CN113331131A - Construction and application of anxiety and depression-like animal model after withdrawal from cocaine addiction - Google Patents
Construction and application of anxiety and depression-like animal model after withdrawal from cocaine addiction Download PDFInfo
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Abstract
The invention discloses a construction and application of an anxiety and depression animal model after withdrawal of cocaine addiction, wherein the method is used for assisting the typing method of the anxiety and depression animal model after withdrawal of cocaine addiction through an ROC curve. The method can accurately determine whether the mouse after cocaine addiction withdraws from shows anxiety-like behavior and/or depression-like behavior and carries out typing, and efficiently distinguish a single anxiety-like behavior cocaine addiction withdrawal model and an anxiety-like behavior cocaine addiction withdrawal model from a cocaine addiction withdrawal model of a mouse model after cocaine addiction withdrawal, so that the accuracy and the stability of subsequent experiments are effectively improved, and the method has extremely important significance for researching potential neurobiological mechanisms of the diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to construction and application of an anxiety and depression-like animal model after withdrawal of cocaine addiction.
Background
Drug addiction is a chronic recurrent brain disease which is mainly characterized by compulsive seeking and using of drugs and incapability of controlling drug administration, and the repeated intake of addictive drugs can induce physical dependence and mental dependence of individuals. Cocaine is widely used as an addictive drug, and unlike addictive substances such as heroin, alcohol and the like, cocaine induces physical dependence for a relatively short time, but anxiety and depression mood which occurs immediately thereafter is an important driving force for inducing cocaine addiction to patients who are abstinent from the drug.
In the related art, cocaine addicted patients have negative emotions such as anxiety, depression, anhedonia and the like during withdrawal, and symptoms often appear in highly diversified combinations. In related studies, those skilled in the art often regard animals with anxiety and depression-like behaviors after Cocaine Withdrawal (CW) as a homogenous population, group-compare the animals after Cocaine withdrawal with control animals (animals without Cocaine withdrawal), calculate anxiety and depression-like behavior detection by using statistical method, and determine whether the indexes have significant difference. However, in the related art, the construction method of the anxiety and depression-like animal model after withdrawal of cocaine addiction is not comprehensive, animals can only be simply divided into an anxiety/depression model group, different animals often have diversity on behavioral phenotypes, a small number of control group individuals can have anxiety/depression-like behavioral manifestations, and if the mice are also listed into a normal control group to be compared with cocaine withdrawal model-building mice in a behavioral manner, the accuracy of experimental results is greatly influenced, so that the experimental conclusion is not meaningful to reference. On the other hand, in combination with actual clinical data, about 50% of the population after withdrawal of cocaine-addicted patients show more or less mood-related mental symptoms, and severe cocaine-addicted patients show even major depressive and anhedonia symptoms (about 8%) after withdrawal, thus indicating that not all cocaine-addicted patients show anxiety/depressive symptoms after withdrawal. Therefore, the development of a mouse model construction method capable of accurately defining whether the withdrawal mouse shows anxiety-like and/or depression-like behaviors after cocaine addiction and a corresponding calculation mode thereof have extremely important significance for researching potential neurobiological mechanisms of the disease.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a typing method of an anxiety and depression-like animal model after withdrawal of cocaine addiction, which can accurately define whether a mouse after withdrawal of cocaine addiction shows anxiety-like and/or depression-like behaviors and carry out typing, thereby effectively improving the accuracy and stability of subsequent experiments and having very important significance for researching potential neurobiological mechanisms of the disease.
In a first aspect of the present invention, there is provided a method for typing an anxiety-depressive animal model after withdrawal from cocaine addiction, comprising the steps of:
(1) obtaining behavioural experimental data of a cocaine addiction withdrawal animal model;
(2) drawing an ROC curve according to the behavioristic experimental data;
(3) and (4) judging the phenotype of the cocaine addiction withdrawal animal model by utilizing the ROC curve.
According to a first aspect of the present invention, in some embodiments of the present invention, in step (3), the criteria for determining the phenotype of the cocaine addiction withdrawal animal model are:
calculating an optimal truncation value of a behavioural experiment and an area under an ROC curve according to the ROC curve;
when the area under the ROC curve is more than 0.8, if the behavior experiment index of the anxiety sample is less than the optimal cutoff value, the anxiety sample is judged to be positive, otherwise, the anxiety sample is judged to be negative;
if the behavior experiment index of the depressive sample is larger than the optimal cutoff value, the depressive sample is judged to be positive, and if not, the depressive sample is judged to be negative;
if the anxiety-like behavioral experiment is positive only, the cocaine addiction withdrawal animal model is of an anxiety-like phenotype;
if the anxiety-like and depression-like behavioral experiments are positive, the cocaine addiction withdrawal animal model is of an anxiety-like and depression-like behavior comorbid phenotype.
