A method for constructing an acute anxiety model for experimental animals and its application

Abstract

This application relates to a method for constructing and applying an acute anxiety model for experimental animals. By injecting exogenous parathyroid hormone-related proteins into the experimental animals, the animals develop anxiety symptoms within a short period of time, thereby obtaining an acute experimental animal model with anxiety symptoms. The acute anxiety model for experimental animals obtained in this application has the advantages of rapid effectiveness, short modeling time, and a distinct anxiety phenotype. This method overcomes the problems of traditional animal acute anxiety models, such as the tendency to incur additional injuries during the modeling process, poor ventilation of experimental animals, differences in restraint pressure, and additional dizziness in experimental animals. This method facilitates the study of the mechanisms of anxiety disorders and drug development.

Description

Technical Field

[0001] The present application relates to the field of animal models, and specifically to a method for constructing and applying an acute anxiety model for experimental animals. Background Art

[0002] Anxiety disorders include generalized anxiety disorder, social anxiety disorder, panic disorder, phobias, post-traumatic stress disorder, and agoraphobia. Anxiety disorders are the most common mental disorders in the population. A systematic review of epidemiological studies in 44 countries found that the global prevalence of anxiety disorders is approximately 7.3%, meaning that 1 in 14 people worldwide suffers from an anxiety disorder. Anxiety disorders have a significant impact on patients' quality of life. First, anxiety can lead to overactivation of the sympathetic nervous system and increase levels of catecholamines in the circulation. Second, anxiety can also affect various behavioral responses, such as increased eating, drinking, and smoking. Furthermore, anxiety disorders increase the risk of coronary heart disease by 26% and the risk of cardiac death by 48%. Anxiety has become an independent risk factor for both coronary heart disease and cardiac death. Therefore, it is imperative to investigate the mechanisms underlying the development and progression of anxiety disorders, identify effective interventions for anxiety disorders, and accelerate the research of potent targeted drugs.

[0003] The mechanisms of anxiety disorders and related drug research require the use of experimental animal anxiety models. Existing anxiety models are mainly divided into chronic anxiety models and acute anxiety models. The chronic anxiety model takes a long time to establish, which has caused certain obstacles to related research. Acute anxiety models often use acute restraint stress (ARS), which is a method of confining mice in a narrow plastic tube for 1-2 hours to complete the construction of the anxiety model. This application proposes an optimized experimental animal acute anxiety model to address the shortcomings of existing acute anxiety models.

[0004] The acute anxiety model involves drilling a vent hole in the wall of a 50ml plastic centrifuge tube, placing the experimental mouse in the tube, tightening the cap, and allowing it to rest for 60-120 minutes before observing or measuring its anxiety phenotype. This modeling method has the following disadvantages:

[0005] (1) Individual differences in mice lead to differences in modeling results: The volume of a 50ml plastic centrifuge tube is fixed, but mice are affected by individual differences. It is possible that the experimental mice cannot be placed in the tube or are overly squeezed in the tube; or there is excess space in the tube, causing the mice to move back and forth in the centrifuge tube, which can easily lead to insufficient restraint. The above situations cause individual mice to feel different restraint pressures, resulting in different modeling effects of the animal anxiety model;

[0006] (2) Insufficient ventilation for experimental animals: The ventilation holes in the wall of the plastic centrifuge tube are drilled subjectively by the experimenter, which may result in insufficient pores, resulting in insufficient ventilation for the mice and causing them to feel excessive breathlessness; or excessive drilling may lead to insufficient restraint, affecting subsequent experimental results;

[0007] (3) Additional dizziness in experimental animals: Plastic centrifuge tubes are light, and the movement of experimental mice in the tubes can easily cause the tubes to roll left and right, which can cause additional dizziness in the experimental mice and thus affect the anxiety results of the experimental mice;

[0008] (4) Additional harm to experimental animals: Mice tend to struggle when placed in centrifuge tubes, which may cause them to bump into the tube mouth and cause additional harm to the experimental mice;

[0009] (5) Time-consuming: When constructing multiple experimental animal models, the acute restraint stress time is long, and the restraint time of a single animal is no less than 60 minutes. When there are too many animals between or within the experimental groups, it is easy to affect the measurement efficiency, and the number of animals measured per day is limited.

