Method for inducing micro-awakening animal model by carbon dioxide

Abstract

The invention discloses a method for inducing a micro-awakening animal model by carbon dioxide, which comprises the following steps: S1, placing a test animal in a recording cage, and introducing air into the recording cage until the test animal enters a slow-wave sleep state; s2, after the test animal enters the slow-wave sleep state for 30 seconds, high-carbonic acid mixed gas is introduced for 30 seconds, and the test animal is slightly awakened; s3, converting and introducing air to reduce the concentration of carbon dioxide to a normal level; and S4, introducing the high-carbonic acid mixed gas every 5 minutes, repeating the steps S2-S3, and continuously monitoring the concentrations of O2 and CO2 in the cage. According to the method for inducing the micro-awakening animal model through the carbon dioxide, micro-awakening is induced in the sleeping process of a mouse through high-concentration CO2, and the influence of the high-concentration CO2 on the change of cortex EEG and hippocampus LFP in the slow-wave sleeping period is observed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of micro-awake animal model, and particularly relates to a method for inducing a micro-awake animal model by carbon dioxide. BACKGROUND

[0002] Sleep apnea refers to that airflow is repeatedly completely or incompletely stopped during sleep, causing intermittent hypoxia and CO2 increase (hypercapnia) of the body, and further stimulating the central nervous system to induce micro-awake to terminate the apnea.

[0003] At present, the research on intermittent hypoxia-induced micro-awake is more, and the research on the neural mechanism of CO2-induced micro-awake is relatively less. The brain circuit related to wakefulness is very complex, and recent researches show that the parabrachial nucleus (PB) is involved in the process of wakefulness, but the role of the parabrachial nucleus neurons in CO2-induced micro-awake has not been fully understood.

[0004] Based on the above understanding, the present application uses high-concentration CO2 to induce micro-awake of mice during sleep, so as to simulate the influence of hypercapnia on sleep in clinical sleep apnea disease. SUMMARY

[0005] In order to solve the above problems, the present application provides a method for inducing a micro-awake animal model by carbon dioxide, which induces a micro-awake mouse model by giving high-concentration CO2, and observes the influence of high-concentration CO2 on the changes of cortical EEG and hippocampal LFP during slow wave sleep.

[0006] In order to achieve the above purpose, the present application provides a method for inducing a micro-awake animal model by carbon dioxide, comprising the following steps: S1, placing a test animal in a recording cage, and introducing air into the recording cage until the test animal enters a slow wave sleep state; S2, after the test animal enters the slow wave sleep state for 30s, high-carbonate mixed gas is introduced for 30s, and the test animal appears micro-awake; S3, introducing air to reduce the concentration of carbon dioxide to a normal level; S4, the high-carbonate mixed gas is introduced every 5 minutes, and steps S2-S3 are repeated, and the concentrations of O2 and CO2 in the cage are continuously monitored.

[0007] Preferably, the high-carbonate mixed gas comprises 10% CO2, 21% O2 and 69% N2.

[0008] Preferably, the cortical EEG, hippocampal LFP and muscle electrical activity of the test animal are monitored to determine whether the test animal enters a micro-awake state.

[0009] Preferably, the monitoring method includes: using stereotactic technology, placing recording electrodes in the frontal cortex and hippocampus of mice to record cortical EEG and hippocampal LFP, and placing silver wire electrodes in the neck and back muscles to record electromyographic activity.

[0010] This invention provides a method for a carbon dioxide-induced microarousal animal model, which utilizes high concentrations of CO2 to induce microarousal during mouse sleep, and observes the effects of high concentrations of CO2 on changes in cortical EEG and hippocampal LFP during slow-wave sleep; the method of this invention can be applied to simulate the effects of hypercapnia on sleep in clinical sleep apnea.

