阅读说明：本技术 一种焦虑联合连续睡眠缺失导致认知下降的动物模型的构建方法及其应用 (Construction method and application of animal model with cognitive decline caused by anxiety combined with continuous sleep deficiency ) 是由 钟倩霞 周勇 丁刘刚 杜志云 潘春杏 李宜鸣 于 2019-12-17 设计创作，主要内容包括：本发明公开了一种焦虑联合连续睡眠缺失导致认知下降的动物模型的构建方法及其应用。该方法包括联合噪音干预、电刺激干预、光干扰干预刺激实验动物,干预结束后对所述实验动物进行动物行为学测试。本发明方法同时关注了焦虑联合连续睡眠缺失导致的认知下降,构建方法操作简单、造模周期短、建模效果好,且对动物不具侵入性。本发明获得的动物模型能更加贴近因压力大、工作负荷重、情绪紧张而产生焦虑和连续睡眠不足状态,进而导致出现认知下降的目标人群健康症状的场景,可用于对生活方式、饮食与药物对焦虑联合连续睡眠缺失导致认知下降的症状进行有效性评价,也为相关食品保健品筛选、药物研究开发等提供可靠的评价模型。(The invention discloses a construction method and application of an animal model with cognitive decline caused by anxiety combined with continuous sleep deficiency. The method comprises the steps of stimulating experimental animals by combining noise intervention, electrical stimulation intervention and light interference intervention, and carrying out animal behavior test on the experimental animals after intervention is finished. The method disclosed by the invention simultaneously focuses on cognitive decline caused by anxiety combined with continuous sleep deficiency, and the construction method is simple to operate, short in modeling period, good in modeling effect and non-invasive to animals. The animal model obtained by the invention can be closer to the situation that anxiety and insufficient continuous sleep are generated due to high pressure, heavy workload and emotional stress, so that the health symptoms of target people with cognitive decline are caused, can be used for evaluating the effectiveness of the cognitive decline caused by the combination of life style, diet and medicine on anxiety and continuous sleep deficiency, and also provides a reliable evaluation model for screening related food health products, researching and developing medicines and the like.)

1. The method for constructing the animal model with cognitive decline caused by anxiety combined with continuous sleep deficiency is characterized by comprising the steps of combining noise intervention, electrical stimulation intervention and light interference intervention to stimulate experimental animals, and carrying out animal behavior test on the experimental animals after intervention is finished.

2. The method as claimed in claim 1, wherein the noise intervention is the application of noise with sound frequency of 0.1-2kHz and sound intensity of 30-200dB to the experimental animal, the noise interference time is 1s-180s each time, the cycle number is more than or equal to 15 times/d, the interference interval is less than or equal to 1.5h, and the interference time period is 0:00-24: 00.

3. The method as claimed in claim 1, wherein the electric stimulation intervention is that 0.4-2mA of stimulation current is applied to the experimental animal, each time of electric stimulation is 0.5-30s, the stimulation times are more than or equal to 15 times/d, the stimulation interval is less than or equal to 1.5h, and the stimulation time period is 0:00-24: 00.

4. The method of claim 1, wherein the light disrupting intervention is a continuous daylight lamp illumination of the experimental animal for a period of 0:00-24: 00.

5. The method of claim 1, wherein the intervention cycle is from 1 to 7 days.

6. The method of claim 1, wherein the animal behavioral test is a Morris behavioral test.

7. The method of claim 6, wherein the Morris behavioural test indicator comprises:

directional navigation indexes are as follows: measuring the time for the experimental animal to reach a specified survival platform;

space exploration indexes are as follows: and (3) determining the number of times that the experimental animal passes through the platform area under the condition of removing the survival platform, and determining the ratio of the time that the experimental animal is active in the quadrant where the platform is located to the total time in one minute of voyage time.

8. The method of any one of claims 1 to 7, wherein the laboratory animal is a rodent.

9. The method of claim 8, wherein the test animal is a mouse.

10. Use of an animal model obtained according to the method of any one of claims 1 to 7 in the screening or identification of a therapeutic agent for reducing or treating anxiety in combination with cognitive decline caused by loss of continuous sleep.

Technical Field

The invention belongs to the technical field of animal model construction. More particularly, relates to a construction method and application of an animal model with cognitive decline caused by anxiety and continuous sleep deficiency.

Background

Due to rapid development of science and technology, work is increasingly tense, the rhythm of life is accelerated, and pressure in intense competition is continuously increased in modern society, so that more and more people are in states of anxiety and sleep deficiency for a long time, particularly mental workers and students at school. The possibility of anxiety and depression onset is increased when the patient is in an anxiety state for a long time; the lack of sleep for a long time can bring many physical and mental injuries such as decreased thinking ability, impaired alertness and judgment, immune dysfunction and the like. The anxiety states and the sleep insufficiency are related and mutually promoted, and the symptoms of insomnia and anxiety are deepened. Existing studies have demonstrated that both loss of sleep and the appearance of anxiety states lead to a decline in cognitive performance. For a mental worker, the decline in cognitive ability significantly reduces their work efficiency. The method finds an animal model which can be close to the state of anxiety and insufficient continuous sleep caused by high pressure, heavy workload and emotional stress so as to further cause the decline of cognitive ability, and is helpful for effectively understanding the occurrence and development rules of diseases and researching prevention and treatment measures.

At present, no research and report concerning animal models with cognitive decline caused by both anxiety and sleep loss are available.

