CN116270643A - Animal model for chronic depression, medicine screening method and application - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a chronic depression animal model, a drug screening method and application. The invention relates to a chronic depression animal model, which is a chronic spontaneous depression induced by long-term low-dose repeated reserpine, and is constructed by the following steps: mice were intraperitoneally injected with low dose reserpine for more than 14 days to obtain an animal model of chronic depression. The animal model is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model, and the depression symptoms of mice in the model are independent of the influence of injected medicines and are more similar to the natural pathogenesis of human depression, so that the animal model has more accurate drug effect evaluation, realizes more accurate medicine screening, and can be applied to screening or identifying alternative medicines for relieving or treating depression.

Description

A kind of chronic depression animal model, drug screening method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an animal model of chronic depression, a drug screening method and its application.

Background technique

Depression, as a major neuropsychiatric disease in the 21st century, is a global public health problem and a prominent social problem. It is characterized by high incidence, high recurrence rate, high suicide rate and high disability rate. At present, there are more than 350 million people with depression in the world, and it has become the second largest disease after cardiovascular disease. At present, the pathological mechanism of depression is still unclear, and antidepressants are still the main means of treatment for depression. In fact, only 50% of people with depression benefit from traditional antidepressant treatment. It cannot be ignored that the diagnosis of depression relies solely on behavioral symptoms, and these medications only provide relief. The important reason is that the existing animal models of depression, which are important means for drug and disease research, have limitations. The development of new animal models of depression is required to accompany the research needs of the progressive development of antidepressant drug resistance.

Common ways of modeling depression animal models can be roughly divided into the following four types: (1) Surgical models, rodent surgical bilateral olfactory bulbectomy (OBX) models have been used to screen antidepressants and are common Injury models; however, the direct link between the OBX model and human depression is controversial. (2) Stress models, stress models mainly include: maternal separation and early traumatic events model, learned helplessness (LH) model, repeated restraint stress model, chronic mild stress (CMS), social isolation model and witnessing failure Model. In general, this type of model is mainly based on the HPA axis's dysregulation of stress. This type of animal model shows persistent behavioral and cognitive deficits, showing an increase in depression-like behavior similar to that in human depression. However, after repeated stress, the HPA response was desensitized and no significant increase in corticosterone levels was observed after 21 days. The validity of this animal model for the etiology of depression in humans is not as clear as other models. Studies have also found that such models have different behavioral and neurobiological outcomes. (3) Drug-induced model, the most common is chronic corticosterone induction, the principle of which is to induce long-term depression-like behavior in rodents through the increase of stress hormones. Reserpine, on the other hand, depletes monoamines (serotonin and catecholamines) and is often used to induce acute depression models. (4) Genetic intervention models, the more classic ones currently used are the Flinders sensitive rat line (FSL) rat model and the Wistar-Kyoto (WKY) rat model, and there are fewer mouse transgenic models; transgenic models simulate the effects of human depression. Some features have certain limitations.

Recently, short-term repeated administration of reserpine has emerged as a progressive animal model of depression, and this short-term reserpine-induced depression model has depression-like symptoms similar to those in humans. Furthermore, short-term reserpine established models have been shown to be identical to depletion of 5-HT, dopamine, and metabolites in the human brain. However, the half-life of reserpine is short (4-6h) and the metabolic cycle is fast (not more than 8h), which results in short-term reserpine-induced acute animal models with short modeling time and high dosage. On the one hand, high-dose reserpine-induced acute model animal mortality is high, and the pathological process of the model is inconsistent with individual depression; on the other hand, it cannot simulate and restore the natural onset process of human depression, and the effective drugs screened by this model Performance is also very different from practical applications.

Therefore, there is an urgent need to develop animal models that can better simulate depression for more effective drug screening.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide an animal model of chronic depression. The animal model of the present invention is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model, which is closer to simulating and restoring the natural onset process of human depression.

Another object of the present invention is to provide a drug screening method based on the above animal model of chronic depression. The animal model of the present invention is a chronic spontaneous depression model, which is closer to simulating and restoring the natural onset process of human depression, so it has more accurate drug efficacy evaluation and more accurate drug screening.

