Obsessive-compulsive disorder animal model as well as construction method and application thereof

Abstract

The invention provides an obsessive-compulsive disorder animal model and a construction method and application thereof, and relates to the technical field of animal models. A construction method of an obsessive-compulsive disorder animal model comprises the following steps: S1, injecting a quantel solution into a rat according to a standard of 0.5 mg / kg, carrying out neck subcutaneous injection twice a week, and continuously injecting for 5 weeks; s2, detecting behavior characteristics of the rats after administration to obtain a basic animal model; and S3, implanting an electrode into the brain of the basic animal model to obtain a target animal model. According to the method, the neurotransmitter activity condition of the corresponding brain region under the disease state can be completely displayed, and an important foundation is laid for exploring the OCD pathogenesis. According to the animal model and the application thereof, the pathophysiological mechanism of OCD and the treatment mechanism of NAc-DBS can be explored from multiple aspects of animal behavioristics, neuroelectrophysiology, histopathology, molecular biology and the like, and a theoretical basis is laid for clarification of OCD pathogenesis and development of new treatment means.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal models, in particular, relates to an obsessive-compulsive disorder animal model, a construction method and application thereof. BACKGROUND

[0002] Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease, and its core symptoms mainly manifest as repeated obsessive ideas and (or) compulsive behaviors. The lifetime prevalence of OCD is about 1-3%, and about 30-40% of the overall patient population will develop into refractory OCD, and the effects of drug treatment and psychological treatment are not ideal. Therefore, developing effective treatment means for OCD patients is becoming increasingly important in today's society. Among them, in addition to conventional drug treatment and psychological treatment, deep brain stimulation (DBS) in physical treatment means opens up a new way for the treatment of OCD due to its precise, reversible and controllable technical characteristics.

[0003] The nucleus accumbens (NAc) is one of the most commonly used targets for DBS treatment of refractory OCD in clinical practice, and is considered to be the "interface" of the limbic system and the motor system, and plays an important role in the brain network related to motivation and reward processing. At present, the mainstream view believes that the dysfunction of NAc is closely related to various neuropsychiatric diseases. However, there are still many problems in the current NAc-DBS treatment of refractory OCD, especially the related pathophysiological mechanisms are not clear, mainly including neural electrophysiological activity, neurotransmitter activity, transcription and post-transcriptional regulation activity, etc.

[0004] A large number of scholars have tried to study ideal OCD animal models, however, like most mental illnesses, the brain parenchyma of OCD patients cannot be observed to have obvious changes in imaging and pathology, so the establishment of its model is much more difficult than that of tumor and other organic diseases. The diagnosis of OCD in clinical practice depends largely on the patient's narration of inner feelings and observation of compulsive behaviors. Animals have the innate defect of being unable to communicate with humans through language, so most OCD animal models are based on repetitive and stereotyped compulsive behaviors rather than obsessive ideas, and cannot fully display the neurotransmitter activity of the corresponding brain area in the disease state, and cannot clearly define the pathogenesis of OCD. Therefore, it is of great significance to establish a new OCD animal model. SUMMARY

[0005] The application aims to provide a construction method of an obsessive-compulsive disorder animal model, which takes a QNP-induced OCD rat model as an experimental object, stimulates the main nucleus NAc by using neuro-electrophysiological stimulation, can completely show the neurotransmitter activity of the corresponding brain area in the disease state, and lays an important foundation for exploring the pathogenesis of OCD.

[0006] Another purpose of the application is to provide an obsessive-compulsive disorder animal model and its application, which can explore the pathophysiological mechanism of OCD and the treatment mechanism of NAc-DBS from the aspects of animal behavior, neuro-electrophysiology, histopathology, molecular biology and the like, and lay a theoretical basis for clarifying the pathogenesis of OCD and developing new treatment methods.

[0007] The application is achieved by the following technical solutions: In a first aspect, the application relates to a construction method of an obsessive-compulsive disorder animal model, comprising the following steps: S1, injecting a rat with a QNP solution at a standard of 0.5 mg / kg, performing subcutaneous injection on the neck twice a week, and continuously injecting for 5 weeks; S2, detecting the behavior characteristics of the rat after administration to obtain a basic animal model; S3, implanting an electrode in the brain of the basic animal model to obtain a target animal model.