Among other things, in some embodiments of the present invention, the optimal cutoff value is the maximum value calculated by the john index.
In some embodiments of the present invention, the index of the anxiety-like behavioural experiment is open arm retention time in the elevated plus maze experiment, and when the open arm retention time is less than the optimal cutoff value, the result of the anxiety-like behavioural experiment is judged to be positive, otherwise, the result is judged to be negative.
In some embodiments of the invention, the indicator of the depressive-like ethology experiment is the immobility time in the tail suspension experiment and the forced swimming experiment, and the result of the depressive-like ethology experiment is judged to be positive when the immobility time is greater than the optimal cutoff value, and is negative otherwise.
If the anxiety-like behavioral experiment is positive only, the cocaine addiction withdrawal animal model is of an anxiety-like phenotype;
if the anxiety-like and depression-like behavioral experiments are positive, the cocaine addiction withdrawal animal model is of an anxiety-like and depression-like behavior comorbid phenotype.
According to the first aspect of the present invention, in some embodiments of the present invention, the behavioural test in step (1) comprises an anxiety-like behavioural test and a depression-like behavioural test.
In some preferred embodiments of the invention, the anxiety-like behavior test comprises an elevated maze test (EPM).
The elevated plus maze has a pair of open arms and a pair of closed arms, rodents tend to move in the closed arms due to darkness but move in the open arms due to curiosity and exploratory property, and when facing a novel stimulus, the animals simultaneously generate an exploratory impulse and fear, which cause conflicting exploration and avoidance behaviors, thereby generating anxiety psychology. In the embodiment of the invention, EPM is mainly used for detecting whether the constructed mouse model has anxiety behavior.
Of course, those skilled in the art can reasonably select other anxiety behavior experiments to replace according to actual use requirements.
In some preferred embodiments of the invention, the depressive-like behavior test comprises a tail-overhang test (TST) and a Forced Swim Test (FST).
In tail suspension experiments, animals struggle and struggle in a tail suspension environment, attempt to escape and cannot escape, so that a non-avoidable compression environment is provided, and after a period of experiments, the animals show a typical 'motionless state' and reflect a behavior despair state, which is similar to depression. In the present examples, TST was mainly used to evaluate depression-like behavior of the constructed mouse model.
Forced swimming, also known as a despair test, is another commonly used test for depression-like behavior in mice. In the present examples, FST was used primarily to further evaluate the depression-like behavior of the constructed mouse models.
Of course, other experiments of behavior like depression can be reasonably selected by those skilled in the art according to actual use requirements for replacement.
According to the first aspect of the present invention, in some embodiments of the present invention, the behavioral experiment in step (1) further comprises activity-based detection.
In some preferred embodiments of the invention, the activity basis detection comprises open field experimentation (OFT).
In the embodiment of the invention, the OFT is mainly used for detecting the basic activity condition of the mouse so as to remove the interference of other factors on result judgment.
According to a first aspect of the invention, in some embodiments of the invention, the animal model comprises a mouse model, a rat model, a monkey model.
Of course, those skilled in the art can reasonably select other animal models to apply the classification method in the embodiment of the present invention according to the actual use requirement.
In a second aspect of the invention, there is provided the use of a typing method according to the first aspect of the invention for the construction of an animal model.
In a third aspect of the invention, there is provided use of the typing method according to the first aspect of the invention in drug screening.
By the parting method, the anxiety and depression animal model after withdrawal of cocaine addiction can be better defined and divided, and different types of medicines can be screened according to the actual subtype, so that the screening result is more accurate and more scientific and effective.
According to a first aspect of the invention, in some embodiments of the invention, the medicament comprises an antidepressant, an anxiolytic and a medicament having both antidepressant and anxiolytic effects.
In a fourth aspect of the present invention, there is provided a cocaine addiction withdrawal anxiety and depression-like animal model obtained by the typing method described in the first aspect of the present invention, wherein the cocaine addiction withdrawal anxiety and depression-like animal model includes a cocaine addiction withdrawal model of simple anxiety-like behavior and a cocaine addiction withdrawal model of anxiety and depression-like behavior co-morbid cocaine addiction.
In a fifth aspect of the invention, the application of the animal model of depression and anxiety after withdrawal of cocaine addiction obtained by the typing method of the first aspect of the invention in drug screening is provided.
