induce anxiety symptoms in rodents. This differs from the temporary anxiety induced by traditional chronic restraint experiments. The anxiety induced by the simulated feed of this invention in rodents exhibits persistent anxiety and physical manifestations, more closely resembling real anxiety disorder. Therefore, it can be widely applied to drug screening, drug mechanism exploration, and research on the mechanisms of anxiety disorders.

[0032] Example 2

[0033] Based on Example 1, this example discloses:

[0034] like Figure 1 As shown, the shell is cylindrical, and the shell wall is provided with several blind holes 4, the inner diameter of which is 1 mm. The blind holes make it more difficult for rats and mice to distinguish the source of food odors, further stimulating the sense of uncertainty and inducing anxiety.

[0035] like Figure 1As shown, the shell and the end cover are made of ABS engineering plastic material, the diameter of the shell is 12mm, the length is 10-30mm, and the outer surface of the shell is doped with yellow dye to make the shell closer to the color of the real feed.

[0036] In this embodiment, the length of the shell is not specifically limited and can be selected between 10-30mm according to the need.

[0037] The simulated feed of the present application is made of ABS engineering plastic (acrylonitrile-butadiene-styrene, abbreviated as ABS), which has a light ivory color in the original appearance, and part of yellow dye is added to make it look more like real feed, and the color is close to the commonly used mouse maintenance feed of Beijing Keaohexiluo Feed Co., Ltd. The simulated feed is made of Shenzhen Xingdeli Industry Co., Ltd., which has very high hardness and is resistant to biting. Mice can hardly cause any wear and tear.

[0038] Embodiment 3

[0039] Based on embodiment 2, the present embodiment discloses:

[0040] A method for modeling and researching anxiety of experimental animals by using simulated feed, including modeling method and research method;

[0041] The modeling method comprises:

[0042] By limiting the feeding time, the mice develop the habit of eating food at a fixed point (this method is a commonly used method and is not described in detail), and the mice develop the habit of eating food immediately after seeing the feed, ensuring that the amount of feed ingested does not affect the normal function of the body; then, during the limited feeding time, the mice are deprived of food (such as taking away half of the food, and this taking away behavior is irregular) under the condition that the mice do not have sufficient food, so that the mice produce anxiety; by limiting the amount of food eaten by the mice (such as only eating 80% of the regular amount of food), the mice's food demand is not met, so that the mice produce anxiety;

[0043] On the basis of the above, the mice produce anxiety through the correlation between uncertainty and anxiety: the real feed and the simulated feed are mixed and fed (including interval mixing and single mixing, interval mixing means that real feed is used for feeding once and simulated feed is used for feeding another time, and this interval is repeated, and single mixing means that real feed and simulated feed are mixed together for feeding each time), because the appearance of the simulated feed is close to that of the real feed and can emit the smell of real feed, the mice cannot distinguish between real and fake feed, and produce uncertainty during the feeding process, and through continuous stress stimulation, the experimental animals produce anxiety symptoms.

[0044] Anxiety in humans is manifested as: no clear objective object of nervous worry, restlessness, and autonomic dysfunction symptoms such as palpitations, hand tremor, sweating, frequent urination, and motor restlessness, and when experimental animals develop symptoms of anxiety, they are similar to these, and the anxiety can be monitored by the following open field (OFT), elevated plus maze (EPM), light-dark box (LDB) and other experiments.

[0045] The research method comprises:

[0046] A. In the process of modeling by simulated feed, the anxiety behavior changes of mice are monitored by one or more combinations of experiments in open field (OFT), elevated plus maze (EPM), and light-dark box (LDB) until the modeling is successful;

[0047] B. The method of modeling by simulated feed is compared with the method of chronic restraint stress-induced anxiety modeling in two stages of 7 days and 14 days; in this process, the anxiety behavior changes of mice are monitored by one or more combinations of experiments in open field (OFT), elevated plus maze (EPM), and light-dark box (LDB);

[0048] C. The experimental animal anxiety model made by simulated feed is intervened by drugs, the anxiety behavior changes of mice are monitored by one or more combinations of experiments in open field (OFT), elevated plus maze (EPM), and light-dark box (LDB), and the therapeutic effect of drugs on the experimental animal anxiety model is evaluated.