Another object of the present invention is to provide the application of the animal model of chronic depression described above.

The object of the present invention is achieved through the following solutions:

An animal model of chronic depression, which is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model, specifically includes the following steps: the method is constructed by intraperitoneally injecting low-dose reserpine into mice for more than 14 days to obtain an animal model of chronic depression .

Further, the low dose means that the dose for injection may be 0.1-0.5 mg/kg.

Further, the injection frequency is once a day.

Further, the mice can be C57 mice. More preferably, it is a 7-week-old male C57 mouse, weighing 20±2 g.

Further, mice were adaptively fed for 1 week before injection.

Further, during the modeling process, the mice were kept in a standard experimental animal environment.

After the modeling, the mice obviously showed symptoms such as body temperature drop, drooping of the upper eyelid, lack of movement, and anhedonia; the injection of reserpine was stopped, and the mice were kept in the standard experimental animal environment for 14 days, and the body temperature of the mice dropped. Symptoms such as ptosis, ptosis, hypokinesia, and anhedonia remained unchanged. Through behavioral tests such as forced swimming immobility time, tail suspension immobility time, sucrose water consumption, and open field experiments, the results showed that the mice exhibited typical depressive behavior, which was in line with the reliability and validity of the depression animal model. , indicating that mice have spontaneously developed into chronic depression, therefore, according to the construction method of the present invention, a mouse model of chronic depression can be successfully obtained.

The animal model of the present invention is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model. In the model, the depressive symptoms of mice do not depend on the influence of injected drugs, and are closer to simulating and restoring the natural onset process of human depression, so they have a more accurate Drug efficacy evaluation to achieve more accurate drug screening.

The animal model of the present invention can be applied to screen or identify candidate drugs for alleviating or treating depression.

The present invention also provides a drug screening method based on the animal model of chronic depression. Use candidate drugs to evaluate drug efficacy based on the model of the present invention, obtain corresponding evaluation indexes, and realize drug screening according to the indexes.

Further, the steps of the screening method specifically include: after continuous intraperitoneal injection of drugs based on the model of the present invention, drug efficacy evaluation is performed, and corresponding evaluation indicators are obtained, and drug screening is realized according to the indicators.

Further, the duration of the continuous administration may be more than 7 days.

Further, the starting time for the drug efficacy evaluation may be 2-14 days after the model is established.

Preferably, the indicators for drug efficacy evaluation may include animal behavior tests, neuron damage, and the like.

Preferably, the animal behavior test may include forced swimming immobility time, tail suspension immobility time, sucrose water consumption, open field test and the like.

Preferably, the damage of the neurons can be assessed by Nissl staining.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The animal model of the present invention is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model, in which the depressive symptoms of mice do not depend on the influence of injected drugs, and are closer to simulating and restoring the natural onset process of human depression, so they have a more accurate Drug efficacy evaluation to achieve more accurate drug screening. The animal model of the present invention can be applied to screen or identify candidate drugs for alleviating or treating depression.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Figure 1 shows the behavioral test results and neuron damage of mice in the chronic depression model induced by long-term low-dose repeated reserpine of the present invention.

Fig. 2 is the indicators for drug efficacy evaluation of vortioxetine and fluoxetine in the acute depression model induced by high-dose reserpine. Among them, A: immobility time of mice in tail suspension test; B: immobility time of mice in forced swimming test; C: sugar water preference test of mice; D: open field test of mice.

Fig. 3 is the index of drug efficacy evaluation of vortioxetine and fluoxetine in the chronic depression model induced by long-term low-dose repeated reserpine of the present invention. Among them, A: immobility time of mice in tail suspension test; B: immobility time of mice in forced swimming test; C: sugar water preference test of mice; D: open field test of mice.

Fig. 4 shows the effects of vortioxetine and fluoxetine on the neuron damage of mice in the chronic depression model induced by long-term low-dose repeated reserpine of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial sources unless otherwise specified. The methods are conventional methods unless otherwise specified.

