Establishment method of animal model for double symptoms of depression accompanied by Parkinson's disease

Abstract

The invention provides a method for establishing a Parkinson-accompanied depression double-symptom animal model, which comprises the following steps of: 1, adaptively culturing an experimental animal, and fasting 24 hours before modeling; and 2, constructing a model by using MPTP injection in combination with chronic unpredictable temperature and stress. According to the modeling method, common modeling is constructed through MPTP and chronic unpredictable temperature stress, typical pathological characteristics of the Parkinson's disease can be simulated, and dyskinesia similar to the Parkinson's disease is induced, so that a two-dimensional evaluation system containing motion coordination and depression-like behaviors is established. Meanwhile, the model established by the method breaks through the limitation of single index research, and a multi-dimensional evaluation system is established for the first time aiming at the condition that the Parkinson's disease has the dual characteristics of dyskinesia and non-dyskinesia at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular to a method for establishing a Parkinson's disease and depression dual-symptom animal model. Background Art

[0002] Parkinson's disease (PD) is a complex neurodegenerative disorder whose clinical manifestations extend beyond motor dysfunction (such as bradykinesia, rigidity, tremor, and postural instability) and are often accompanied by significant non-motor symptoms, among which depressive symptoms are particularly common and seriously impact patients' quality of life. However, current preclinical research has significant limitations.

[0003] Existing rodent PD models mainly focus on simulating a single dimension of movement disorders, which is usually achieved by selectively damaging substantia nigra dopaminergic neurons with neurotoxins (such as MPTP), and evaluated through behavioral methods such as the rotarod test and gait analysis. Although such models can effectively replicate the core motor phenotype of PD, they generally ignore the simulation and systematic evaluation of key affective disorders such as depression. This single research dimension results in the model being unable to fully reflect the complex clinical pathological characteristics of PD patients, especially the reality of the coexistence of motor and non-motor symptoms, which limits researchers' understanding of the overall mechanism of the disease and the exploration of effective treatment strategies (especially for non-motor symptoms). Traditional behavioral paradigms used to assess depression (such as the sucrose preference test) lack standardization in the application of PD models, and their association with the specific pathological mechanisms of PD is insufficiently verified.

[0004] Therefore, there is an urgent need to develop a dual-symptom animal model that can simultaneously simulate PD motor dysfunction and depressive-like behavior, and to establish a multidimensional, standardized assessment system that integrates motor coordination and emotional state.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first purpose of the present invention is to provide a method for establishing an animal model of Parkinson's disease with depression. The method uses MPTP plus injection combined with chronic unpredictable mild stress to construct a joint model. This modeling method can simulate the typical pathological characteristics of Parkinson's disease and induce movement disorders similar to Parkinson's disease, thereby establishing a two-dimensional evaluation system including motor coordination and depressive-like behavior.

[0007] The second purpose of the present invention is to provide a model established by the above-mentioned method for establishing a dual-symptom animal model of Parkinson's disease with depression. This model breaks through the limitations of single-indicator research and for the first time establishes a multidimensional evaluation system for Parkinson's disease, which has the dual characteristics of motor dysfunction (forelimb grip strength, bradykinesia) and non-motor dysfunction (depressive-like behavior).

[0008] The third object of the present invention is to provide the application of the above-mentioned Parkinson's disease and depression bi-symptom animal model in drug screening or research on Parkinson's disease and depression comorbidity.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0010] The present invention provides a method for establishing an animal model of Parkinson's disease with depression, comprising the following steps:

[0011] Step 1: Adaptively cultivate experimental animals and fast for 22-26 hours before modeling;

[0012] Step 2: Use MPTP injection combined with chronic unpredictable mild stress to construct a model.

[0013] Preferably, the animals are fasted for 24 hours before modeling in step 1.

[0014] MPTP is fat-soluble and can penetrate the blood-brain barrier. It is metabolized into the toxic product MPP by monoamine oxidase B (MAO-B) in glial cells. + .MPP + It enters dopaminergic neurons through the dopamine transporter, inhibits mitochondrial complex I, blocks ATP synthesis and promotes the generation of reactive oxygen species (ROS), leading to acute neuronal necrosis. It can simulate the rapid dopaminergic neuron loss in PD, with the striatal dopamine content decreasing by 90% within a few days.

[0015] MPTP preferentially induces dopaminergic damage, and CUMS subsequently activates neuroinflammation (microglial Iba1↑, TNF-α↑). The dual pathways synergistically simulate the "motor-emotional comorbidity" mechanism of PD depression, solving the defect that a single model cannot simulate "motor-emotional comorbidity", thereby establishing a dual-symptom animal model of Parkinson's disease and depression.