[0010] Therefore, existing methods for creating experimental animal anxiety models suffer from numerous drawbacks, such as wide variability in modeling results, insufficient ventilation, excessive dizziness, excessive reactions, and time-consuming procedures. These drawbacks increase the workload and create significant challenges in research into the mechanisms of anxiety disorders. Summary of the Invention

[0011] This application demonstrates for the first time the role of parathyroid hormone-related protein (PTHrp) on mood and creates a method for exogenously injecting PTHrp to induce an anxiety phenotype in experimental animals. The purpose of this application is to overcome the shortcomings of existing technologies and provide a more concise and efficient method for establishing an acute anxiety model in experimental animals. This method addresses the problems existing in existing methods for establishing anxiety models in experimental animals and facilitates the study of the mechanisms of anxiety disorders and drug development.

[0012] To achieve the above objectives, this application provides the following technical solutions:

[0013] The first aspect of the present application is to provide a method for constructing an acute anxiety model in experimental animals, comprising the following steps:

[0014] S1. Stabilize the emotions of experimental animals;

[0015] S2. Inject parathyroid hormone-related protein into the body of experimental animals.

[0016] S3. Wait for the experimental animals to develop anxiety, thereby obtaining an acute anxiety model of the experimental animals.

[0017] Preferably, the parathyroid hormone-related protein is injected into the tail vein of the experimental animal.

[0018] Preferably, when the experimental animals are mice, the injection dose of the parathyroid hormone-related protein is 1 ng / mouse.

[0019] Preferably, the method for stabilizing the emotions of the experimental animals in step S1 is touch adaptation, in which the mice are touched within a specific time range and a fixed time period every day.

[0020] Furthermore, in step S3, after the parathyroid hormone-related protein acts on the experimental animal, the anxiety phenotype of the experimental animal is detected.

[0021] Preferably, the anxiety phenotype of the experimental animals is detected by an open field test, and if the anxiety phenotype of the experimental animals meets the standard, the construction is completed.

[0022] Preferably, the waiting time in step S3 is 15 minutes, and the mice develop anxiety.

[0023] The second aspect of the present application is to provide an experimental animal acute anxiety model, which is prepared by the above method.

[0024] Preferably, the experimental animal in step S1 is any one of rats, mice, and rabbits.

[0025] The third aspect of the present application is to provide an application of an acute anxiety model of experimental animals in studying the mechanism of anxiety disorders, and to study the therapeutic effect of drugs on mood disorders by detecting the anxiety phenotype of the acute anxiety model of experimental animals.

[0026] Compared with the prior art, the advantages of this application include:

[0027] The present application provides a method for constructing and applying an acute anxiety model for experimental animals. The method creatively obtains an experimental animal model with anxiety symptoms by injecting exogenous parathyroid hormone-related proteins into the experimental animals. This method has the advantages of simplicity and high efficiency, and the anxiety phenotype of the obtained animal anxiety model is obvious. At the same time, the present application also avoids the problems of additional injuries, impact on free ventilation, differences in restraint pressure, and additional dizziness in experimental animals that exist in the modeling process of traditional anxiety animal models. At the same time, the present application is fast-acting and has a short modeling time, which facilitates the mass production of multiple animal anxiety models. Behavioral function can be measured shortly after drug injection, greatly reducing the modeling time cost of researchers. This provides a more concise and efficient method for modeling acute anxiety models for experimental animals, which is beneficial to the research of anxiety disorders and the development of drugs for treating mood disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0029] Figure 1 This is a flow chart of the method for constructing an acute anxiety model for experimental animals;

[0030] Figure 2 Bar graph showing the anxiety level of mice injected with exogenous PTHrp as measured by open field test. DETAILED DESCRIPTION

[0031] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0032] Parathyroid hormone-related protein (PTHrp) is an active molecule secreted by various tissue cells, including granulocyte CSF and stem cell factor. PTHrp has multiple biological functions and participates in the growth and development of multiple organs. It can promote the infiltration and metastasis of breast cancer, prostate cancer, and non-small cell lung cancer, and is also one of the most important regulators of osteolysis.