The invention has the beneficial effects that:
1. the method for parting the animal model of anxiety and depression after withdrawal of cocaine addiction can accurately determine whether a mouse which is withdrawn after cocaine addiction shows anxiety-like and/or depression-like behaviors and perform parting, thereby effectively improving the accuracy and stability of subsequent experiments and having very important significance for researching potential neurobiological mechanisms of the disease.
2. The model animal obtained by the method for classifying the anxiety and depression-like animal model after withdrawal of cocaine addiction can effectively improve the screening accuracy of the medicine and reduce the risk of unclear medicine effect caused by the unclear division of a single anxiety-like behavior cocaine addiction withdrawal model and an anxiety and depression-like behavior cocaine addiction withdrawal model.
Drawings
FIG. 1 is a timeline of an experimental procedure for constructing mice with 14-day AL and ADL behavioral phenotypes on cocaine withdrawal in an example of the present application;
FIG. 2 is a timeline of an experimental procedure for constructing mice with 28-day AL and ADL behavioral phenotypes on cocaine withdrawal in an example of the present application;
FIG. 3 is a graph showing comparison of OFT, EPM, FST and TST results of mice with AL and ADL behavioral phenotypes after cocaine withdrawal for 14 days in the present application example and a control group, wherein A is a graph showing comparison of EPM results, B is a graph showing comparison of TST results, C is a graph showing comparison of FST results, and D is a graph showing comparison of OFT results;
FIG. 4 is a graph showing comparison of OFT, EPM, FST and TST results of mice with AL and ADL behavioral phenotypes after cocaine withdrawal for 14 days in the present application and a control group, wherein A is a graph showing comparison of EPM results, B is a graph showing comparison of TST results, C is a graph showing comparison of FST results, and D is a graph showing comparison of OFT results.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Experimental Material
The experimental mice used in the examples of the present invention were all C57BL/6J male mice (purchased from southern medical university animal center) with a week age of about 8 weeks, and were maintained in the following conditions: keeping the temperature constant at 22-25 ℃, keeping the water and grain sufficient in the animal house with the circadian rhythm of 12h during the feeding period, and adopting the conventional mouse basic grain in the field for feeding the grain.
Construction of anxiety and depression-like mouse model after withdrawal from cocaine addiction
(1) Construction of 14-day cocaine addiction model:
the experimental mice are taken and injected with cocaine with the dose of 45mg/kg into the abdominal cavity of the mice every day (the injection is divided into 3 times every day, the injection is 15mg/kg every time, and the interval of the injection is 1 hour), and the injection is continuously carried out for 14 days, so that the mice achieve a continuous crazy drinking state.
Mice in the control group were injected daily with an equivalent dose of 0.9% physiological saline.
After 14 days of injection, the drug supply of the experimental group and the control group is stopped, cocaine withdrawal is carried out for 14 days, then open field experiments (OFT), elevated maze Experiments (EPM), forced swimming experiments (FST) and suspended tail ethology experiments (TST) are carried out on all experimental mice, EPM, FST and TST data are collected, ROC curves are drawn, and anxiety and depression sample (CWADL) phenotype mice after cocaine addiction withdrawal are screened out.
The experimental procedure is shown in FIG. 1 or 2.
(2) Construction of 28-day cocaine addiction model:
the method is the same as the step (1) and is characterized in that: in this example, the cocaine withdrawal time was extended to 28 days.
In the steps (1) and (2), the specific experimental operations OF the open field experiment (OF), the elevated maze Experiment (EPM), the forced swimming experiment (FST) and the tail suspension behavioural experiment (TST) are as follows:
(a) open field experiment (OFT):
OFT is mainly used for detecting basic activity of mice.
The specific experimental steps are as follows:
experimental mice were placed in the test chamber 1h before the start of the experiment, making them fully familiar with the environment. After the experiment, the experimental mouse is placed in the center of an open field device made of polyvinyl chloride material with the thickness of 50cm × 50cm × 40cm, and the total free movement distance of the experimental mouse within 10 minutes is recorded by the device. After the information collection was completed, the experimental mice were removed from the open field, the equipment was wiped with 75% alcohol, and after sufficient removal of residual odor, the mice were placed in the next round for testing.
(b) Elevated maze Experiment (EPM):
the elevated plus maze has a pair of open arms and a pair of closed arms, rodents tend to move in the closed arms due to darkness but move in the open arms due to curiosity and exploratory property, and when facing a novel stimulus, the animals simultaneously generate an exploratory impulse and fear, which cause conflicting exploration and avoidance behaviors, thereby generating anxiety psychology. In this example, EPM was used to test whether the constructed mouse model has anxiety-like behavior.