[0016] Preferably, as a further specific implementation, the step 2 specifically includes the following steps:

[0017] MPTP was dissolved in sterile physiological saline to prepare a solution with a concentration of 1 mg / mL-4 mg / mL, and intraperitoneally injected into the experimental animals at a fixed time every day for 3-7 consecutive days;

[0018] A dynamic stimulation paradigm was then rotated, with 1-2 stressors randomly applied daily, and the experiment lasted for 20-25 complete day-night cycles.

[0019] In the modeling of chronic unpredictable mild stress, dynamic stimulation paradigm rotation is a key technical link for the success of the model. Its core lies in avoiding the animals' adaptation through randomized and non-repetitive stress stimulus combinations, thereby more realistically simulating the chronic stress environment of human depression.

[0020] Preferably, MPTP is prepared into a solution with a concentration of 3 mg / mL;

[0021] Preferably, the duration of continuous injection is 5 days;

[0022] Preferably, the experiment lasts for 21 complete day-night cycles.

[0023] Preferably, as a further specific embodiment, the intraperitoneal injection is any one of left intraperitoneal injection or right intraperitoneal injection;

[0024] After injection, gently massage the injection site and observe for 20-35 minutes to monitor for acute toxic reactions;

[0025] Preferably, the observation is continued for 30 minutes.

[0026] Preferably, as a further specific embodiment, the stimulus factor in the stress is any one or two of fasting, water deprivation, cage tilting, wet cage, strobe light, day and night reversal, restraint, intermittent lighting or noise.

[0027] Preferably, as a further specific embodiment, the decibel level of the noise is 85dB-90dB, and the duration is 30min-35min;

[0028] The intermittent lighting method is to alternate between light and dark every 1.5 hours to 2 hours;

[0029] The stroboscopic method is to use 1Hz-2Hz light flashes for 6h-6.5h;

[0030] The restraint method is to place the experimental animal in a fixator and restrain it for 2 hours to 4 hours.

[0031] Preferably, the intermittent lighting method is to alternate between light and dark every 2 hours;

[0032] Preferably, the noise level is 85dB and the duration is 30 minutes;

[0033] Preferably, the angle of the tilting cage is 45° to 50°;

[0034] Preferably, the stroboscopic method is to use a 2 Hz light flash for 6 hours;

[0035] Preferably, the day and night reversal is a 12-hour day and night reversal;

[0036] Preferably, the wet cage method is to pour water into the bedding of the experimental animals to keep the bedding moist.

[0037] In the present invention, the stimulation factors are completely random, and the time of applying the stimulation is not fixed every day. The purpose is to block the process of establishing the conditioned reflex of the subject to the stimulation timing, thereby effectively avoiding predictability.

[0038] Preferably, as a further specific embodiment, the specific steps of adaptively cultivating experimental animals are:

[0039] The experimental animals were raised at a temperature of 21° C.-23° C. and a humidity of 40% RH-60% RH for 7 days, and were weighed at regular intervals every day.

[0040] Preferably, as a further specific embodiment, the weight changes of the experimental animals need to be recorded daily during the modeling period, individuals with severe diarrhea or sudden weight loss are eliminated, and spare animals are added.

[0041] Preferably, as a further specific embodiment, the experimental animal is a rodent;

[0042] Preferably, the experimental animal is any one of rats or mice;

[0043] Preferably, the experimental animal is a rat.

[0044] The present invention also provides a Parkinson's disease and depression comorbidity model established by the method for establishing the above-mentioned Parkinson's disease and depression dual-symptom animal model.

[0045] The present invention also provides application of the Parkinson's disease and depression comorbidity model in drug screening or research on Parkinson's disease and depression comorbidity.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) By combining MPTP with chronic unpredictable mild stress, we can simulate the typical pathological characteristics of Parkinson's disease and induce movement disorders similar to those of Parkinson's disease, thereby establishing a two-dimensional evaluation system that includes motor coordination and depressive-like behavior.

[0048] (2) The model established broke through the limitations of single-indicator research and established a multidimensional evaluation system for the first time for Parkinson's disease, which has the dual characteristics of motor dysfunction (forelimb grip strength, bradykinesia) and non-motor dysfunction (depressive-like behavior). BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : This is the result diagram of the falling speed in the rotating rod experiment;

[0050] Figure 2 : The time on the rod in the rotarod experiment;

[0051] Figure 3 : Results of the sucrose preference experiment. DETAILED DESCRIPTION

[0052] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or the conditions recommended by the manufacturer. Those whose reagents or instruments are not specified by the manufacturer are conventional products that can be purchased commercially.

[0053] Example

[0054] MPTP was selected as the chemical drug to induce Parkinson's-like symptoms.