[0011] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0012] Figure 1 To illustrate the effect of high CO2 concentration on microarousal during NREM sleep in mice, A is a representative image of high CO2-induced microarousal; B is a spectrum of power density in cortical EEG (top) and hippocampal LFP (bottom) as a function of CO2 level; C is a magnified view from the dashed box in A. Figure 2 The graphs show the power of different frequency bands in cortical EEG during high-concentration CO2-induced microarousing. A represents the influence of high-concentration CO2 on the power of different frequency bands in cortical EEG during microarousing; B shows the statistical changes in the power of cortical EEG δ during microarousing; C shows the statistical changes in the power of cortical EEG θ during microarousing; D shows the statistical changes in the power of cortical EEG α during microarousing; E1 shows the statistical changes in the power of cortical EEG β during microarousing. , , , ; Figure 3 The power of hippocampal LFP at various frequency bands during high-concentration CO2-induced micro-awakening is shown in Figure A. A representative image of the effect of high-concentration CO2 on the power of hippocampal LFP at various frequency bands during micro-awakening is shown. Figure B shows the statistical changes in power of hippocampal LFP δ during micro-awakening. Figure C shows the statistical changes in power of hippocampal LFP θ during micro-awakening. Figure D shows the statistical changes in power of hippocampal LFP α during micro-awakening. Figure E shows the statistical changes in power of hippocampal LFP β during micro-awakening. Figure F shows the statistical changes in power of hippocampal LFP γ during micro-awakening. Figure G shows the statistical changes in power of hippocampal LFP ripples during micro-awakening. , , , ; Figure 4The changes in power spectral density of each frequency band of cortical EEG and hippocampal LFP during high-concentration CO2-induced microarousing are shown in Figure 1. In Figure 2, A represents the change in power spectral density of each frequency band of cortical EEG during high-concentration CO2-induced microarousing, and B represents the change in power spectral density of each frequency band of hippocampal LFP during high-concentration CO2-induced microarousing. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the embodiments of the present invention and are not intended to limit the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout.

[0014] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0015] Similar labels and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0016] Example 1 A method for creating a carbon dioxide-induced microarousal animal model includes: using stereotactic techniques to place recording electrodes in the frontal cortex and hippocampus of mice to record cortical EEG and hippocampal LFP, and placing silver wire electrodes in the neck and back muscles to record electromyographic activity. High concentrations of CO2 are introduced during slow-wave sleep in mice to induce microarousal, and the power and power density of each frequency band of cortical EEG and hippocampal LFP are analyzed.

[0017] The mice were male C57BL / 6J mice, 8-10 weeks old and weighing 23-25g. The mice were housed in an environment with a constant temperature of 24±1℃, with free access to food and drink, and a light exposure time of 6:00-18:00. The mice were allowed one week to acclimatize to their environment before the experiment began.

[0018] (1) Electrode implantation surgery Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.4 mL / 100 g), and their heads were fixed to a stereotaxic apparatus. The skull was exposed, and insulated tungsten wire electrodes were positioned in the dorsal hippocampus (AP=-2.0 mm, ML=-1.5 mm, DV=-1.8 mm) to record local field potentials (LFP) in the hippocampus, referring to the Franklin and Paxinos mouse brain atlas. Grounded electrodes and cortical EEG electrodes were placed in the left and right prefrontal lobes, and a common EEG electrode (negative electrode) was placed in the parietal cortex. Two silver wire electrodes were inserted into the muscles on both sides of the neck and back of the mouse to record electromyography (EMG). All electrodes were connected to a recording cap, and dental cement was used to fix all electrodes and the cap. The mice were housed individually post-surgery and allowed to recover for 7 days.

[0019] (2) Signals collected and digitized using a pinnacle preamplifier system and an 8-channel PowerLab recording system were filtered to bandpass (cortical EEG: 0.1-30Hz; hippocampal LFP: 0.2-300Hz; EMG: 0.1-200Hz) and sampled at a rate of 1kHz.

[0020] The cortical EEG frequency bands were divided into: δ (1-4Hz), θ (4-8Hz), α (8-13Hz), and β (13-30Hz). The hippocampal LFP frequency bands were δ (1-4Hz), θ (4-8Hz), α (8-13Hz), β (13-30Hz), γ (30-80Hz), and ripples (80-200Hz). The EMG spectra were measured in 4-second time units, and the average power of each frequency band in the cortex and hippocampus was evaluated using Fast Fourier Transform (FFT).