[0008] Further, the concentration of the QNP solution is 0.5 mg / mL. The preparation method of the QNP solution is as follows: weighing 10 mg of QNP powder stored in a-20℃ refrigerator, dissolving the QNP powder in 20 mL of physiological saline, avoiding light treatment after dispensing, and storing in a 4℃ refrigerator for short-term preservation.

[0009] Further, the operation of S3 is as follows: S31, anesthetizing the basic animal model, cutting the scalp to expose the skull and the anterior halo, opening the bone window with a burr, implanting the electrode into the target point of the brain of the basic animal model, and welding a silver wire on the skull screw; wherein, the electrode end is connected with the cable of the in-vivo multi-channel neuro-electrophysiological recording and stimulation system at the same time of implanting the electrode, and a reference electrode and a ground wire are also connected. When the electrode tip reaches the surface of the cerebral cortex, the in-vivo multi-channel neuro-electrophysiological system is turned on to measure the impedance value to ensure the integrity of the electrode, and then the electrode is slowly lowered to the area position of the target point. The signal state prompted by the electrophysiological system working interface needs to be observed during the lowering process to preliminarily judge the spatial structure passed by the electrode path, and the change of the impedance value is also checked. The target point can be the nucleus of the nucleus accumbens, the substantia nigra region or the striatum region.

[0010] S32, after the electrode is lowered to the region close to the target point, the gelatin sponge or agar gel is filled in the gap to close the opening and protect the brain tissue surface, the excess water on the skull surface is cleaned and dried by using a cotton swab, and then the electrode and the skull nail are fixed by using dental cement, the skull nail is completely covered, the tail end of the thin silver wire with a proper length is leaked as a reference electrode wire, and the fixation is completed after the dental cement is fully dried and firm.

[0011] S33, the animal model with stable postoperative function recovery is anesthetized, the microelectrode metal adapter on the head is connected with the extension cable of the in-vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, and the neuroelectrophysiological activity signal is recorded.

[0012] Further, in S33, after the microelectrode metal adapter on the head is connected with the extension cable of the in-vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, a stimulation program with a starting time length of 1h is started, the stimulation is performed once a day, the stimulation is continuously performed for 5 days, and the neuroelectrophysiological activity signal is recorded after the completion.

[0013] Further, the electrode is welded by the electrode wire and the metal adapter.

[0014] Further, the length of the electrode wire is 1.0-1.5cm, and the outer diameter of the metal adapter is 1mm. The length of the electrode wire is preferably 1.5cm.

[0015] Further, in S31, the length of the thin silver wire is 0.5-2cm. The length of the thin silver wire is preferably 1cm.

[0016] The second aspect relates to an obsessive-compulsive disorder animal model constructed by the construction method of the obsessive-compulsive disorder animal model.

[0017] The third aspect relates to the application of the obsessive-compulsive disorder animal model in screening drugs for treating obsessive-compulsive disorder diseases.

[0018] The fourth aspect relates to the application of the obsessive-compulsive disorder animal model in the research on the pathological mechanism of obsessive-compulsive disorder diseases.