[0055] Before modeling, all animals were adaptively housed under standard conditions (temperature 22±1°C, humidity 50±10% RH, 12h day and night cycle) for 7 days. They were weighed regularly every day. Fasting was performed 24 hours before formal modeling to reduce gastrointestinal stress response. MPTP was dissolved in sterile saline, the concentration was 3 mg / mL, and stored at 4°C in the dark. Intraperitoneal injection was performed at a fixed time every day (09:00-11:00). The specific process included: the dosage volume was converted according to the dose of 30 mg / kg, and the injection was given into the lower left abdomen. After the injection, the injection site was gently massaged to promote absorption. The acute toxicity reaction (tremor, erect hair, etc.) was monitored for 30 minutes of continuous observation. The injection was continued for 5 days. During the modeling period, the weight changes were recorded daily. Individuals with severe diarrhea or sudden weight loss were eliminated and supplemented with spare animals.

[0056] After MPTP modeling is completed, a dynamic stimulation paradigm rotation is used according to the stressor parameter configuration scheme shown in Table 1 below to ensure that the daily stress mode has non-repetitive characteristics. The experimental period is set to three weeks (21 complete day-night cycles). Systematic stimulation exposure is implemented through a multimodal stressor combination intervention program. The randomized rotation strategy of the stress paradigm can effectively block the subject's conditioned reflex establishment process to the stimulation timing. Note that if two stimulations are performed on the same day, they are not performed simultaneously and the two stimulations are separated by more than 3 hours; the stimulation arrangement is completely randomized.

[0057] Table 1: Excitation source parameter configuration scheme

[0058]

[0059] In the present invention, the specific stress factors and dates used are shown in Table 2.

[0060] Table 2: Stress factors used in the present invention

[0061]

[0062] Experimental Example 1: Rotarod Test Verification

[0063] The rotarod test, also known as the fatigue rotarod test, is a behavioral test primarily used to assess motor coordination and balance in experimental animals. It is used to evaluate the potential effects of drugs or other treatments on motor function. This simple and reliable method is widely used in neuroethological studies of rodents, such as mice and rats.

[0064] Before each experiment, rats were placed in the experimental apparatus's laboratory environment for at least two hours to minimize stress caused by environmental changes. Before each experiment, the rotarod was wiped with 75% alcohol to remove residue from the previous rat and prevent interference from odors and other factors. Rats were placed on the rotarod apparatus and the speed was increased from 5 rpm to 40 rpm over a 120-second acceleration period. Rats were forced to walk forward to prevent falling. Training sessions were conducted three times daily for two consecutive days before the experiment. A standardized rodent motor function assessment system was developed, using a sensor array to monitor animal kinematic parameters in real time: the motor coordination latency measured the time from movement initiation to fall; the critical termination speed was the instantaneous rotarod speed. The experimental procedure consisted of five consecutive cycles of testing, with cubic spline interpolation used to eliminate outliers and then calculate the best three-way mean. The experimental system set a maximum observation time of 180 seconds. The test automatically terminated when the rodent's movement duration reached this threshold, and the experimental result was recorded as the maximum duration of 180 seconds. The animal's contact with the rotarod was observed in real time. The experiment was terminated when a persistent lack of progressive movement occurred, and the corresponding parameters before the cessation of progressive movement were recorded. Environmental parameters were maintained at a constant temperature of 23 ± 0.5°C and a background noise level of less than 45 dB to minimize uncertainty in the experimental results due to environmental changes.

[0065] The experimental results are as follows Figure 1 and Figure 2As shown, the primary purpose of the rotarod test is to assess motor function by measuring the time an animal maintains balance on an accelerating rotating rod or the speed at which it falls. Independent-sample t-tests were performed on the data from both groups, and both groups met the criteria for homogeneity of variance. The MPTP group's time on the rod (74.7±5.1s) was significantly shorter than that of the healthy control group (126.4±10.7s) (t=4.970, ***p<0.001). The falling speed in the MPTP group (25.8±1.5rpm) was significantly lower than that in the healthy control group (35.6±1.3rpm), with significant differences (t=4.264, ***p<0.001). These results indicate that MPTP impairs motor coordination in rats, a finding highly consistent with the characteristic bradykinesia and balance impairment of Parkinson's disease. The shortened time on the rod indicates impaired muscle coordination and motor learning in the model animals, consistent with motor dysfunction caused by dopaminergic neuron damage.

[0066] Experimental Example 2:

[0067] The sucrose preference test (SPT) is a classic method for assessing the anhedonia phenotype in rodent models of depression. Rodents have an innate chemotactic tendency toward sweet substances and exhibit a significant preference for sucrose solutions over pure water when exposed to them simultaneously.