The specific experimental steps are as follows:
the test mouse was placed in the center of the elevated maze with the head facing the open/close arm (center area: 5X 5cm), and the time for freely searching the open arm (area: 29X 5cm) and the closed arm (area: 29X 5cm) within 10min was recorded.
The anxiety-like behavior of the mice was less exploratory than normal mice, and therefore, if the stay time of the experimental mice on the open/close state is significantly reduced, it can be said that the experimental mice have anxiety-like behavior.
(c) Tail suspension experiment (TST):
animals struggle in the tail suspension environment and attempt to escape and fail to escape, thus providing a non-avoidable stress environment, and after a period of experimentation, the animals will exhibit a typical "immobility state" reflecting a so-called "behavioral despair state", which is similar to depression. In this example, TST was used mainly to evaluate the depression-like behavior of the constructed mouse model.
The specific experimental steps are as follows:
the tail 1cm of the experimental mouse is fixed on a cross bar 40cm away from the ground, and the experimental mouse can not climb onto other objects in the experimental process. The immobility time of the experimental mice within 6min was recorded.
The longer the immobility time is, the more obvious the depression tendency of the experimental rat can be shown.
(d) Forced Swimming Test (FST):
forced swimming, also known as a despair test, is another commonly used test for depression-like behavior in mice. In this example, FST was used primarily to further evaluate the depression-like behavior of the constructed mouse model.
The specific experimental steps are as follows:
the experimental mice were placed in a glass drum (height 35cm, inner diameter 22cm) with water depth of 22cm and water temperature of 25 ℃ and the swimming conditions and immobility time of the experimental mice were recorded within 6 min.
In conventional practice in the art, mice tend to struggle vigorously during the initial phase of the FST test and, over time, switch to a more passive floating state. Since most mice were abnormally active 2min before testing, the experimental procedure only analyzed mice for immobility time 4min later.
(3) Screening CWADL model by using ROC curve
From the behavioral results, it can be seen that the individual behaviors of the cocaine addiction withdrawal group and the control group mice show significant differences, and after the withdrawal of cocaine addiction, not all individuals show this clinical manifestation of mood-related mental symptoms, making it difficult to correctly evaluate whether the cocaine withdrawal mice develop anxious-depressive-like behaviors. The prevalence rate of anxiety and depression of patients after withdrawal of cocaine addiction is only about 50% and the prevalence rate of anxiety and depression is about 60%. Thus, if only a simple one-way anova is statistically significant, it is difficult to find "positive" mice in the cocaine withdrawal group that truly have the CWADL phenotype.
To determine whether the particular behavioral phenotype detected in EPM, FST and TST was "positive" or "negative" for each mouse, ROC curves were introduced for additional evaluation. And calculating the Youden index (Youden index), the optimal cutoff value, the area AUC (area Under the ROC curve), and other related statistics according to the ROC curve. Jotan index, also known as correct index, is a method of assessing the authenticity of screening tests, indicating the overall ability of screening methods to find true patients and non-patients. The larger the index, the better the screening experiment and the greater the authenticity. The optimal cutoff value is the maximum value of the john's index, which is the optimal threshold for identifying patients and non-patients. The AUC can be obtained by summing the areas of all parts under the ROC curve, the area under the comparison curve is used as an index of the model, and the closer the AUC is to 1.0, the higher the authenticity of the detection method is.
And when the area under the ROC curve is more than 0.8, calculating the maximum value (the optimal cutoff value) according to the jotan index, and in the elevated plus maze experiment, if the open arm retention time is less than the optimal cutoff value, judging the open arm retention time to be positive, otherwise, judging the open arm retention time to be negative. In tail suspension experiment and forced swimming experiment, if the immobility time is larger than the optimal cut-off value, the result is judged to be positive, otherwise, the result is negative. If the anxiety-like behavioral experiment is positive only, the cocaine addiction withdrawal animal model is of an anxiety-like phenotype; if the anxiety-like and depression-like behavioral experiments are positive, the cocaine addiction withdrawal animal model is of an anxiety-like and depression-like behavior comorbid phenotype.
The results are shown in FIGS. 3 and 4.