[0033] By detecting the anxiety phenotype of the experimental animal acute anxiety model, the therapeutic effect of the drug on mood disorders can be detected. The modeling method of the experimental animal anxiety model in the prior art has many disadvantages, for example, large differences in modeling results, insufficient air permeability of the experimental animals, additional dizziness of the experimental animals, and long modeling time. The present application discovered for the first time that exogenous injection of parathyroid hormone-related protein causes acute anxiety symptoms in mice, thereby successfully constructing an acute anxiety model. By stabilizing the emotions of the experimental animals, then injecting parathyroid hormone-related protein into the body of the experimental animals, and waiting for the experimental animals to develop anxiety, an experimental animal anxiety model is obtained. This method overcomes the shortcomings of the prior art, has a short modeling time, and a clear anxiety phenotype, which provides convenience for the study of the mechanism of anxiety disorders and drug development. Therefore, the present application is conducive to the study of the mechanism of anxiety and the development of drugs for treating mood disorders.

[0034] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. Wherein, the methods used in the examples are all conventional methods unless otherwise specified.

[0035] Example 1: Touch adaptation of experimental animals

[0036] Three days before the formal experiment, the mice were stroked at a fixed time every day.

[0037] The specific operation is as follows: Place a cotton ball / towel of the same size as the experimenter's palm on the palm of your left hand, place the mouse on the cotton ball / towel and wrap it slightly to prevent the mouse from falling due to struggling. Gently stroke the experimental mouse with your right hand for 3 minutes. After the stroke, remove the excrement on the cotton ball or replace the towel, then replace the next animal and repeat the above operation.

[0038] Example 2: Tail vein injection of PTHrp

[0039] In this example, PTHrp was injected into the tail vein of experimental mice. Fifteen minutes after the drug injection, the mice developed anxiety, thereby obtaining an experimental animal anxiety model.

[0040] The experiment was divided into three groups: saline group, PTHrp group, and PTHrp and anti-PTHrp mixed drug group. The saline group received 0.1 ml of saline per animal, the PTHrp group received 1 ng of PTHrp per animal, and the mixed drug group received 1 ng of PTHrp per animal and 50 μg of anti-PTHrp per animal.

[0041] The steps for tail vein injection are as follows:

[0042] (1) Place the mouse in a fixture, secure it, and leave its tail exposed;

[0043] (2) Wipe the tail with alcohol or apply hot water to dilate the blood vessels;

[0044] (3) Straighten and tighten the tail, and try to insert the needle from far to near. If necessary, insert the needle multiple times.

[0045] (4) Slowly introduce PTHrp into the tail vein of the mouse.

[0046] Example 3: Detecting anxiety phenotypes in experimental animals

[0047] This example uses mice as an example to detect the anxiety phenotype of experimental animals through an open field test. The injection concentration of the drug is 1 ng / mouse. 15 minutes after the drug injection, the mice develop anxiety, thus obtaining an experimental animal anxiety model, and then their anxiety phenotype is tested.

[0048] The specific experimental steps are as follows:

[0049] (1) Pretreatment: Before the experiment begins, clean the bottom of the experimental box from the feces, urine, and other waste left by the animals in the previous experiment;

[0050] (2) Set the corresponding parameters in the software and record the animal's number, date, status and other information;

[0051] (3) The three groups of experimental animals obtained in Example 2 were taken out of the breeding cages and placed with their backs facing the experimenter;

[0052] (4) The experimental animal is quickly placed in the corner of the experimental box, facing the wall, and the researcher leaves immediately;

[0053] (5) Open the animal behavior analysis software and automatically record the activities of the experimental animals in the box for 6 minutes;

[0054] (6) After the experiment, place the experimental animals in other cages prepared in advance;

[0055] (7) Spray the instrument with alcohol to remove odor and wipe it dry with a paper towel;

[0056] (8) Use the any-maze system to analyze data and analyze the time the three groups of experimental animals spent in the central area and display it.