[0068] The experimental paradigm is usually conducted using a two-bottle free-drinking apparatus, where the ratio of sucrose solution to pure water intake is measured as a quantitative indicator of anhedonia. When given free choice, healthy rats consume approximately 80% of their fluid intake from sucrose solution. The significant decrease in preference observed after animals experience pathological interventions such as chronic stress is believed to reflect impaired responsiveness of their reward system. This lack of pleasure is consistent with the core symptoms of depression. In a chronic unpredictable stress depression model, animals in the experimental group showed a statistically significant decrease in sucrose solution intake compared to the control group, a phenomenon interpreted as behavioral evidence of stress-induced anhedonia.

[0069] The sucrose preference experiment protocol is as follows. Day one is the acclimation period. Animals are acclimated to the experimental room, housed individually, and have free access to both water bottles and clean tap water. Days two and three are the training period. One bottle contains water, and the other contains a 1% sucrose solution. Day three swaps the water and 1% sucrose solution, allowing animals to freely consume either solution. Days four and five are the testing period. One bottle contains water, and the other contains a 1% sucrose solution. Animals have free access to either solution, with the water bottles swapped every 24 hours.

[0070] Weigh and record the water bottles before testing. After 24 hours, reposition the water bottles. After 48 hours, weigh and record the water bottles again. Calculate the daily consumption of water and 1% sucrose solution. Sucrose preference (%) = sucrose solution consumed / (sucrose solution consumed + drinking water consumed) × 100% to assess the symptoms of anhedonia in the CUMS model.

[0071] The experimental results are as follows Figure 3 As shown, sucrose preference in the MPTP group (75.0±2.5%) was significantly lower than in the healthy group (91.0±0.8%) (***p<0.001). This metric directly reflects dysfunction in the dopamine reward pathway, suggesting anhedonia, a core symptom of depression, in the model animals. Combined with the results of the rotarod test, the MPTP group exhibited both motor deficits and mood disturbances, successfully mimicking the clinical features of Parkinson's disease accompanied by depression.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a dual-symptom animal model of Parkinson's disease and depression, characterized in that: The steps include: Step 1: Adaptively cultivate experimental animals and fast for 22-26 hours before modeling; Step 2: Use MPTP injection combined with chronic unpredictable mild stress to construct a model.

2. The method for establishing a Parkinson's disease and depression dual symptom animal model according to claim 1, characterized in that: The step 2 specifically includes the following steps: MPTP was dissolved in sterile physiological saline to prepare a solution with a concentration of 1 mg / mL-4 mg / mL, and intraperitoneally injected into the experimental animals at a fixed time every day for 3-7 consecutive days; A dynamic stimulation paradigm was then rotated, with 1-2 stressors randomly applied daily, and the experiment lasted for 20-25 complete day-night cycles.

3. The method for establishing a bi-symptom animal model of Parkinson's disease and depression according to claim 2, characterized in that: The intraperitoneal injection is any one of left intraperitoneal injection or right intraperitoneal injection; After injection, gently massage the injection site and observe for 20-35 minutes to monitor acute toxic reactions.

4. The method for establishing a Parkinson's disease and depression bi-symptom animal model according to claim 2, characterized in that: The stimulating factors in the stress are any one or two of fasting, water deprivation, cage tilting, wet cage, strobe light, day and night reversal, restraint, intermittent lighting or noise.

5. The method for establishing a bi-symptom animal model of Parkinson's disease and depression according to claim 4, characterized in that: The decibel level of the noise is 85dB-90dB and the duration is 30min-35min; The intermittent lighting method is to alternate between light and dark every 1.5 hours to 2 hours; The stroboscopic method is to use 1Hz-2Hz light flashes for 6h-6.5h; The restraint method is to place the experimental animal in a fixator and restrain it for 2 hours to 4 hours.

6. The method for establishing a bi-symptom animal model of Parkinson's disease and depression according to claim 1, characterized in that: The specific steps of the adaptive training of experimental animals are: The experimental animals were raised at a temperature of 21° C.-23° C. and a humidity of 40% RH-60% RH for 7 days, and were weighed at regular intervals every day.

7. The method for establishing a bi-symptom animal model of Parkinson's disease and depression according to claim 1, characterized in that: During the modeling period, the weight changes of the experimental animals need to be recorded daily, and individuals with severe diarrhea or sudden weight loss should be eliminated and replaced with spare animals.

8. The method for establishing a bi-symptom animal model of Parkinson's disease and depression according to claim 1, characterized in that: The experimental animals are rodents; Preferably, the experimental animal is any one of rats or mice; Preferably, the experimental animal is a rat.

9. A Parkinson's disease and depression comorbidity model established by the method for establishing a Parkinson's disease and depression bi-symptom animal model according to any one of claims 1 to 8.

10. Use of the Parkinson's disease and depression comorbidity model according to claim 9 in drug screening or research on Parkinson's disease and depression comorbidity.

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