Construction method and application of PD sleep disorder mouse model based on ML brain region

Abstract

The invention discloses a construction method and application of a PD sleep disorder mouse model based on an ML brain region, belongs to the technical field of neuroscience, and discloses a potential neural mechanism of PD sleep disorder by deeply discussing the regulating effect of the ML brain region on PD model mouse sleep disorder through a multi-layer and multi-technology experimental design. As a key brain region for sleep-awakening regulation, the ML brain region may become a potential target spot for treating PD-related sleep disorders, and a new thought and theoretical basis are provided for future clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of neuroscience technology, and in particular to the construction method and application of a mouse model of PD sleep disorder based on the ML brain region. Background Technology

[0002] Sleep disorders are one of the early non-motor symptoms of Parkinson's disease (PD), mainly manifested as three types: sleep maintenance insomnia, rapid eye movement sleep behavior disorder, and excessive daytime sleepiness, which seriously affect the quality of life of PD patients.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disease, encompassing both motor and non-motor symptoms. Currently, PD is often diagnosed clinically after significant motor impairment has become apparent, at which point the disease is already in its middle to late stages. Early symptoms, such as sleep disturbances, are easily overlooked. However, the neural mechanisms regulating sleep disorders in PD are not fully understood.

[0004] This invention explores the regulatory role of the lateral nucleus of the medial mammillary body (ML) on sleep disorders in patients with Parkinson's disease (PD) from the perspective of brain functional state transition. This invention has the potential to reveal a novel neural mechanism regulating sleep-wake cycles and sleep disorders in PD patients, thereby opening up new theoretical understanding for exploring the mechanisms of sleep-wake cycles. Furthermore, it provides a new perspective for exploring the etiology, pathogenesis, and intervention measures of sleep disorders in clinical PD patients, aiming to provide new intervention methods for the clinical treatment of PD sleep disorders. Summary of the Invention

[0005] The present invention aims to provide a method for constructing and applying a mouse model of PD sleep disorder based on the ML brain region, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for constructing and applying a mouse model of PD sleep disorder based on the ML brain region, the specific steps of which include:

[0008] S1. Mouse selection: Select C57BL / 6 mice aged 12-16 weeks, weighing 20-25g;

[0009] S2. Stereoscopic injection of virus into the brain: 1 μL of virus was injected into the substantia nigra of the right midbrain of mice using stereoscopic injection. The injection rate was 100 nL / min and the injection time was 10 min. After the virus was completely injected, the needle was left in place for 8 min before the glass electrode was lifted.

[0010] S3. Use a scalp needle to suture the skin on the surface of the skull, and disinfect with povidone-iodine.

[0011] S4. Place the surgical mouse on a heating pad to allow it to recover until it is fully awake.

[0012] S5. Ten weeks after virus expression, animal behavioral tests such as cylinder experiments and pole climbing experiments were conducted to screen out mice that successfully developed the model.

[0013] S6. Take some mice for cardiac perfusion and brain extraction, and perform tyrosine hydroxylase (TH) immunofluorescence staining on the substantia nigra (SNc) and striatum (CPU).

[0014] Preferably, the application of a method for constructing a mouse model of PD sleep disorder based on the ML brain region includes its use in evaluating drugs for treating Parkinson's disease, in evaluating methods for treating Parkinson's disease, in evaluating compositions with potential therapeutic effects on Parkinson's disease, and in screening potential targets for treating PD-related sleep disorders.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, through a multi-level, multi-technology experimental design, delves into the regulatory role of the ML brain region on sleep disorders in PD model mice, revealing the potential neural mechanisms underlying PD sleep disorders. As a key brain region for sleep-wake regulation, the ML brain region may become a potential target for treating PD-related sleep disorders, providing new ideas and theoretical basis for future clinical treatment. Furthermore, the gene expression differences in the ML brain region revealed by this invention provide important clues for developing new drug targets. Attached Figure Description

[0017] Figure 1 A flowchart illustrating the steps involved in constructing a mouse model of PD sleep disorder based on the ML brain region;

[0018] Figure 2 A diagram showing the behavioral test results of PD mouse modeling;

[0019] Figure 3 A diagram showing the histopathological examination results of PD mouse model;

[0020] Figure 4 A graph showing the results of the dynamic changes in the 24-hour sleep-wake phase in PD mice;

[0021] Figure 5 A diagram showing the changes in sleep status after significant motor impairment appeared in PD mice;

[0022] Figure 6 A diagram illustrating the electrical activity characteristics of ML neurons in PD mice during the sleep-wake cycle;

[0023] Figure 7 The image shows the results of chemogenetic activation of the ML brain region to improve sleep in PD mice. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments:

[0025] like Figure 1 The method for constructing a PD sleep disorder mouse model based on the ML brain region, as shown, includes the following specific steps:

[0026] S1. Mouse selection: Select C57BL / 6 mice aged 12-16 weeks, weighing 20-25g;

[0027] S2. Stereoscopic injection of virus into the brain: 1 μL of virus was injected into the substantia nigra of the right midbrain of mice using stereoscopic injection. The injection rate was 100 nL / min and the injection time was 10 min. After the virus was completely injected, the needle was left in place for 8 min before the glass electrode was lifted.