[0049] Example 4

[0050] Experimental verification:

[0051] 1. Purpose of the experiment

[0052] The anxiety triplets: open field experiment + elevated plus maze experiment + light-dark box experiment (OFT + EPM + LDB) mouse behavior model are used to verify the effectiveness and scientificity of an experimental method of a simulated feed-induced anxiety model, and compared with the method of chronic restraint stress-induced anxiety modeling (the existing recognized modeling method for preparing anxiety model), to prove the feasibility of the model for exploring the efficacy and mechanism of anti-anxiety drugs.

[0053] 2. Materials and methods

[0054] 2.1 Materials

[0055] 2.1.1 Instruments and consumables: SuperMaze animal behavior analysis system, open field experiment system, elevated plus maze experiment system, light-dark box experiment system: Shanghai Xinsuo Information Technology Co., Ltd. Mouse gavage device: Jinan Yiyandian Technology Development Co., Ltd. Mouse restraint device: Shanghai Yurui Scientific Instrument Co., Ltd.

[0056] 2.1.2 Drug: Diazepam, Beijing Yimin Pharmaceutical Co., Ltd., specification: 2.5 mg / tablet (20 tablets / plate), batch number: 20170802.

[0057] 2.1.3 Animals: SPF male C57BL / 6J mice, body weight 20±2g, 120. Provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number: SCXK(Jing)2021-0006.

[0058] 2.2 Methods

[0059] 2.2.1 Feeding conditions and grouping

[0060] 5 mice per cage, free access to food and water, and raised in a light and temperature and humidity controlled room: temperature 21±2℃, humidity 50±10%, 12h / 12h light-dark cycle (reversed light-dark cycle, 20:00 lights on, 8:00 lights off), all animal experimental operations were carried out in accordance with the NIH promulgated guidelines for animal welfare and use.

[0061] 2.2.2 Experimental methods

[0062] 2.2.2.1 Experiment 1: simulation feed adaptation feeding experiment

[0063] After the mice arrived, they were adapted for one week before entering the experiment.

[0064] Grouping: normal control group, simulation feed group (simulation group).

[0065] Modeling method: normal group was normally fed, and the simulation group started a 7-day diet training, with real feed given at 9:00 every day, real feed removed at 9:30, real feed given at 4:30 pm, and real feed removed at 5:00. After training, open field test, elevated plus maze test and light-dark box test were performed, as follows:

[0066] Open field test: the experiment was conducted in an open field box with a length of 50 cm, a width of 50 cm and a depth of 50 cm. The bottom of the open field box was divided into 9 squares, with the central area in the middle and the remaining area as the peripheral area. The bottom and the four sides of the open field box were all white. The experiment was conducted in a quiet weak red light environment. The mice were placed in the center of the open field box at the beginning, and the activity within 6 min was observed. The total distance traveled by the mice, the distance traveled in the central area and the number of uprights were recorded.

[0067] Elevated plus maze test: The EPM test system includes two 35 cm x 6 cm opposite open arms and two 35 cm x 6 cm opposite closed arms, the upper part of the closed arm is open, the open arm is open on all sides, and there is a 5 cm x 5 cm relative open part between the open arm and the closed arm. The maze is 50 cm high from the ground. The mouse is placed in the central open part with its head facing the open arm. The Supermaze software records the number of times the mouse enters the open arm and the closed arm and the time spent in the two arms (all four limbs enter or exit the arm as the standard) within 5 minutes. Calculate the percentage of the number of times the mouse enters the open arm and the time, respectively, accounting for the total number of times (the sum of the number of times in both arms) and the total time (the sum of the time spent in both arms).

[0068] Light-dark box test: The light-dark box is composed of two identical size (L x W x H = 25 cm x 25 cm x 30 cm) compartments (light and dark) separated by a middle partition door (6.5 cm x 6.5 cm). The light compartment is illuminated by white light, and the dark compartment is illuminated by red light. At the beginning of the experiment, each mouse is placed in the center of the light compartment facing the partition wall, and the middle partition door is removed. Each time the animal's four paws enter the compartment is counted as one entry. The analysis software records the mouse's light compartment distance, light compartment stay time, and light compartment entry number within 5 minutes.

[0069] 2.2.2.2 Experiment two: 7-day stress modeling experiment

[0070] Grouping: normal group, simulation group, restraint group.

[0071] The simulation group established a mouse anxiety model by simulated feed stress (Simulated feed, SF). The modeling method is as follows: the normal group is normally fed, the simulation group is stimulated by simulated feed for 7 days, the modeling time is during 9:00-10:30 and 16:00-17:30 every day, as shown in the table below, for 7 consecutive days.

