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| ORIGINAL ARTICLE |
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| Year : 2019 | Volume
: 3
| Issue : 2 | Page : 55-62 |
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Sleep electroencephalography power spectral response to transcutaneous auricular vagus nerve stimulation on insomnia rats
Man Luo1, Liang Li1, Jinling Zhang1, Xiao Guo1, Bin Zhao1, Shaoyuan Li1, Yong Yang2, Shiqin Liu2, Yu Wang1, Suxia Li3, Yue Jiao1, Yufeng Zhao4, Peijing Rong1
1 Department of Physiology, Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing, China
2 Institute of Biomedical Engineering, College of Automation, Hangzhou Dianzi University, Hangzhou, China
3 Department of Psychiatry, National Institute on Drug Dependence, Peking University, Haidian, China
4 National Data Center of Traditional Chinese Medicine, Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, China
| Date of Submission | 07-Sep-2019 |
| Date of Acceptance | 11-Oct-2019 |
| Date of Web Publication | 25-Nov-2019 |
Correspondence Address:
Prof. Peijing Rong
No. 16, Nanxiao Street, Dongzhimen Nei, Dongcheng District, Beijing
China
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/hm.hm_51_19
Background: Insomnia is a prevalent sleep disorder and strong risk factor for poor quality of life, depression, and other lifestylerelated diseases. Objectives: To investigate the effect of the transcutaneous vagus nerve stimulation (taVNS) on sleep electroencephalographic (EEG) in the para-chlorophenylalanine (PCPA) insomnia rats. Methods: Rats were divided into control, model, taVNS and sham taVNS (stnVNS) (stimulate the auricular margin, as transcutaneous none VNS, stnVNS) group (n=6 in each group). A week before the experiment, the electrodes were fixed to the skull of all the rats for recording the sleep EEG. PCPA was used to establish insomnia models. The rats of taVNS and stnVNS group were treated via an electroacupuncture apparatus for seven consecutive days, and simultaneously, the sleep EEG were recorded for all groups after the treatment daily. And the power spectrum analysis was used in this experiment. Results: After modeling, the percentage of power spectrum of delta frequency band significantly decreased, while the theta, alpha, and beta frequency bands significantly increased in the model group compared to the control group. After intervention, the percentage of the delta frequency band significantly increased in the taVNS group as compared to the stnVNS group. Conclusion: These results suggest that taVNS can significantly modulate the power spectrum of the delta frequency band and may constitute a potential low-cost alternative for the treatment of insomnia. Keywords: Auricular acupuncture, electroencephalography spectral analysis, insomnia, transcutaneous auricular vagus nerve stimulation, vagus nerve
How to cite this article:
Luo M, Li L, Zhang J, Guo X, Zhao B, Li S, Yang Y, Liu S, Wang Y, Li S, Jiao Y, Zhao Y, Rong P. Sleep electroencephalography power spectral response to transcutaneous auricular vagus nerve stimulation on insomnia rats. Heart Mind 2019;3:55-62 |
How to cite this URL:
Luo M, Li L, Zhang J, Guo X, Zhao B, Li S, Yang Y, Liu S, Wang Y, Li S, Jiao Y, Zhao Y, Rong P. Sleep electroencephalography power spectral response to transcutaneous auricular vagus nerve stimulation on insomnia rats. Heart Mind [serial online] 2019 [cited 2023 May 29];3:55-62. Available from: http://www.heartmindjournal.org/text.asp?2019/3/2/55/271533 |
| Introduction | |  |
Insomnia is a prevalent sleep disorder characterized by difficulties in falling asleep at bedtime, frequent waking at night, waking up too early in the morning, and insufficient sleep duration or quality.[1],[2],[3] Literature suggests that between 6% and 12% of the general population was diagnosed with insomnia.[4] In addition, insomnia is a strong risk factor for poor quality of life, depression, and other lifestyle-related diseases.[5] For example, studies indicate that insomniacs are two to four times more likely than those without insomnia to have a comorbid cardiovascular, chronic pain disorder, gastrointestinal, or respiratory.[6]
Pharmacological therapies such as benzodiazepines are widely used to increase sleep duration. Nevertheless, pharmacological treatments are frequently accompanied by side effects such as psychomotor impairment.[7] Other treatment modalities, such as cognitive behavior therapy[8] and physiotherapy (such as electric/magnetic therapy[9] or light therapeutic[10] and musicotherapy[11]), can be an effective and nondrug therapy for primary insomnia. However, in clinical practice, the number of physicians who have received specialized training is relatively small, and these therapies have not fully played their clinical roles or they are recommended to be combined with other physical therapy and drug therapy.[12],[13] Therefore, it is of great significance to seek a novel, safe, simple, practical, and economical alternative therapy for both patients and doctors.
Recently, transcutaneous auricular vagus nerve stimulation (taVNS) has drawn the attention of investigators. taVNS is inspired by vagus nerve stimulation (VNS) and based on the auricular theory of traditional Chinese medicine (TCM).[14] Meanwhile, as auriculotherapy, the benefits of taVNS are not only safety but also economy, and it also means that VNS has been developed from invasive implant to taVNS, which is a noninvasive[15] alternative therapy as compared with VNS.