Example 1

An animal model of chronic depression, which is a long-term low-dose repeated reserpine-induced chronic spontaneous depression model, specifically includes the following steps: the method is constructed by intraperitoneally injecting low-dose reserpine into mice for more than 14 days to obtain an animal model of chronic depression . The specific process can be as follows:

(1) Experimental animals: male C57BL/6 mice (about 7 weeks old, initial weight 20±2g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (animal license number: NO.SCXK (Liao) 2020-0001), Raised in the Animal Center of Sun Yat-sen University (SPF grade), 12-hour circadian rhythm, relative humidity of 60±10%, constant room temperature of 24°C, quiet and ventilated environment, mouse feed and litter were all sterilized and sterilized. After one week of adaptive feeding, they were used for experiments.

(2) Model construction: 0.5 mg/kg reserpine was intraperitoneally injected into mice once a day for 14 days. During the modeling period, the mice experienced loss of appetite, weight loss, and body temperature drop. The mice were given nutritional support and warming treatment by gavage of 1-2mL sugar saline and egg whites.

After 14 days of modeling, observe the mice and conduct behavioral tests, including forced swimming immobility time, tail suspension immobility time, sucrose water consumption, open field experiment and other behavioral tests; the injection of reserpine was stopped, and the mice The mice were kept in a standard experimental animal environment for 14 days, and the above-mentioned detection was also carried out.

Methods as below:

(1) Observe the mental state of the mice and measure their body temperature;

(2) Tail suspension test (TST): the mice were randomly divided into non-stress control group, stress control group and medication group. Stress stimulation was given at an appropriate time after drug or solvent control. The rear 1/3 of the tail of the mouse was fixed with adhesive tape, hung on the bracket, and the head was 58cm away from the table, and video recording was performed. The background of the video was white. Stop after 6 minutes of timing, clean up each mouse after the test is completed and use 75% alcohol to remove the smell left by the last mouse. The immobility time of the mice for the last four minutes was counted using the small animal movement trajectory tracking system software. Depression can lead to increased immobility time of mice within 4min.

(3) Forced swimming test (FST): A transparent columnar bucket of 45cm×35cm×60cm was selected as the experimental material. During the experiment, the water temperature was kept at about 25°C, and the water depth should not be lower than 15cm, so as to prevent the rat’s toes from touching the bottom of the bucket. On the day before the experiment, each mouse swam for 15 minutes, and after 24 hours, measured the immobility time of the mice within 6 minutes and 4 minutes later. During each measurement, the water was changed and 75% alcohol was used to remove the odor left by the previous mouse. The immobility time of the mice for the last four minutes was counted using the small animal movement trajectory tracking system software.

(4) Open-field test (OFT): Use a 50cm×50cm×50cm square open carton, and use a white paint pen to divide the inner wall into 25 small squares of 10cm×10cm. Take pictures and time, and use 75% alcohol to clean the inner wall and bottom of the square box, so as not to affect the next test result due to the smell left by the animals last time. Observe the residence time of the mice in the central area and the times of crossing the central area within 15 minutes. The trajectory of the mice was analyzed with the small animal trajectory tracking system software.

(5) Sucrose preference test (SPT): 1% sucrose water was selected, and the specific method was as follows: on the first day, 2 bottles of 1% sucrose water were filled with 200 mL; on the second day, 1 bottle of 1% sucrose water was mixed with 1 Bottles of purified water, 200mL each; on the third day, no food and water; on the fourth day, one bottle of 1% sucrose water and one bottle of purified water, each 200mL; on the fifth day, measure the consumption of sugar water and purified water. Sugar water preference coefficient = sugar water consumption / (sugar water + pure water consumption) × 100%. The sugar water preference coefficient of the mice was calculated by measuring the amount of sugar water and purified water consumed on the 5th day, so as to compare the difference between the normal group and the model group.