[0072] Table 1, simulation feed modeling flowchart

[0073]

[0074]

[0075] The restraint group places the mouse in the restraint barrel for 8 hours continuously for 7 consecutive days.

[0076] After 7 days of modeling, the open field test, elevated plus maze test and light-dark box test are performed, and the test method is the same as experiment one.

[0077] 2.2.2.3 Experiment three: 14-day stress modeling experiment

[0078] Grouping and experimental method same as experiment two (only the modeling period is extended by 7 days).

[0079] 2.2.2.4 Experiment four: drug intervention for 7 days

[0080] Grouping: normal group, positive drug group, simulation group, simulation + positive drug group, restraint group, restraint + positive drug group.

[0081] Experimental method same as experiment two (modeling continues);

[0082] Drug intervention: normal group intragastrically administered with pure water, positive drug group intragastrically administered with diazepam, simulation group intragastrically administered with pure water, simulation + positive drug group intragastrically administered with diazepam, restraint group intragastrically administered with pure water, restraint + positive drug group intragastrically administered with diazepam.

[0083] Intragastric administration: the volume of intragastric administration of pure water is 0.1 mL / 10 g, i.e. 0.01 ml of pure water per gram of body weight is administered intragastrically;

[0084] Diazepam dose: the concentration of intragastric administration of diazepam is 2 mg / kg, and the volume of intragastric administration is also 0.1 mL / 10 g;

[0085] Intragastric administration once a day, continuous administration for 7 days, 1 hour after the last intragastric administration, behavior determination: open field test, elevated plus maze test and light-dark box test, test method same as experiment one.

[0086] 2.2.3 Statistical method

[0087] Data were analyzed using GraphPad Prism 8.01 software (GraphPad Software, Inc., San Diego, California, USA). Before testing, all groups were tested for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene test). All test parameters were compared between groups using unpaired T test (one-tailed) for pairwise comparison, with a test level set at p<0.05.

[0088] 3. Experimental results

[0089] (1) Experiment one:

[0090] ① Mice open field test

[0091] Table 2: Results of total distance of mice in open field test

[0092] Group Number of animals Total path (mm) Normal group 10 28549±2147 Simulation group 12 29569±1308

[0093] As shown in the figure, the effect of 7-day simulated feed adaptation on the total distance of mice in the open field test. Figure 2

[0094] ​In the mouse open field experiment, compared with the normal control group, the simulation feed adaptive feeding had no effect on the total distance of the mouse open field.

[0095] Table 3 Mouse open field central area distance result record

[0096] Group Number of animals Central area path (mm) Normal group 10 1876±225.4 Simulation group 12 1981±211.7

[0097] As Figure 3 shown, the effect of simulation feed adaptive feeding on the central area distance of the mouse open field experiment after 7 days.

[0098] In the mouse open field experiment, compared with the normal control group, the simulation feed adaptive feeding had no effect on the central area distance of the mouse open field.

[0099] Table 4 Mouse open field experiment central area residence time result record

[0100] Group Number of animals Stay time (s) Normal group 10 13.53±1.485 Simulation group 12 13.17±1.686

[0101] As Figure 4 shown, the effect of simulation feed adaptive feeding on the central area residence time of the mouse open field after 7 days.

[0102] In the mouse open field experiment, compared with the normal control group, the simulation feed adaptive feeding had no effect on the central area residence time of the mouse open field experiment.

[0103] Table 5 Mouse open field experiment central area entry times result record

[0104] Group Number of animals Central area entry times Normal group 10 10.78±1.211 Simulation group 12 11.36±1.012

[0105] As Figure 5 shown, the effect of simulation feed adaptive feeding on the central area entry times of the mouse open field after 7 days.

[0106] In the mouse open field experiment, compared with the normal control group, the simulation feed adaptive feeding had no effect on the central area entry times of the mouse open field experiment.

[0107] ②Mouse elevated plus maze experiment:

[0108] Table 6 Elevated plus maze experiment entry time percentage (OT%)

[0109] Group Number of animals OT% Normal group 9 0.1794±0.02501 Simulation group 10 0.1747±0.02531

[0110] As Figure 6 shown, the effect of simulation feed adaptive feeding on the central area entry times of the mouse open field after 7 days.

[0111] In the mouse elevated plus maze experiment, compared with the normal control group, the simulated feed adaptive feeding had no effect on the percentage of entering time of mice in the elevated plus maze experiment.