[0021] (3) Mice that had recovered from head surgery were allowed to adapt to the recording system for 3 days. On the 4th day, they were placed in the recording cage at 9:00 AM. Between 12:00 PM and 4:00 PM, gas stimulation was administered to the mice after they entered slow-wave sleep for 2 hours. The specific procedures are as follows:

[0022] Air (21% O2, 79% N2) was continuously and slowly circulated into the cages, and the electroencephalogram (EEG) waveforms of the mice were observed. After the mice entered slow-wave sleep for 30 seconds, a hypercarbonated mixture (10% CO2, 21% O2, 69% N2) was introduced for 30 seconds, during which the mice exhibited micro-awakening. Then, air (21% O2, 79% N2) was continuously circulated to reduce the carbon dioxide concentration to normal levels. The hypercarbonated mixture was circulated at 5-minute intervals. Throughout the recording process, the concentrations of O2 and CO2 in the cages were continuously monitored.

[0023] The results are as follows Figure 1As shown, when mice enter stable slow-wave sleep, cortical EEG and hippocampal LFP are slow-wave with no electromyographic activity; this is consistent with the findings in In vivo cell type-specific CRISPR knockdown of dopamine betahydroxylase reduces locus coeruleus evoked wakefulness.

[0024] CO2 was then slowly introduced. When the CO2 concentration in the environment reached 6.81±2.38%, the cortical EEG and hippocampal LFP switched from slow waves to fast waves, followed by brief electromyographic activity, indicating the appearance of micro-awakening.

[0025] After reducing the carbon dioxide concentration to normal levels, cortical EEG and hippocampal LFP shifted from fast wave to slow wave, electromyographic activity disappeared, and the mice re-entered a slow wave sleep state. Figure 1 (A). This can be seen from the spectrum view ( Figure 1 During CO2-induced microarousing, the power density of cortical EEG and hippocampal LFP is concentrated in the low-frequency band (4-8Hz).

[0026] like Figure 2 As shown, the power of each frequency band of cortical EEG was analyzed. During high-concentration CO2-induced microarousal, the power of each frequency band of cortical EEG was significantly reduced. This result is consistent with the results in Beta2-containing nicotinic receptors contributing to the organization of sleep and regulating putative micro-arousals in mice.

[0027] Compared to pre-CO2 exposure levels, the power in the δ, θ, α, and β frequency bands was significantly reduced during high-concentration CO2-induced microarousing (δ: t=13.97, p <0.0001; θ: t=7.148, p=0.0011; α: t=9.195, p =0.0002; β: t=6.599, p =0.0018).

[0028] Compared to before micro-arousal, the power of the δ, θ, α, and β bands was significantly reduced during high-concentration CO2-induced micro-arousal (δ: t=7.079, p =0.0012; θ: t=4.408, p =0.0188; α: t=5.184, p =0.0077; β: t=6.162,p =0.0028).

[0029] Compared to post-CO2 exposure, during high-concentration CO2-induced microarousing, the δ, α, and β band powers were all significantly reduced (δ: t=6.014, p = 0.0032; α: t=6.059, p =0.0031; β: t=7.998, p =0.0005).

[0030] Analysis of the power of each frequency band in the hippocampal LFP revealed that during high-concentration CO2-induced microarousing, the power of the α, β, γ, and ripple bands of the hippocampal LFP decreased significantly, while the power of the δ and θ bands showed no significant change. The results are as follows: Figure 3 As shown.

[0031] Compared to pre-CO2 exposure levels, during high-concentration CO2-induced microarousing, the power of the α, β, γ, and ripple bands was significantly reduced (α: t=11.15, p <0.0001; β: t=17.42, p <0.0001; γ: t=6.750, p =0.0016; ripples: t=9.324, p =0.0002), the power in the δ and θ bands showed no significant change (δ: t=2.654, p =0.1965; θ: t=0.2878, p >0.9999).

[0032] Compared to before micro-arousal, the power of the α, β, and ripples bands was significantly reduced during high-concentration CO2-induced micro-arousal (α: t=5.149, p =0.0080; β: t=10.05, p =0.0001; ripples: t=10.20, p =0.0001), and the power in the δ and θ bands did not change significantly (δ: t=2.129, p =0.4245; θ: t=0.2166, p >0.9999).

[0033] Compared to post-CO2 exposure, the power of the α, β, γ, and ripple bands was significantly reduced during high-concentration CO2-induced microarousing (α: t=13.68, p <0.0001; β: t=13.31, p <0.0001; γ: t=9.536, p=0.0002; ripples: t=14.45, p <0.0001), the power in the δ and θ frequency bands showed no significant change (δ: t=2.865, p =0.1450; θ: t=1.334, p >0.9999).