[0028] S3. Use a scalp needle to suture the skin on the surface of the skull, and disinfect with povidone-iodine.

[0029] S4. Place the surgical mouse on a heating pad to allow it to recover until it is fully awake.

[0030] S5. Ten weeks after virus expression, animal behavioral tests such as cylinder experiments and pole climbing experiments were conducted to screen out mice that successfully developed the model.

[0031] S6. Take some mice for cardiac perfusion and brain extraction, and perform tyrosine hydroxylase (TH) immunofluorescence staining on the substantia nigra (SNc) and striatum (CPU).

[0032] Applications of the method for constructing a mouse model of PD sleep disorder based on the ML brain region include: applications in evaluating drugs for treating Parkinson's disease, applications in evaluating methods for treating Parkinson's disease, applications in evaluating compositions with potential therapeutic effects on Parkinson's disease, and applications in screening potential targets for treating PD-related sleep disorders.

[0033] The specific implementation process is as follows:

[0034] Test materials

[0035] Experimental subjects: SPF grade C57BL / 6J adult male mice, PD model mice and control mice, aged 12-16 weeks, and other experimental mice, aged 8-12 weeks, weighing 25-33g;

[0036] All mice used were housed in an SPF-grade animal facility and acclimatized to the environment for one week prior to behavioral testing. A circadian rhythm of 12-hour light (7:00 am to 7:00 pm) and 12-hour darkness was maintained, with an ambient temperature of 20 ± 2°C, relative humidity of 50%–60%, lighting of 15–20 Lx, and noise levels below 60 dB. No more than five mice were placed in each cage.

[0037] This invention uses stereotactic injection into the brain to construct a PD mouse model. Figure 2 A).

[0038] The specific method is as follows: inject rAAV-CMV-SYN-SNCA(A53T)-WPRE-BghpolyA virus into the unilateral substantia nigra pars compacta (SNc) of mice.

[0039] After 12 weeks of viral expression, the effectiveness of the model construction was evaluated through behavioral experiments:

[0040] (1) The cylinder test was used to detect asymmetry in the use of the forelimbs in mice in order to assess the motor asymmetry caused by unilateral substantia nigra damage. Figure 2 (DE);

[0041] (2) The pole climbing test was used to assess motor coordination and further verify PD-related motor dysfunction. Figure 2 ,BC).

[0042] The above behavioral experimental results show that mice injected with rAAV-CMV-SYN-SNCA(A53T) virus exhibit significant motor dysfunction, successfully mimicking the core behavioral characteristics of PD, and confirming the effective construction of the PD mouse model.

[0043] To verify the histopathological characteristics of the PD mouse model, the present invention involved perfusion of the heart in some mice to harvest the brain, and immunofluorescence staining of the substantia nigra (SNc) and striatum (CPU) with tyrosine hydroxylase (TH). The results showed (e.g.) Figure 3 (A) Immunofluorescence staining of TH in the substantia nigra pars compacta (SNc) of the midbrain in PD mice and control mice. (B) Statistical graph of the number of TH-positive neurons in the substantia nigra; one-way ANOVA, ***P<0.001, scale bar: 100μm. (C) Immunofluorescence staining of TH in the striatum (CPU) of PD mice and control mice. (D) Statistical graph of the mean fluorescence intensity of TH-positive neurons in the striatum; one-way ANOVA, ***P<0.001, scale bar: 100μm. The number of TH-positive neurons in the substantia nigra of the model group mice was significantly reduced (one-way ANOVA, ***P<0.001), and the mean fluorescence intensity of TH-positive neurons in the striatum was significantly reduced (one-way ANOVA, ***P<0.001).

[0044] The above results indicate that the model group mice showed significant loss of dopaminergic neurons in the substantia nigra and a significant reduction in striatal dopaminergic nerve fiber projections, successfully mimicking the typical pathological features of PD and further confirming the effectiveness of the PD mouse model.