[0112] Table 7 Percentage of entering times of elevated plus maze experiment (OE%)

[0113] Group Number of animals OE% Normal group 9 0.2812±0.01699 Simulation group 10 0.2711±0.02367

[0114] As shown in the figure, the effect of simulated feed adaptive feeding on the percentage of entering times of mice in the elevated plus maze experiment after 7 days. Figure 7

[0115] In the mouse elevated plus maze experiment, compared with the normal control group, the simulated feed adaptive feeding had no effect on the percentage of entering times of mice in the elevated plus maze experiment.

[0116] ③Mice light-dark box experiment:

[0117] Table 8 Light area entering time of light-dark box experiment

[0118] Group Number of animals Bright area entry time (s) Normal group 9 125.9±3.980 Simulation group 10 138.7±4.995

[0119] As shown in the figure, the effect of simulated feed adaptive feeding on the light area entering time of mice in the light-dark box experiment after 7 days. Figure 8

[0120] In the mouse light-dark box experiment, compared with the normal control group, the simulated feed adaptive feeding had no effect on the light area entering time of mice in the light-dark box experiment.

[0121] Table 9 Light area entering times of light-dark box experiment

[0122] Group Number of animals Bright area entry times Normal group 9 14.40±0.8459 Simulation group 10 15.00±0.9309

[0123] As shown in the figure, the effect of simulated feed adaptive feeding on the light area entering times of mice in the light-dark box experiment after 7 days. Figure 9

[0124] In the mouse light-dark box experiment, compared with the normal control group, the simulated feed adaptive feeding had no effect on the light area entering times of mice in the light-dark box experiment.

[0125] (2) Experiment two:

[0126] ①Mice open field experiment

[0127] Table 10 Total distance results record of mice open field experiment

[0128] Group Number of animals Total path (mm) Normal group 7 31725±776.1 Simulation group 9 30032±1132 Restraint group 10 29086±925.9

[0129] As shown in the figure, the effect of simulated feed adaptive feeding on the total distance of mice in the open field experiment after 7 days. Figure 10 ​​​Figure 7 shows the effect of 7-day simulated feed modeling on the total distance of mice in the open field test.

[0130] In the open field test of mice, compared with the normal control group, 7-day simulated feed modeling and 7-day chronic restraint stress had no effect on the total distance of mice in the open field test.

[0131] Table 11 records the results of the central area distance of mice in the open field test.

[0132] Group Number of animals Central area path (mm) Normal group 7 2425±131.9 Simulation group 9 1412±122.8**** Restraint group 10 1356±111.6****

[0133] As shown in Figure 8, the effect of 7-day simulated feed modeling on the central area distance of mice in the open field test is shown. Figure 11 Compared with the control group, ****p<0.0001.

[0134] In the open field test of mice, compared with the normal control group, 7-day simulated feed modeling and 7-day chronic restraint stress can significantly reduce the central area distance of mice in the open field test.

[0135] Table 12 records the results of the central area residence time of mice in the open field test.

[0136] Group Number of animals Stay time (s) Normal group 7 17.54±1.450 Simulation group 9 10.52±1.069*** Restraint group 10 9.712±1.199***

[0137] As shown in Figure 9, the effect of 7-day simulated feed modeling on the central area residence time of mice in the open field test is shown. Figure 12 Compared with the control group, ***p<0.001.

[0138] In the open field test of mice, compared with the normal control group, 7-day simulated feed modeling and 7-day chronic restraint stress can significantly reduce the central area residence time of mice in the open field test.

[0139] Table 13 records the results of the central area entry times of mice in the open field test.

[0140] Group Number of animals Central area entry times Normal group 7 13.33±0.9320 Simulation group 9 8.167±0.6256**** Restraint group 10 8.833±1.242**

[0141] As shown in Figure 10, the effect of 7-day simulated feed modeling on the central area entry times of mice in the open field test is shown. Figure 13 Compared with the control group, **p<0.01, ****p<0.0001.

[0142] In the open field test of mice, compared with the normal control group, 7-day simulated feed modeling and 7-day chronic restraint stress can significantly reduce the central area entry times of mice in the open field test.

[0143] ②Mice elevated plus maze test:

[0144] Table 14 Open field test entry time percentage (OT%)

[0145] Group Number of animals OT% Normal group 11 0.2301±0.02750 Simulation group 10 0.1389±0.02603** Restraint group 13 0.1830±0.02091

[0146] As Figure 14 shown, the effect of simulation feed modeling for 7 days on the open field test entry time percentage of mice was observed. Compared with the control group, **p<0.01.