[0019] The technical scheme of the present application has at least the following advantages and beneficial effects: For the target area NAc core part of mediating target-oriented behavior and obsessive-compulsive behavior adjustment control, the present application takes the QNP-induced OCD rat model as the experimental object, implants a microelectrode into the core part of NAc, and adopts NAc nerve electrophysiological stimulation to record the change of nerve electrophysiological signals. The firing mode and dynamics system of NAc neurons SPK in the OCD state have characteristic differences compared with the normal case, and can be restored to the normal direction after NAc-DBS intervention. Meanwhile, there are high-dimensional chaotic complex components in the LFP activity of NAc in the OCD state, and these pathological change trends can be alleviated to a certain extent under the intervention of NAc-DBS. Therefore, the animal model provided by the present application can explore the pathophysiological mechanism of OCD and the treatment mechanism of NAc-DBS from animal behavior, nerve electrophysiology, histopathology, molecular biology and the like, and can lay an important foundation and theoretical basis for clarifying the pathogenesis of OCD and developing new treatment methods. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The flow chart of the construction method of the obsessive-compulsive disorder animal model provided by the embodiment of the present application is shown in the figure. Figure 2 The motion trajectory comparison chart of the animal models of the control group and example 2 in the open field related to obsessive-compulsive behavior is shown in the figure. Figure 3 The 2D motion heat comparison chart of the animal models of the control group and example 2 in the open field related to obsessive-compulsive behavior is shown in the figure. Figure 4 The statistical result comparison chart of the number of times of returning home and the total motion distance of the animal models of the control group and example 2 is shown in the figure. Figure 5 The schematic diagram of implanting an electrode into the animal model of the embodiment of the present application is shown in the figure. Figure 6 The in-vivo multi-channel nerve electrophysiological recording chart of the animal model of example 1 of the present application is shown in the figure. Figure 7 The distribution chart of the overall and each wave band signal waveform of the local field potential of the animal models of the control group, example 1 and example 2 of the present application is shown in the figure. Figure 8Cross-frequency coupling co-modulation distribution diagram of animal models of the application control group, example 1, example 2, wherein A is the cross-frequency coupling co-modulation distribution diagram between the low-frequency phase (1-30 Hz) and the high-frequency amplitude (1-100 Hz) of the LFP signal of the nucleus accumbens of the Control (control) group, the Sham stimulation (sham stimulation-example 1) group and the NAc-DBS (example 2) group; B is the cross-frequency coupling co-modulation distribution diagram between the lower frequency phase (1-7 Hz) and the high-frequency amplitude (1-100 Hz) of the LFP signal of the nucleus accumbens of the Control (control) group, the Sham stimulation (sham stimulation-example 1) group and the NAc-DBS (example 2) group. DETAILED DESCRIPTION

[0021] The application will be further described below in conjunction with the specific embodiments, but the embodiments do not limit the application in any form. Unless otherwise specified, the raw materials used in the embodiments of the application are conventionally purchased raw materials.

[0022] Example 1 A method for constructing an obsessive-compulsive disorder animal model, comprising the following steps: S1, injecting rats with quinpirole solution at a standard of 0.5 mg / kg, performing neck subcutaneous injection twice a week, and continuously injecting for 5 weeks; S2, detecting the behavior characteristics of the rats after administration to obtain a basic animal model; S3, intervention treatment: S31, welding an electrode wire with a length of 1.5 cm and a metal adapter with an outer diameter of 1 mm; anesthetizing the basic animal model of S2, cutting the scalp to expose the skull and the anterior horn, opening the bone window with a drill, implanting the electrode into the target point (nucleus of the nucleus accumbens) of the brain of the basic animal model, and welding a silver wire with a length of 1 cm on the skull screw; S32, after the electrode is lowered to the area close to the target point (nucleus of the nucleus accumbens), filling the gap with gelatin sponge or agarose gel to close the opening and protect the surface of the brain tissue, cleaning and drying the excess water on the surface of the skull with a cotton swab, and then using dental cement to fix the electrode and the skull screw, and after the skull screw is completely covered, the tail end of the silver wire with an appropriate length is exposed as a reference electrode connection, and after the dental cement is fully dried and firm, the fixation is completed; S33, anesthetizing the animal model with stable postoperative function, connecting the microelectrode metal adapter on the top of the head with the extension cable of the in vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, obtaining the target animal model, and recording the neuroelectrophysiological activity signal of the target animal model.