[0057] See also Figure 2 , Figure 2 Bar graph showing the anxiety level of mice injected with exogenous PTHrp as measured by open field test. Figure 2 The horizontal axis represents the saline group, the PTHrp group, and the PTHrp and anti-PTHrp mixed drug group, respectively. The vertical axis represents the time each group of mice spent in the center of the open field. The experimental results showed that 15 minutes after drug injection, the PTHrp group spent significantly less time in the center of the open field, indicating that the mice developed anxiety and developed an anxious phenotype. However, after tail vein injection of the PTHrp and anti-PTHrp mixed drug, the time the mice spent in the center of the open field was not statistically different from that in the saline group, indicating that the antagonism of PTHrp significantly alleviated the mice's anxious phenotype. The experiments in this example confirm the highly effective anxiogenic effect of PTHrp.

[0058] In the process of studying the mechanism of mood disorders, drugs are applied to the experimental animal acute anxiety model obtained in this application, and then the anxiety phenotype of the experimental animal acute anxiety model is detected to study the therapeutic effect of the drug on mood disorders. This experimental animal acute anxiety model has a short modeling time and a clear anxiety phenotype, which greatly improves the work efficiency of researchers and also reduces the time cost of modeling. Therefore, this application is conducive to the research of the mechanism of anxiety and the development of drugs for treating mood disorders.

[0059] In summary, the present application provides a method for constructing an acute anxiety model for experimental animals and its use. By injecting exogenous PTHrp into the experimental animals, an experimental animal model with an anxiety phenotype is obtained. This method has the advantages of simplicity and efficiency, and the anxiety phenotype of the obtained animal anxiety model is obvious. It also avoids the problems of additional injuries, impact on free ventilation, differences in restraint pressure, and additional dizziness in experimental animals that exist in the modeling process of traditional anxiety animal models. Behavioral function can be measured within a short period of time after drug injection, greatly reducing the time and cost of modeling for researchers. Therefore, the present application facilitates the study of the mechanism of anxiety and the development of drugs for treating mood disorders.

[0060] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A method for constructing an acute anxiety model for experimental animals, characterized in that: The following steps are involved: S1. Stabilizing the emotions of the experimental animals, wherein the method for stabilizing the emotions of the experimental animals in step S1 is touch adaptation, and the experimental animals are touched within a fixed time period every day; S2. injecting parathyroid hormone-related protein into the experimental animals, wherein the injection dose of the parathyroid hormone-related protein is 1 ng / animal; S3. Waiting for the experimental animal to develop anxiety, thereby obtaining an acute anxiety model of the experimental animal; wherein, the judgment condition for constructing the acute anxiety model includes detecting the anxiety phenotype of the experimental animal through an open field test. If the anxiety phenotype of the experimental animal meets the standard, the construction is completed. wherein, the waiting time in step S3 is 15 minutes, and the experimental animal develops anxiety.

2. The construction method according to claim 1, characterized in that The injection step in step S2 is to inject the parathyroid hormone-related protein into the tail vein of the experimental animal.

3. The construction method according to any one of claims 1 to 2, characterized in that In step S3, after the parathyroid hormone-related protein acts on the experimental animal, the anxiety phenotype of the experimental animal is detected.

4. The method for constructing an acute anxiety model for experimental animals according to claim 1, wherein The experimental animal in step S1 is any one of rats, mice, and rabbits.

5. An application of the experimental animal acute anxiety model according to the method for constructing the experimental animal acute anxiety model of claim 1 in studying the mechanism of anxiety disorders, characterized in that: By detecting the anxiety phenotype of the acute anxiety model of the experimental animals, the therapeutic effect of the drug on mood disorders is studied.

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