[0147] In the open field test of mice, compared with the normal control group, simulation feed modeling for 7 days can significantly reduce the open field test entry time percentage of mice.

[0148] Table 15 Open field test entry number percentage (OE%)

[0149] Group Number of animals OE% Normal group 11 0.3567±0.01186 Simulation group 10 0.2894±0.02044** Restraint group 13 0.3246±0.01215*

[0150] As Figure 15 shown, the effect of simulation feed modeling for 7 days on the open field test entry number percentage of mice was observed. Compared with the control group, *p<0.05, **p<0.01.

[0151] In the open field test of mice, compared with the normal control group, simulation feed modeling for 7 days and chronic restraint stress for 7 days can significantly reduce the open field test entry number percentage of mice.

[0152] ③Light-dark box test of mice:

[0153] Table 16 Light-dark box test light area entry time

[0154] Group Number of animals Bright area entry time (s) Normal group 6 156.8±6.219 Simulation group 7 142.2±3.962* Restraint group 9 138.8±4.355*

[0155] As Figure 16 shown, the effect of simulation feed modeling for 7 days on the light-dark box test light area entry time of mice was observed. Compared with the control group, *p<0.05.

[0156] In the light-dark box test of mice, compared with the normal control group, simulation feed modeling for 7 days and chronic restraint stress for 7 days can significantly reduce the light area entry time of mice.

[0157] Table 17 Light-dark box test light area entry number

[0158] Group Number of animals Bright area entry times Normal group 6 19.33±0.6667 Simulation group 7 15.70±1.023** Restraint group 9 17.43±0.6494*

[0159] As Figure 17 shown, the effect of simulation feed modeling for 7 days on the light-dark box test light area entry number of mice was observed. Compared with the control group, *p<0.05, **p<0.01.

[0160] In the light-dark box experiment in mice, compared with the normal control group, 7 days of simulated diet modeling and 7 days of chronic restraint stress significantly reduced the number of times mice entered the light area.

[0161] (III) Experiment 3:

[0162] ① Mouse open field experiment

[0163] Table 18 Record of total distance traveled in mouse open field experiment

[0164] Group Number of animals Total path (mm) Normal group 9 24389±1875 Simulation group 12 24014±1176 Restraint group 12 24585±1134

[0165] like Figure 18 The figure shown is a graph illustrating the effect of simulated feed on the total distance traveled in the open field experiment of mice 14 days after modeling.

[0166] In the mouse open field experiment, compared with the normal control group, 14 days of simulated diet modeling and 14 days of chronic restraint stress had no effect on the total distance of the mouse open field experiment.

[0167] Table 19 Record of distance traveled in the central region during mouse open field experiments

[0168] Group Number of animals Central area path (mm) Normal group 9 11367±3132 Simulation group 12 4682±2343* Restraint group 12 3623±25208*

[0169] like Figure 19 As shown, this graph illustrates the effect of simulated feed on the central region distance in mice after 14 days of modeling. Compared with the control group, *p<0.05.

[0170] In the mouse open field experiment, compared with the normal control group, 14 days of simulated diet modeling and 14 days of chronic restraint stress significantly reduced the central zone distance in the mouse open field experiment.

[0171] Table 20 Record of mouse dwell time in the central region during open field experiment

[0172] Group Number of animals Stay time (s) Normal group 9 17.54±1.450 Simulation group 12 10.52±1.069*** Restraint group 12 9.712±1.199***

[0173] like Figure 20 As shown in the figure, the effect of simulated feed on the time spent in the central region of the mouse open field experiment 14 days after modeling. Compared with the control group, ***p<0.001.

[0174] In the mouse open field experiment, compared with the normal control group, 14 days of simulated diet modeling and 14 days of chronic restraint stress significantly reduced the time spent in the central area of ​​the mouse open field experiment.

[0175] Table 21 Record of mouse open field experiment number of entries into the central area

[0176] Group Number of animals Central area entry times Normal group 9 12.63±0.9246 Simulation group 12 9.100±0.9000** Restraint group 12 8.615±0.7969**

[0177] As Figure 21 shown, the effect diagram of the number of times of entering the central area of the mouse open field test after 14 days of simulation feed modeling, compared with the control group, **p<0.01.

[0178] In the mouse open field test, compared with the normal control group, simulation feed modeling for 14 days and chronic restraint stress for 14 days can significantly reduce the number of times of entering the central area of the mouse.