[0023] Example 2 A method for constructing an obsessive-compulsive disorder animal model, comprising the following steps: S1, injecting rats with quinpirole solution at a standard of 0.5 mg / kg, performing subcutaneous injection in the neck twice a week, and continuously injecting for 5 weeks; S2, detecting the behavior characteristics of the rats after administration to obtain a basic animal model; S3, intervention treatment: S31, welding an electrode wire with a length of 1.0 cm and a metal adapter with an outer diameter of 1 mm; anesthetizing the basic animal model of S2, cutting the scalp to expose the skull and the anterior horn, opening the bone window with a burr, implanting the electrode into the target point (nucleus accumbens) of the brain of the basic animal model, and welding a silver wire with a length of 2 cm on the skull screw; S32, after the electrode is lowered to the area close to the target point (nucleus accumbens), filling the gap with gelatin sponge or agarose gel to close the opening and protect the surface of the brain tissue, cleaning and drying the excess water on the surface of the skull with a cotton swab, and then using dental cement to fix the electrode and the skull screw, and after the skull screw is completely covered, the tail end of the silver wire with an appropriate length is exposed as a reference electrode connection, and after the dental cement is fully dried and fixed, the fixation is completed; S33, after the animal model is stable after the operation, the metal adapter of the microelectrode on the top of the head is connected with the extension cable of the in vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, a stimulation program with a duration of 1 h is started, the stimulation is performed once a day, and the stimulation is continuously performed for 5 days to obtain a target animal model, and the neuroelectrophysiological activity signal is recorded after the completion.

[0024] Control group Injecting rats with physiological saline at a standard of 0.5 mg / kg, performing subcutaneous injection in the neck twice a week, and continuously injecting for 5 weeks.

[0025] Test example 1, behavior evaluation of mine site experiment The open field test (OFT) related to obsessive-compulsive behavior is used to simulate and evaluate the repetitive checking and other obsessive-compulsive behavior symptoms of OCD model rats.

[0026] Experimental Methods: A 1m×1m open field with a black background was selected and surrounded by transparent plexiglass panels. Four 8cm×8cm×8cm transparent plexiglass boxes with only one opening were placed in the open field. Two of the boxes were placed in the middle of the field, and the other two were placed in two diagonally opposite corners of the field. The camera device was located directly above the open field at a height of 160cm. The real-time data collected was transmitted to a dedicated workstation via a dedicated signal transmission cable for storage and analysis. The number of effective visits to the open field by the rats (the head and at least one forepaw entered the box, and the corresponding part remained in the box for at least 1 second) was recorded within a certain observation and recording time range (30 minutes). The main behavioral indicators of interest included: number of home visits, total movement distance, movement trajectory map, 2D movement heat map, etc.

[0027] 2. Elevated plus maze behavioral assessment An elevated plus-maze consisting of one open arm and one closed arm (with closed arm baffles 30 cm high) was used. Each arm was 50 cm long, 10 cm wide, and 80 cm above the ground. A camera was mounted directly above the elevated plus-maze, with a height difference of 130 cm. A dedicated signal transmission cable transmitted real-time data to a dedicated workstation for storage and analysis. Based on the biological characteristics of rodents preferring dark environments and their natural impulse to explore novel objects (at high altitude), a conflict between exploratory and avoidant behaviors was created, simulating a state of anxiety in rats. This behavioral experiment was designed with a test duration of 300 seconds. Key metrics of interest included the number of entries and dwell time in open arms 1, open arm 2, closed arm 1, and closed arm 2. The proportion of closed arm entries, the proportion of closed arm dwell time, movement trajectory maps, and 2D movement heat maps were calculated.

[0028] An open field test was conducted to compare the model group with the control group to determine whether the QNP-induced OCD rats showed a significant increase in the number of home visits and total distance traveled. An elevated plus maze was also used to determine the conflict between exploratory and avoidant behaviors between the two groups, thereby simulating a state of anxiety. After batch modeling and repeated behavioral assessments, these findings will serve as indicators of modeling success and symptom severity.

[0029] according to Figures 2-4 It can be seen that compared with the control group, the number of home returns and the total distance traveled by the rats in the model group increased significantly.

[0030] 3. Brain electrical stimulation electrode implantation test results according to Figures 5-6It can be known that after the modeling is successful, the prepared electrode is implanted into the target NAc by means of the stereotaxic instrument, and the impedance is tested in real time to ensure the integrity of the electrode, and the external amplification speaker is used to assist in reaching the target area, so that the state of the neuron discharge signal is judged by the sound characteristics, and when the neuron discharge interval is obvious, the characteristic similar to the firecracker sound can appear.