[0045] Dynamic sleep monitoring in PD mice

[0046] To investigate the temporal characteristics of sleep disorders in PD mice, this invention conducted continuous sleep monitoring on PD model mice for 6 weeks. The 24-hour sleep-wake cycle was recorded weekly, and the results showed (e.g.) Figure 4 As shown), (AC) 24-hour sleep phase changes: (A) Total sleep duration, (B) Non-rapid eye movement (NREM) sleep duration, (C) Rapid eye movement (REM) sleep duration; (DF) Daytime (7:00-19:00) sleep phase changes: (D) Total sleep duration, (E) NREM sleep duration, (F) REM sleep duration; (GI) Nighttime (19:00-7:00) sleep phase changes: (G) Total sleep duration, (H) NREM sleep duration, (I) REM sleep duration. In summary, this experiment shows that: (1) Total sleep duration began to decrease significantly 6 weeks after viral injection; (2) Non-rapid eye movement (NREM) sleep duration was significantly reduced compared to the control group.

[0047] The above results indicate that sleep disorders in PD model mice develop progressively.

[0048] To investigate sleep changes in a Parkinson's disease (PD) model mouse, 24-hour sleep monitoring was performed on mice that exhibited significant motor impairment 12 weeks after viral injection. Compared with the control group, the PD model mice showed significant circadian rhythm disruption: significantly increased daytime (7:00-19:00) wakefulness duration (**P<0.05) and significantly decreased rapid eye movement (REM) sleep duration (***P<0.001).

[0049] The results show (e.g.) Figure 5(As shown), (AC) 24h Wake state phase changes: (A) Representative EEG and EMG graphs of mice in the Wake state. (B) Line graph of 24h Wake state phase changes. (C) Statistical bar chart of daytime and nighttime wake (wake) duration, unpaired t-test, **P<0.01. (DF) 24h NREM state phase changes. (D) Representative EEG and EMG graphs of mice in the NREM sleep state; (E) Line graph of 24h NREM sleep state phase changes; (F) Statistical bar chart of daytime and nighttime wake (NREM) duration, unpaired t-test, *P<0.05. (GI) 24h REM state phase changes. (G) Representative EEG and EMG diagrams of mice in REM sleep state; (H) Line graph of 24-hour REM sleep phase changes; (I) Histogram of REM sleep duration. Unpaired t-test, ***P<0.01. PD model mice exhibited significant daytime sleeplessness and REM sleep abnormalities simultaneously with the onset of motor disorders. Compared with the control group, PD mice showed a significantly increased number of transitions between different sleep states and a significantly increased number of awakenings.

[0050] Changes in the electrical activity of ML neurons in PD mice

[0051] To investigate the role of the ML brain region in sleep-wake regulation in PD mice, this invention performed in vivo multichannel recording and simultaneous EEG / EMG polysomnography on successfully modeled PD mice. A total of 252 neuronal activities were recorded. Compared with the control group, the results showed (e.g.) Figure 6(A) Schematic diagram of the experimental modeling sites and multi-channel recordings, EEG / EMG recordings in PD mice. (B) Schematic diagram of multi-channel electrode sites in PD mice. (C) Scatter plot of neuronal firing frequency distribution in the ML brain region of PD mice. (D) Proportion of neurons related to different states in the ML brain region of PD mice. Red represents highly active REM neurons, pink represents highly active REM-Wake neurons, green represents highly active Wake neurons, orange represents highly active NREM neurons, and gray represents neurons not involved in sleep-wake cycles. A total of 252 neurons were recorded. (E) Proportion of different types of neurons. Gray represents the control group, and purple represents the PD group. (F) Statistical graph of firing frequency of different types of neurons. In summary, this experiment showed that: (1) the proportion of highly active neurons during REM sleep was significantly increased (22.54% vs. 11.69% in the control group), while their average firing frequency did not change significantly (P>0.05); (2) the proportion of highly active neurons during Wake sleep was significantly decreased (6.15% vs. 10.39% in the control group), but their average firing frequency decreased significantly (P<0.01); (3) the proportion of highly active neurons during REM-Wake sleep did not change significantly (14.75% vs. 16.02% in the control group); (4) highly active neurons during NREM sleep accounted for 4.92%, which was higher than that in the control group, and their average firing frequency decreased significantly (P<0.05). Highly active neurons during NREM-REM sleep accounted for 1.64%, and the remaining 50% of neurons did not show obvious circadian rhythm correlation; (5) the average firing frequency of neurons in the ML brain region decreased significantly (P<0.001).