(6) Nissl staining: Put animal tissue slices into Nissl staining solution for 5 minutes, wash with water, differentiate slightly with 1% glacial acetic acid, wash with tap water to terminate the reaction, control the degree of differentiation under a microscope, wash with tap water, put the slices in an oven Toast dry. Seal the slides transparently, put the slices into clean xylene for 5 minutes, and seal the slides with neutral gum. Microscopic examination, image acquisition and analysis. Scan with a digital pathology scanner for imaging analysis.

data analysis

SPSS16.0 was used to statistically analyze the experimental data, and GraphPad Prism 9.0 was used to conduct one-way analysis of variance (One-Way ANOVA) between groups, and the Tukey test was selected for pairwise comparison between multiple samples, and P<0.05 was considered to be statistically different. The results are expressed as Mean±SEM.

The results showed that after 14 days of modeling, the mice had symptoms such as body temperature drop, ptosis of the upper eyelid, lack of movement, and anhedonia. The determination of effectiveness; and the mice that continue to be fed for 14 days still continue to maintain the above symptoms (the test results are shown in Figure 1), indicating that the mice have spontaneously developed into chronic depression. Therefore, according to the construction method of the present invention, the mouse model of chronic depression can be successfully obtained, and after the mouse model of the present invention is successfully constructed, it can maintain a depressed state for a long time, which is more conducive to simulating the actual application of drug administration in the evaluation process. , so as to better screen for drugs.

Example 2

A drug screening method based on the chronic depression animal model of the present invention. Use candidate drugs to evaluate drug efficacy based on the model of the present invention, obtain corresponding evaluation indexes, and realize drug screening according to the indexes. Drug efficacy evaluation indicators include forced swimming immobility time, tail suspension immobility time, sucrose water consumption, open field test and neuron damage. The drugs were Vortioxetine and Fluoxetine.

(1) Comparative model, acute depression mouse model

The above-mentioned C57BL/6 mice after one week of adaptive feeding were randomly divided into 3 groups (8 mice in each group), control group: intraperitoneal administration of normal saline (Anhui Shuanghe Pharmaceutical Co. Xitine (purchased from Sigma Company) was administered by intraperitoneal injection at a dose of 10 mg/kg for 7 consecutive days; on the 8th day, the dose of reserpine 4 mg/kg was injected intraperitoneally once to the mice in the 3 groups, and 3 to 48 hours after the injection. The behavioral tests of TST, FST, SPT and OFT were completed within a period of time, and the behavioral evaluation was carried out. The results are shown in Figure 2.

It can be seen from the figure that in the tail suspension and forced swimming experiments, compared with the normal mice (Control), the immobility time of the control mice (Depression) was significantly increased, while the animals treated with vortioxetine and fluoxetine for 7 days The immobility time of the mice was significantly reduced (Fig. 2A-B). The results of the sugar water preference experiment showed that the consumption of sucrose water in the mice in the control group was significantly reduced, while vortioxetine and fluoxetine could significantly increase the sugar water preference of the mice (Fig. 2C). In the open field test, the activity time and movement distance in the central area of control mice were significantly reduced, while vortioxetine and fluoxetine significantly reversed these phenomena (Fig. 2D).

Nissl staining results showed that, compared with normal mice, the CA1 region of the hippocampus of the acutely depressed mice showed obvious neuron loss and the destruction of the hierarchical structure of pyramidal neurons, and the CA3 region showed an amorphous shape and was opaque Disorderly arrangement of cytoplasmic pyramidal neuron arrays and constricted nuclei in the hippocampus of depressed mice. Treatment with vortioxetine and fluoxetine significantly reduced depression-induced loss of pyramidal neurons and attenuated these pathological changes in the CA1 and CA3 regions of the hippocampus (Fig. 2E–G).

Therefore, judging from the existing mouse models of acute depression, both vortioxetine and fluoxetine have significant curative effects on depression. However, in the actual clinical application process, the effect of fluoxetine is not obvious, and the therapeutic effect is only produced after 4-9 weeks of continuous medication, and the side effects are relatively large, such as suicide risk, sexual dysfunction and cognitive impairment. However, vortioxetine can produce therapeutic effect within 1 week after taking the medicine, and its adverse reactions are significantly lower than that of fluoxetine.