[0031] According to Figure 7 It can be known that LFP is the macroscopic sum of the postsynaptic potential, and 8-34 effective signals are recorded in three groups in the early experiment, different waveforms of different wave bands are filtered out by using a band-pass filter, and in the present study, 1-3 Hz is defined as the Delta band (delta wave), 3-7 Hz is defined as the Theta band (theta wave), 7-12 Hz is defined as the Alpha band (alpha wave), 12-30 Hz is defined as the Beta band (beta wave), and 30-80 Hz is defined as the Gamma band (gamma wave).

[0032] According to Figure 8 It can be known that the cross-frequency coupling mechanism of the electroencephalogram signal is considered to be able to play an important coordinating role in the time scale and the spatial scale on the intrinsic dynamic characteristics of the nervous system. In the pre-experiment, it is found that for the nucleus accumbens, most of the neurons in the neuron cluster oscillate and synchronize in a lower frequency range, and only a small part of the neurons present an active state in a higher frequency range. Therefore, the cross-frequency coupling model between the low-frequency phase and the high-frequency amplitude has strong applicability in the analysis of the local field potential of the nucleus accumbens.

[0033] In summary, there are differences in the signal distribution of each frequency band of LFP and SPK in the NAc area under the OCD state, and a certain degree of improvement is obtained after the NAc-DBS intervention, based on the tissue transparency technology, the information interaction between neurons can be presented, more theoretical basis is provided for elucidating the pathogenesis of OCD and the treatment mechanism of NAc-DBS, the possibility of DBS treatment of other mental diseases is explored, and a new idea is provided for the surgical treatment of mental diseases.

[0034] The above is only a preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an obsessive-compulsive disorder animal model, characterized in that: The following steps are involved: S1. Rats were injected with quinpirole solution at a standard of 0.5 mg / kg, subcutaneously in the neck twice a week for 5 consecutive weeks; S2. Detect the behavioral characteristics of rats after drug administration to obtain a basic animal model; S3. Implant electrodes into the brain of the basic animal model to obtain the target animal model.

2. The construction method according to claim 1, characterized in that The concentration of the quinpirole solution is 0.5 mg / mL.

3. The construction method according to claim 1, characterized in that The operation of S3 is as follows: S31. Anesthetize the basic animal model, cut the scalp to expose the skull and bregma, use a drill to open a bone window, implant electrodes into the target site in the brain of the basic animal model, and solder fine silver wires to the skull pins. S32. After the electrode is lowered to an area close to the target, fill the gap with gelatin sponge or agarose gel to seal the opening and protect the brain tissue surface. Use a cotton swab to clean and dry excess moisture from the skull surface, then use dental cement to secure the electrode to the skull pin. After completely covering the skull pin, leave an appropriate length of thin silver wire at the end as the reference electrode connection. Once the dental cement is fully dry and firm, the fixation is complete. S33. Anesthetize the animal model whose function has recovered stably after surgery, connect the metal adapter of the microelectrode on the top of the head to the extension cable of the in vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, and record the neuroelectrophysiological activity signals.

4. The construction method according to claim 3, characterized in that In S33, after connecting the microelectrode metal adapter on the top of the head to the extension cable of the in vivo multi-channel neuroelectrophysiological recording and stimulation system AlphaLab SNR, it also includes starting a stimulation program with a duration of 1 hour, stimulating once a day for 5 consecutive days, and recording the neuroelectrophysiological activity signals after completion.

5. The construction method according to claim 3, characterized in that The electrode is formed by welding an electrode wire and a metal adapter.

6. The construction method according to claim 3, characterized in that: The length of the electrode wire is 1.0-1.5 cm, and the outer diameter of the metal adapter is 1 mm.

7. The construction method according to claim 3, characterized in that: In S31, the length of the thin silver wire is 0.5-2 cm.

8. An animal model of obsessive-compulsive disorder, characterized in that: The obsessive-compulsive disorder animal model is constructed by the method for constructing the obsessive-compulsive disorder animal model according to any one of claims 1 to 7.

9. Use of the obsessive-compulsive disorder animal model according to claim 8 in screening drugs for treating obsessive-compulsive disorder.

10. Use of the obsessive-compulsive disorder animal model according to claim 8 in the study of the pathological mechanism of obsessive-compulsive disorder.

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