[0179] ②Mice elevated plus maze experiment:

[0180] Table 22 Elevated plus maze experiment entering time percentage (OT%)

[0181] Group Number of animals OT% Normal group 12 0.2242±0.03816 Simulation group 10 0.1161±0.02640* Restraint group 11 0.1509±0.02015*

[0182] As Figure 22 shown, the effect diagram of the number of times of entering the central area of the mouse open field test after 14 days of simulation feed modeling, compared with the control group, **p<0.01.

[0183] In the mouse open field test, compared with the normal control group, simulation feed modeling for 14 days and chronic restraint stress for 14 days can significantly reduce the number of times of entering the central area of the mouse.

[0184] Table 23 Elevated plus maze experiment entering time percentage (OT%)

[0185] Group Number of animals OE% Normal group 12 0.3334±0.02203 Simulation group 10 0.2630±0.01934* Restraint group 11 0.3193±0.01784

[0186] As Figure 23 shown, the effect diagram of the number of times of entering the central area of the mouse open field test after 14 days of simulation feed modeling, compared with the control group, **p<0.01.

[0187] In the mouse open field test, compared with the normal control group, simulation feed modeling for 14 days and chronic restraint stress for 14 days can significantly reduce the number of times of entering the central area of the mouse.

[0188] ③Mice light-dark box experiment:

[0189] Table 24 Light-dark box experiment light area entering time

[0190] Group Number of animals Bright area entry time (s) Normal group 6 135.4±6.347 Simulation group 7 117.3±5.335* Restraint group 9 122.8±2.616*

[0191] As Figure 24 shown, the effect diagram of the number of times of entering the central area of the mouse open field test after 14 days of simulation feed modeling, compared with the control group, **p<0.01.

[0192] In the light-dark chamber experiment in mice, compared with the normal control group, 14 days of simulated diet modeling and 14 days of chronic restraint stress significantly reduced the time mice spent entering the light zone.

[0193] Table 25 Number of times the light area was entered in the dark-light box experiment

[0194] Group Number of animals Bright area entry times Normal group 6 15.44±0.9444 Simulation group 7 12.17±0.9198* Restraint group 9 12.25±0.8627*

[0195] like Figure 25 As shown, this graph illustrates the effect of simulated feed on the number of times mice entered the light area of ​​the light-dark chamber experiment 14 days after modeling. Compared with the control group, *p<0.05.

[0196] In the light-dark chamber experiment in mice, compared with the normal control group, 14 days of simulated diet modeling and 14 days of chronic restraint stress significantly reduced the number of times mice entered the light zone.

[0197] (4) Experiment 4:

[0198] ① Mouse open field experiment

[0199] Table 26 Record of total distance traveled in mouse open field experiment

[0200]

[0201]

[0202] like Figure 26 The figure shown is a graph illustrating the effect of gavage intervention on the total distance traveled in the open field experiment in mice after 7 days.

[0203] In the mouse open field experiment, compared with the normal control group, gavage intervention for 7 days had no effect on the total distance traveled in the open field of mice.

[0204] Table 27 Record of mouse open field experiment central zone distance results

[0205] Group Dose Number of animals Central area path (mm) Control group 0 11 835.6±95.97 Positive drug group 3.33 mg / kg 13 1129±132.3* Simulation group 0 11 496.8±72.18** Simulation + positive drug group 3.33 mg / kg 12 751.7±89.01# Restraint group 0 8 550.5±89.01* Restraint + positive drug group 3.33 mg / kg 12 949.6±132.0&

[0206] like Figure 27 As shown, this is a graph illustrating the effect of gavage intervention on the central region distance in mice after 7 days. Compared with the normal group, *p<0.05, **p<0.01, #p<0.05 compared with the simulation group, and &p<0.05 compared with the restraint group.

[0207] In the mouse open field experiment, after 7 days of gavage intervention, the positive control group significantly increased the central distance of the mouse open field compared to the normal control group. Compared to the simulation group, the simulation + positive control group significantly increased the central distance of the mouse open field. Compared to the restraint group, the restraint + positive control group significantly increased the central distance of the mouse open field.

[0208] Table 28: The results of the central area stay time of the mouse open field test

[0209] Group Dose Number of animals Central area stay time (s) Control group 0 11 5.286±0.9398 Positive drug group 3.33 mg / kg 13 7.943±0.6624* Simulation group 0 11 3.117±0.5120* Simulation + positive drug group 3.33 mg / kg 12 4.434±0.4504# Restraint group 0 8 1.792±0.3903* Restraint + positive drug group 3.33 mg / kg 12 5.319±0.9039&&

[0210] As shown in Figure 28 Figure 7, the effect of gavage intervention for 7 days on the central area stay time of the mouse open field test. Compared with the normal group, *p<0.05, compared with the simulation group, #p<0.05, compared with the restraint group, &&p<0.01.