[0052] The above results indicate that the neuronal activity patterns in the ML brain region of PD model mice were significantly altered, with an increased proportion of REM sleep-related neurons and an inhibition of Wake and NREM-related neuronal activity, suggesting that the ML brain region may play an important role in PD-related sleep-wake regulation disorders.

[0053] The regulatory role of the ML brain region in sleep-wake cycles in PD mice

[0054] To verify the role of the ML brain region in sleep-wake regulation in PD mice, this invention uses a chemogenetic manipulation experiment. A non-cell-dependent chemogenetic inhibitory virus, rAAV-hSyn-hM3D(Gq)-EGFP-WPRE-hGHpolyA, is injected into the ML brain region of PD mice. Two weeks later, EEG / EMG electrodes are implanted in the top of the mouse skull, and EEG / EMG is recorded after another week of recovery.

[0055] This experiment employed a self-controlled pre- and post-controlled design to observe the sleep-wake phases in PD model mice. At 8:30 AM daily, mice were injected intraperitoneally with either saline or CNO (2 mg / kg). Sleep-wake cycles were recorded for 3 hours starting 30 minutes after injection. The first day, with saline as the control, and the second day, with CNO as the experimental group. To control for the influence of circadian rhythms, the injection and recording procedure was repeated daily at 7:00 PM. The effect of CNO on the sleep-wake cycle was assessed by comparing the total time of wakefulness (Wake), non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep before and after CNO administration.

[0056] Experimental results show (e.g.) Figure 7 (A) Flowchart of the chemogenetic activation manipulation of PD mice. (BE) Bar chart of the duration of different sleep-wake states in 3 hours of daytime activated PD mice. Gray represents the control group, and blue represents the experimental group. (B) Bar chart of total sleep duration in 3 hours, n=5. Paired t-test, *P<0.05. (E) Bar chart of total wakefulness duration in 3 hours. Paired t-test, *P>0.05. (FI) Bar chart of the duration of different sleep-wake states in 3 hours of nighttime activated PD mice. (F) Bar chart of total sleep duration in 3 hours. Paired t-test, P>0.05. (I) Bar chart of total wakefulness duration in 3 hours. Paired t-test, *P<0.05. Compared with the control group injected with physiological saline, the sleep-wake phase of mice in the CNO-treated group was significantly altered. Specifically, the total daytime sleep duration was significantly increased (P<0.05), while the wakefulness duration of mice in the CNO-treated group was significantly decreased (P<0.05). There was no significant difference in the duration of rapid eye movement (REM) sleep (P>0.05), and the wakefulness duration of mice in the CNO-treated group was also significantly decreased (P<0.05). The total nighttime sleep duration was significantly increased (P<0.05), with no significant difference in the duration of non-rapid eye movement (NREM) sleep (P>0.05) or rapid eye movement (REM) sleep (P>0.05). Meanwhile, the wakefulness duration of mice in the CNO-treated group was significantly decreased (P<0.05).

[0057] These results demonstrate that chemogenetic activation of ML neurons significantly improves sleep disorders, particularly NREM sleep, in PD mice while suppressing wakefulness. This further confirms the crucial role of the ML brain region in PD-related sleep-wake regulation, providing important experimental evidence for the neural regulatory mechanisms of PD sleep disorders.

[0058] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing and applying a mouse model of PD sleep disorder based on the ML brain region, characterized in that: The specific steps include: S1. Mouse selection: Select C57BL / 6 mice aged 12-16 weeks, weighing 20-25g; S2. Stereoscopic injection of virus into the brain: 1 μL of virus was injected into the substantia nigra of the right midbrain of mice using stereoscopic injection. The injection rate was 100 nL / min and the injection time was 10 min. After the virus was completely injected, the needle was left in place for 8 min before the glass electrode was lifted. S3. Use a scalp needle to suture the skin on the surface of the skull, and disinfect with povidone-iodine. S4. Place the surgical mouse on a heating pad to allow it to recover until it is fully awake. S5. Ten weeks after virus expression, animal behavioral tests such as cylinder experiments and pole climbing experiments were conducted to screen out mice that successfully developed the model. S6. Take some mice for cardiac perfusion and brain extraction, and perform tyrosine hydroxylase (TH) immunofluorescence staining on the substantia nigra (SNc) and striatum (CPU).

2. The application of the method for constructing a mouse model of PD sleep disorder based on the ML brain region according to claim 1, characterized in that: This includes applications in evaluating medicines for treating Parkinson's disease, applications in evaluating methods for treating Parkinson's disease, applications in evaluating compositions with potential therapeutic effects on Parkinson's disease, and applications in screening potential targets for treating PD-related sleep disorders.

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