[0034] The power spectral density of each frequency band in the cortical EEG and hippocampal LFP was analyzed, and the results are as follows: Figure 4 As shown.

[0035] Compared with pre-CO2 exposure, pre-micro-awakening, and post-CO2 exposure, during high-concentration CO2-induced micro-awakening, the power spectral density of the cortical δ, θ, α, and β bands decreased, and the power spectral density of the hippocampal α, β, γ, and ripple bands decreased, while the δ and θ bands showed no significant differences.

[0036] Therefore, this invention utilizes high concentrations of CO2 to simulate hypercapnia in clinical sleep apnea by inducing a mouse model of micro-arousal through administration of high concentrations of CO2.

[0037] During wakefulness, hippocampal theta oscillations are crucial for the encoding and subsequent consolidation of spatial memories during NREM sleep, suggesting that reduced theta power may lead to aberrant memory encoding.

[0038] Alpha and beta waves are the dominant wavebands in the waking state. Alpha oscillations are associated with wakefulness before falling asleep and with sleep after falling asleep; moreover, in the latter case, alpha oscillations are an expression of brain synchronization processes rather than a marker of sustained wakefulness. Alpha rhythms are generally considered to be related to cognitive functions such as attention and inhibitory control, suggesting that a decrease in their power may lead to cognitive impairment. Beta rhythms play an important role in maintaining attention to environmental stimuli, suggesting that a decrease in their power may impair the ability to perceive stimuli.

[0039] Changes in brainwave rhythms are strongly correlated with psychological effects. When the intensity of thought increases, the amplitude of the alpha rhythm typically tends to decrease; when the task requires more focused attention, the amplitude of the theta band in the frontal lobe tends to increase. The difficulty of the cognitive task is closely related to changes in the amplitude of the gamma rhythm, manifesting as an enhancement of the gamma rhythm in the cognitive state, suggesting that a decrease in gamma power may cause cognitive impairment. Hippocampal ripples may be coordinators of brain plasticity activation or reactivation, suggesting that a decrease in ripple power may affect brain plasticity activation. Changes in the power of the delta, theta, alpha, beta, gamma, and ripple frequency spectrum bands may, to some extent, affect learning and cognitive functions.

[0040] The power spectrum represents the signal power within a unit frequency band, indicating the distribution of signal power in the frequency domain. A higher power spectral density value indicates a larger power distribution within that frequency band. Our experiments show that during high-concentration CO2-induced micro-arousal, the power spectral density in the cortical δ, θ, α, and β bands all decreases, as do the power spectral densities in the hippocampal α, β, γ, and ripple bands. While the power spectral densities in the δ and θ bands show a decreasing trend, the differences are not significant, indicating that a smaller power spectral density value corresponds to a smaller power distribution.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for a carbon dioxide-induced microarousal animal model, characterized in that, Includes the following steps: S1. Place the experimental animal in a recording cage and introduce air into the recording cage until the experimental animal enters a slow-wave sleep state. S2. After the experimental animals entered a slow-wave sleep state for 30 seconds, a high-carbonic acid mixture was introduced for 30 seconds, and the experimental animals showed slight awakening. S3. Convert the air inlet to reduce the carbon dioxide concentration to a normal level; S4. The high carbonic acid mixed gas is ventilated at 5-minute intervals. Steps S2-S3 are repeated, and the concentrations of O2 and CO2 in the cage are continuously monitored.

2. The method for a carbon dioxide-induced microarousal animal model according to claim 1, characterized in that: The high-carbonate mixture consists of 10% CO2, 21% O2, and 69% N2.

3. The method for a carbon dioxide-induced microarousal animal model according to claim 1, characterized in that: Cortical EEG, hippocampal LFP, and electromyographic activity were monitored in the experimental animals to determine whether they had entered a state of micro-arousal.

4. The method for a carbon dioxide-induced microarousal animal model according to claim 3, characterized in that, The monitoring methods include: using stereotactic localization technology, placing recording electrodes in the frontal cortex and hippocampus of mice to record cortical EEG and hippocampal LFP, and placing silver wire electrodes in the neck and back muscles to record electromyographic activity.

5. The application of a method for creating a carbon dioxide-induced microarousal animal model as described in any one of claims 1 to 4 in simulating the effect of hypercapnia on sleep in clinical sleep apnea for non-medical purposes.

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