[0211] In the mouse open field test, after gavage intervention for 7 days, compared with the normal control group, the positive drug group can significantly increase the central area stay time of the mouse open field test. Compared with the simulation group, the simulation+positive drug group can significantly increase the central area stay time of the mouse open field test. Compared with the restraint group, the restraint+positive drug group can significantly increase the central area stay time of the mouse open field test.

[0212] Table 29: The results of the central area entry times of the mouse open field test

[0213] Group Dose Number of animals Central area entry times Control group 0 11 5.750±0.6046 Positive drug group 3.33 mg / kg 13 7.556±0.7286* Simulation group 0 11 3.385±0.4875** Simulation + positive drug group 3.33 mg / kg 12 3.385±0.7311# Restraint group 0 8 3.571±0.7825* Restraint + positive drug group 3.33 mg / kg 12 6.545±1.155&

[0214] As shown in Figure 29 Figure 8, the effect of gavage intervention for 7 days on the central area entry times of the mouse open field test. Compared with the control group, *p<0.05, **p<0.01, compared with the simulation group, #p<0.05, compared with the restraint group, &p<0.05.

[0215] In the mouse open field test, after gavage intervention for 7 days, compared with the normal control group, the positive drug group can significantly increase the central area entry times of the mouse open field test. Compared with the simulation group, the simulation+positive drug group can significantly increase the central area entry times of the mouse open field test. Compared with the restraint group, the restraint+positive drug group can significantly increase the central area entry times of the mouse open field test.

[0216] ②Elevated plus maze test of mice:

[0217] Table 30: The percentage of entry time (OT%) of the elevated plus maze test

[0218] Group Dose Number of animals OT% Control group 0 14 0.1764±0.03496 Positive drug group 3.33 mg / kg 12 0.1710±0.01321 Simulation group 0 7 0.09654±0.01762* Simulation + positive drug group 3.33 mg / kg 8 0.1594±0.02540# Restraint group 0 9 0.1055±0.01225* Restraint + positive drug group 3.33 mg / kg 10 0.1645±0.01578&&

[0219] As shown in Figure 30 Figure 9, the effect of gavage intervention for 7 days on the percentage of entry time (OT%) of the elevated plus maze test of mice. Compared with the control group, *p<0.05, compared with the simulation group, #p<0.05, compared with the restraint group, &&p<0.01.

[0220] In the elevated cross maze test in mice, after 7 days of gavage intervention, the positive control group significantly increased the percentage of time mice took to enter the elevated cross maze compared to the normal group. Compared to the simulation group, the simulation + positive control group significantly increased the percentage of time mice took to enter the elevated cross maze. Compared to the restraint group, the restraint + positive control group significantly increased the percentage of time mice took to enter the elevated cross maze.

[0221] Table 31 Percentage of Entry Times in the Elevated Cross Maze Experiment (OE%)

[0222] Group Dose Number of animals OE% Control group 0 14 0.2570±0.02127 Positive drug group 3.33 mg / kg 12 0.2599±0.01191 Simulation group 0 7 0.1904±0.02008* Simulation + positive drug group 3.33 mg / kg 8 0.2529±0.01421# Restraint group 0 9 0.1980±0.01454* Restraint + positive drug group 3.33 mg / kg 10 0.2510±0.01885&

[0223] like Figure 31 As shown in the figure, the effect of gavage intervention on the percentage of mice entering the elevated cross maze test after 7 days is as follows: compared with the control group, *p<0.05; compared with the simulation group, #p<0.05; compared with the restraint group, &p<0.05.

[0224] In the elevated cross maze test in mice, after 7 days of gavage intervention, the simulation + positive control group significantly increased the percentage of mice entering the elevated cross maze compared to the simulation group. Compared to the restraint group, the restraint + positive control group significantly increased the percentage of mice entering the elevated cross maze.