(2) chronic depression mouse model of the present invention

The above-mentioned C57BL/6 mice after one week of adaptive feeding were randomly divided into 3 groups (8 in each group), and the chronic depression mouse model was established according to the steps of Example 1 respectively (the mice were intraperitoneally injected with reserpine 0.5mg/ kg, continuous injection for 14 days), stop injecting reserpine, and continue feeding for 7 days, the control group: give normal saline (Anhui Shuanghe Pharmaceutical Company) intraperitoneally every day; experimental group: vortioxetine and fluoxetine respectively The intraperitoneal injection of the dosage per kg was administered continuously for 7 days; TST, FST, SPT and OFT behavioral tests were performed on the second day after the administration, and the behavioral evaluation was performed. The results are shown in Figure 3.

It can be seen from the figure that in the tail suspension and forced swimming tests, the immobility time of the mice treated with vortioxetine for 7 days was similar to that of the normal mice, while the immobility time of the mice in the fluoxetine group and the control group increased significantly (Fig. 3A -B). The results of the sugar water preference experiment showed that compared with the normal mice, the consumption of sucrose water in the control group was significantly reduced, and both vortioxetine and fluoxetine could significantly increase the sugar water preference of the mice, and vortioxetine Ting works better (Fig. 3C). In the open field test, compared with normal mice, the activity time and movement distance in the central area of the mice in the control group were significantly reduced, and the vortioxetine group significantly reversed this phenomenon, while the fluoxetine group had no obvious reversal effect (Fig. 3D).

The above experimental results show that the general acute depression mouse model cannot accurately evaluate the drug efficacy, but the evaluation result of the chronic depression mouse model of the present invention is more accurate and closer to the actual clinical effect. It can be seen that the mice in the chronic depression mouse model of the present invention have spontaneously developed into chronic depression, which is closer to simulating and restoring the natural onset process of human depression, so it has more accurate drug efficacy evaluation and more accurate drug screening.

The Nissl staining test was performed on the mice after the experiment in (2) above, and the results are shown in FIG. 4 . The results showed that compared with normal mice, the CA1 region of the hippocampus of the mouse in the chronic depression mouse model of the present invention showed the destruction of the hierarchical structure of pyramidal neurons with obvious neuron loss, and the CA3 region showed a disorganization of the pyramidal neurons. Shape, disordered arrangement of pyramidal neuron arrays with opaque cytoplasm, and shrunk nuclei in the hippocampus of depressed mice. While treatment with vortioxetine significantly reduced depression-induced loss of pyramidal neurons and attenuated these pathological changes in the CA1 and CA3 regions of the hippocampus, fluoxetine did not (Fig. 4). Therefore, it can be considered that vortioxetine has a potential protective effect.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. An animal model of chronic depression is a model of inducing and inducing chronic spontaneous depression by long-term low-dose repeated reserpine, and is characterized by comprising the following steps: mice were intraperitoneally injected with low dose reserpine for more than 14 days to obtain an animal model of chronic depression.

2. The animal model of chronic depression according to claim 1, wherein: the low dose refers to the injected dose of 0.1-0.5mg/kg.

3. The animal model of chronic depression according to claim 1, wherein: the frequency of the injections was once a day.

4. The animal model of chronic depression according to claim 1, wherein: the mice are C57 mice.

5. A drug screening method based on the animal model of chronic depression according to any one of claims 1 to 4.

6. The drug screening method according to claim 5, comprising the steps of: the method is characterized in that after continuous administration of the medicine by intraperitoneal injection based on the animal model of the chronic depression as claimed in any one of claims 1-4, the medicine effect is evaluated, corresponding evaluation indexes are obtained, and the screening of the medicine is realized according to the indexes.

7. The method of claim 6, wherein: the duration of administration is 7 days or more.

8. The method of claim 6, wherein: the starting time for the drug effect evaluation is 2-14 days after the model establishment.

9. Use of the animal model of chronic depression according to any one of claims 1-4 for screening or identifying alternative drugs for alleviating or treating depression.

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