The existing methods for constructing the sleep deficiency animal model include a chronic tail clamping stimulation and intraperitoneal injection chlorpropamide compound factor modeling method, a caffeine intraperitoneal injection superimposed multi-platform water environment continuous sleep deprivation method, a platform water environment method and a forced exercise method. The existing method for constructing the anxiety animal model comprises a method combining a light and shade box experiment and a tail suspension experiment, a chronic constraint stress model, a social isolation model, an uncertain empty bottle stress model and a natural enemy exposure model. Although the existing model building methods are numerous, the model building process period is long, the model building method is invasive, the goal of simultaneously paying attention to cognitive decline caused by anxiety and sleep deficiency cannot be achieved, and the research requirement cannot be met.

Disclosure of Invention

The invention aims to solve the technical problems that the existing animal model cannot pay attention to the cognitive decline caused by sleep deficiency and anxiety states at the same time, and the existing construction method has the defects of long modeling period and invasiveness, and provides the construction method of the animal model with the cognitive decline caused by anxiety combined with continuous sleep deficiency.

The invention aims to provide a construction method of an animal model with cognitive decline caused by anxiety combined with continuous sleep deficiency.

The invention also aims to provide application of the constructed animal model with cognitive decline caused by combination of anxiety and continuous sleep deficiency.

The above purpose of the invention is realized by the following technical scheme:

an animal model construction method for cognitive decline caused by anxiety combined with continuous sleep deficiency is characterized in that noise intervention, electrical stimulation intervention and light interference intervention are combined to stimulate experimental animals, and animal behavioural tests are carried out on the animal model after intervention is finished.

The invention stimulates the experimental animal in high frequency and multiple dimensions in a combined noise intervention, electrical stimulation intervention and light interference intervention mode to cause the emotional anxiety of the experimental animal and the scene that the experimental animal can not fall asleep continuously, and maximally simulates the scene that people in real life are anxious and lack of continuous sleep to further cause cognitive decline under the interference of various external factors such as large working pressure, long working time and heavy working load. And after intervention is finished, judging whether the modeling is successful or not through animal behavioral testing, wherein the standard for judging the success of modeling is the appearance of cognitive decline of the experimental animal. The method has no invasion to experimental animals, is simple and convenient to operate, has a short molding period, and can greatly reduce the experimental cost.

Preferably, the noise intervention is to apply noise with sound frequency of 0.1-2kHz and sound intensity of 30-200dB to the experimental animal, the noise circulation frequency can be self-simulated according to the experimental requirements, the interference time of each noise is 1s-180s, the circulation frequency is more than or equal to 15 times/d, the interference interval is less than or equal to 1.5h, and the interference time period is 0:00-24: 00.

Preferably, the electric stimulation intervention is to apply 0.4-2mA stimulation current to the experimental animal, the stimulation current is obtained after 10-200VAC multi-voltage rectification, the electric stimulation cycle frequency can be self-simulated according to the experimental requirements, the electric stimulation time is 0.5-30s each time, the stimulation frequency is more than or equal to 15 times/d, the stimulation interval is less than or equal to 1.5h, and the stimulation time period is 0:00-24: 00.

Preferably, the light interference intervention is continuously irradiating the experimental animal by a fluorescent lamp for 0:00-24:00, and the continuous illumination period can be self-planned according to experimental requirements.

According to the invention, the animal model with animal behavior and related physiological indexes conforming to cognitive decline caused by anxiety combined with continuous sleep deficiency is obtained by optimizing the intervention time and intensity of noise, electric stimulation and light interference, and the established animal model has better effect.

Preferably, the intervention cycle is 1-7 days.

Preferably, the animal behavioural test is a Morris behavioural test. The method can be used for comparing whether the experimental animal has the symptom of cognitive decline or not through a Morris behavioural test, and if the Morris behavioural test index of the animal model is obviously lower than that of a normal control (p <0.05), the animal model is successfully modeled.

Further, preferably, the Morris behavioural test indicator comprises:

directional navigation indexes are as follows: measuring the time for the experimental animal to reach a specified survival platform;

space exploration indexes are as follows: determining the number of times that the experimental animal passes through a platform area under the condition that the survival platform is removed; the ratio of the time the experimental animal was active in the quadrant of the platform to the total time during one minute of the flight time with the survival platform removed was determined.

Preferably, the laboratory animal is a rodent.

Further, preferably, the experimental animal is a mouse.

Use of an animal model obtained according to the above method for screening or identifying a therapeutic agent for reducing or treating anxiety in combination with cognitive decline caused by loss of continuous sleep.

Wherein the screening or identification process comprises the steps of:

(1) dividing the animal model obtained by the method into a control group and a test group, applying a test compound to the test group, and respectively performing the Morris behavioural test on the test group and the control group;

(2) comparing the animal model behaviors of the test group and the control group, and if the Morris behavioristics test index of the animal model of the test group is improved, the test compound can be used as a therapeutic agent for cognitive decline caused by anxiety combined with continuous sleep deficiency.

In addition, the animal behavior experiment can be used for evaluating the effect of each test compound on resisting cognitive decline after multidimensional stimulation is carried out and an animal model process of cognitive decline caused by anxiety combined with continuous sleep deficiency is constructed by presetting the administration period for the experimental animal.

The invention has the following beneficial effects:

the animal modeling method disclosed by the invention has the advantages of non-invasiveness, short modeling period, simplicity in operation, low cost and good modeling effect. The animal model constructed by the invention can be effectively used for the research of food or drugs for resisting cognitive decline and refreshing, for example, the animal model can be used for screening or identifying the food or drugs for relieving or treating cognitive decline caused by sleep deficiency and anxiety, and meanwhile, the animal model can well evaluate the cognitive ability improvement and the refreshing effect of the food or drugs.

Drawings

FIG. 1 is a diagram showing the directional course of a mouse in comparative example 1;

FIG. 2 is a graph showing the time taken for the directional navigation of the mouse in comparative example 1.

Detailed description of the preferred embodiments

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.