In the mouse model with cocaine withdrawal for 14 and 28 days in the embodiment of the invention, AUC is greater than 0.8 in an ROC curve drawn by EPM, TST and FST three sets of ethological data, which indicates that the detection method is real and reliable. EPM, TST and FST in the day 14 withdrawal group were statistically different (P <0.05) from the control group, and TST and FST in the day 28 withdrawal group were statistically different (P < 0.05). Simply comparing whether the control group and the withdrawal group have statistical differences, the CWADL model mouse which is truly 'positive' is difficult to screen, and the optimal cutoff value (shown by a dotted line in the figure) is calculated according to the drawn ROC curve, so that whether the behaviors of the three groups of EPM, TST and FST are 'positive' can be judged. In the EPM experiment, the mice with arm-opening retention time less than the optimal critical value in the withdrawal group had anxiety-like behavior. In both the TST and FST experiments, mice in the withdrawal group with immobility times greater than the optimal threshold had depressive-like behavior. Furthermore, after this data processing method, it was found that the control mice had "positive" phenotype individuals in the EPM, TST and FST behavioral tests at different withdrawal time (14 days and 28 days), but the number of "positive" phenotype individuals was significantly less than that of "negative" phenotype individuals. The incidence rate of anxiety and depression of the cocaine withdrawal patients is extremely consistent with that of the clinical cocaine withdrawal patients, which shows that the model mouse fully simulates the behavior of anxiety and depression of the cocaine withdrawal patients.
The basic activities of two groups of mice at different withdrawal time are detected by open field experiments, and the results show that the basic activities of the control group and the mouse model are not different, which indicates that the TST and FST immobility time of the mouse model is prolonged and is not caused by the self activity difference. Further combining with ROC curve analysis, the mouse behavioral phenotype can be effectively further classified from a mouse model, a mouse which is only positive in EPM experiment is classified into a simple Anxiety-like behavior (AL) and a mouse which is all positive in EPM, TST and FST is classified into two subtypes of Anxiety-and depression-like behaviors (ADL), so that the experiment can be carried out by using a mouse with a specific phenotype according to the experimental requirements.
Wherein the experimental results are expressed as mean ± SEM. p <0.05 was considered to have significant differences.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for typing an anxiety and depression-like animal model after withdrawal from cocaine addiction comprises the following steps:
(1) obtaining behavioural experimental data of a cocaine addiction withdrawal animal model;
(2) drawing an ROC curve according to the behavioristic experimental data;
(3) and (4) judging the phenotype of the cocaine addiction withdrawal animal model by utilizing the ROC curve.
2. The typing method as claimed in claim 1, wherein in the step (3), the criteria for determining the phenotype of the cocaine addiction withdrawal animal model are:
calculating an optimal truncation value of a behavioural experiment and an area under an ROC curve according to the ROC curve;
when the area under the ROC curve is more than 0.8, if the behavior experiment index of the anxiety sample is less than the optimal cutoff value, the anxiety sample is judged to be positive, otherwise, the anxiety sample is judged to be negative;
if the behavior experiment index of the depressive sample is larger than the optimal cutoff value, the depressive sample is judged to be positive, and if not, the depressive sample is judged to be negative;
if the anxiety-like behavioral experiment is positive only, the cocaine addiction withdrawal animal model is of an anxiety-like phenotype;
if the anxiety-like and depression-like behavioral experiments are positive, the cocaine addiction withdrawal animal model is of an anxiety-like and depression-like behavior comorbid phenotype.
3. The typing method according to claim 1, wherein the behavioral tests in the step (1) include anxiety-like behavioral tests and depression-like behavioral tests;
wherein the anxiety-like behavior experiment comprises an elevated maze experiment;
the depression-like behavior test includes tail suspension test and forced swimming test.
4. The typing method according to claim 1, wherein the behavioral test in the step (1) further comprises an activity-based test, and the activity-based test comprises an open field test.
5. The typing method according to claim 1, wherein said animal model comprises mouse model, rat model, monkey model.
6. Use of the typing method according to any one of claims 1 to 5 for the construction of an animal model.
7. Use of the typing method according to any one of claims 1 to 5 in drug screening.
8. The use according to claim 7, wherein the drugs include antidepressants, anxiolytics and drugs with both antidepressant and anxiolytic effects.
9. The cocaine addiction withdrawal anxiety-depression-like animal model obtained by the typing method of any one of claims 1 to 5, wherein the cocaine addiction withdrawal anxiety-depression-like animal model comprises a single anxiety-like behavior cocaine addiction withdrawal model and an anxiety-depression-like behavior comorbid cocaine addiction withdrawal model.
10. Use of the animal model of depression-like anxiety after withdrawal from cocaine addiction obtained by the typing method of any one of claims 1 to 5 in drug screening.
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