[0225] ③ Mouse light-dark box experiment:

[0226] Table 32 Light-Dark Box Experiment: Entry Time to the Light Zone

[0227] Group Dose Number of animals Bright area entry time (s) Control group 0 13 137.4±5.107 Positive drug group 3.33 mg / kg 3.33 mg / kg 7 139.0±2.551 sham group 0 10 118.1±6.912* sham + positive drug group 3.33 mg / kg 11 135.3±5.150# restraint group 0 12 125.8±3.469* restraint + positive drug group 3.33 mg / kg 9 125.2±4.349

[0228] like Figure 32 As shown in the figure, the effect of gavage intervention on the entry time of mice into the light area of ​​the light-dark box experiment after 7 days is shown. Compared with the control group, *p<0.05, and compared with the simulation group, #p<0.05.

[0229] In the mouse light-dark box experiment, after 7 days of gavage intervention, the positive control group significantly increased the time for mice to enter the light area in the light-dark box experiment compared to the normal control group. Compared to the simulation group, the simulation + positive control group significantly increased the time for mice to enter the light area in the light-dark box experiment.

[0230] Table 33 Number of times the light area was entered in the dark-light box experiment

[0231] group dose number of animals number of times of entering the light area control group 0 13 20.00±2.566 positive drug group 3.33 mg / kg 7 26.67±1.406* sham group 0 10 15.00±0.9189* sham + positive drug group 3.33 mg / kg 11 19.21±1.039## restraint group 0 12 14.00±0.7862* restraint + positive drug group 3.33 mg / kg 9 20.60±1.586&&&

[0232] like Figure 33 As shown in the figure, the effect of gavage intervention on the number of times mice entered the light area of ​​the light-dark box experiment after 7 days of intervention was *p<0.05 compared with the control group, ##p<0.01 compared with the simulation group, and &&&p<0.001 compared with the restraint group.

[0233] In the mouse light-dark box experiment, after 7 days of intragastric intervention, compared with the normal control group, the positive drug group can significantly increase the number of entering the light area of the mouse light-dark box experiment. Compared with the simulation group, the simulation + positive drug group can significantly increase the number of entering the light area of the mouse light-dark box experiment. Compared with the restraint group, the restraint + positive drug group can significantly increase the number of entering the light area of the mouse light-dark box experiment.

Claims

1. A method for modeling anxiety disorder in laboratory animals using simulated feed, characterized by: A simulated feed is used, comprising a shell with an end cap screwed to one end. The shell wall has several through holes, and real feed is placed inside. The real feed cannot leak out through the through holes, but its odor can escape. The shell is hard, and experimental animals cannot break the real feed by tearing at it. The shape of the shell is consistent with the feed that experimental animals normally eat, and both the shell and the end cap are non-toxic and odorless. The method includes a modeling method and a research method. The modeling method includes: first, by subjecting rodents to timed feeding to develop a habit of rapid, fixed-point eating; then, during timed feeding, depriving the rodents of sufficient food, causing them to become hungry and unable to eat, thus inducing anxiety; second, by restricting the amount of food consumed by the rodents through timed and quantitative feeding, causing them to become hungry but unable to eat to satiety, thus inducing anxiety; third, by mixing real feed with simulated feed, as the simulated feed resembles real feed in appearance and emits the same smell, the rodents cannot distinguish between the real and simulated feed, creating uncertainty during feeding, and inducing anxiety through continuous stimulation; finally, through the combination of hunger without food, insufficient food intake, insufficient satiety, and uncertainty, the rodents develop anxiety. The research methods described include: A. During the process of modeling with simulated feed, the anxiety behavior of rodent experimental animals is monitored through a combination of one or more experiments, such as open field, elevated cross maze, and light and dark box, until the model is successfully established. B. Compare experimental animals obtained by the simulated feed modeling method with those obtained by the chronic restraint stress-induced anxiety modeling method in two phases of 7 days and 14 days; during this process, monitor the changes in anxiety behavior of rodent experimental animals by one or more of the following experiments: open field, elevated cross maze, and light-dark box. C. Drug intervention was applied to experimental animal anxiety models created using simulated feed. Changes in anxiety behavior in rodent experimental animals were monitored through a combination of one or more experiments, such as open field, elevated cross maze, and light-dark chamber, and the therapeutic effect of drugs on the experimental animal anxiety model was evaluated.

2. The method for establishing an anxiety model in experimental animals using simulated feed as described in claim 1, characterized in that: The shell is cylindrical, and the shell wall is provided with several blind holes, the inner diameter of which is 1mm.

3. The method for establishing an anxiety model in experimental animals using simulated feed as described in claim 2, characterized in that: The shell and end cap are both made of ABS engineering plastic. The shell has a diameter of 12mm and a length of 10-30mm. The outer surface of the shell is also doped with yellow dye to make the shell more similar to the color of real feed.

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