In our previous studies, we found that taVNS can significantly reduce sleep disturbance in patients with depression[16] and increase sleep quality in patients with epilepsy.[17] In addition, clinical trials on auricular acupuncture have shown that stimulating at points on the ear with vagus nerve distribution can significantly improve sleep quality.[18] A systematic review based on the GRADE system[19] had evaluated that auricular acupuncture has favorable effects on insomniacs and no reports of adverse events were described in all studies. These results demonstrate the potential of taVNS in the treatment of insomnia.
As we all known, sleep is a spontaneous and reversible resting state of the higher vertebrate cycle, and it appears to be a temporary interruption of the body's responsiveness to external stimuli and consciousness.[20] Here, we tried to establish the insomnia model by intraperitoneal (i.p.) injection of para-chlorophenylalanine (PCPA). PCPA has been identified as new brain serotonin (5-HT)-specific consumer and has been found to inhibit sleep.[21],[22] When rats were treated with PCPA, rats' brain 5-HT content decreased to 10%–12% of normal.[23],[24] When the brain is deprived of 5-HT by PCPA, ascending systems that release histamine, noradrenaline, acetylcholine, and other neurotransmitters are able to maintain wakefulness.[25],[26] Experiments have shown that the percent of sleep-wakefulness began to change on the 3rd day after PCPA microinjection, and the effect was most significant on the 6th day. It can cause almost complete insomnia, which starts to return on the 7th day and returns to normal on the 9th day.[27],[28] In another experiment, Sprague–Dawley (SD) rats were given PCPA (300 mg/kg, i.p.) continuously for 2 days. On the 7th day after the treatment, 5-HT level recovered to a certain extent, but the recovery degree was relatively low. Therefore, it is reasonable to conclude that PCPA can induce insomnia.
Electroencephalography (EEG) is a widely applied tool to investigate the mechanism and treatment of sleep disorders. EEG can amplify the electrophysiological effects of brain activity,[29] which can be divided into different frequency bands according to different brain states, functions, or pathology. Delta frequency band, for example, is characteristic of the deep sleep phase. EEG signals are the most obvious and intuitive signals during sleep.[30] In 1938, Bailey and Bremer first reported that VNS could cause EEG changes. Studies have also shown that VNS has the function of regulating cerebral cortical electrical activity (delta quantity increased), directly or indirectly regulating sleep.[31] Thus, we tried to stimulate the auricular vagus nerve endings by an electroacupuncture apparatus, so as to observe the changes of brain electrical activity of SD rats before and after intervention and to provide further scientific basis for taVNS treatment of insomnia.
| Materials and Methods | | |
This study has been approved by the Institutional Ethics Committee of the China Academy of Chinese Medical Sciences. All experimental procedures comply with the guidelines of the “Principles of Laboratory Animal Care” (NIH publication number 80-23, revised 1996) and the legislation of the People's Republic of China for the use and care of laboratory animals (ethical approval number: 201403152).
Specific pathogen-free male SD rats weighing 200 ± 20 g were used in this study. The rats were purchased from the Chinese National Institutes for Food and Drug Control (Certificated No. SCXK [Jing] 2014-0013). The rats were divided into four groups (n = 6 for each group): control group, model group, taVNS group (treatment group), and stnVNS (sham transcutaneous non-VNS, stimulating the auricular margin, an area without vagus nerve distribution group). Animals were kept in ventilated glass cages at 24°C ± 2°C and humidity at 50% ±5%. Light/dark cycles of 12 h during which the lights were turned off at night to provide dark environment (19:00 to 7:00). Rats had access to standard rodent pellets and water adlibitum. Before the experiment, the rats were adapted to laboratory environment for 1 week.
Operations
All rats were anesthetized with 1% pentobarbital sodium (1 mL/100 g). The anesthetized rats were fixed on a stereotaxic apparatus for operation. Surgical steps included (1) exposing the skull; an incision about 4 cm in length was cut from the intersecting point of the rat's two eyes to the midline of the cranium; (2) drilling holes; three holes were drilled with a small cranial drill at the level of the fronto-occipital cortex as electrode positions, and the location of the holes were related to the bregma, lambda, and midline sutures.[32],[33] The hole for the reference electrode was at the right frontal cortex, and the holes for the recording electrode were at the left and right occipital cortex [Figure 1]; (3) burying electrode; three flat head stainless steel screws (diameter = 1.4 mm, thread length = 6 mm) with sockets were screwed into the skull on the drilled holes. The depth was measured by penetrating the skull to the duramater to record the EEG. All electrodes were fixed with dental cement (Shanghai New Century Dental Materials Co. Ltd.) on the skull. | Figure 1: Location of electrodes. The reference electrode was at the right frontal cortex (2.0 mm lateral to the midsagittal line, 1.5 mm anterior to the bregma), and the recording electrode was at the left and right occipital cortex (2.0 mm lateral to the midsagittal line, 3.5 mm posterior to the bregma)
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The two sets of electrical signal data were averaged when the power spectral analysis was conducted. 3 days after the operations, 40,000 units of benzylpenicillin sodium[34] for injection was administered by i.p. One week after surgical procedures, the sockets with the wire were connected to the Neural Signal Processor (Neurotechnology Systems, Inc., Sale Lake City, UT, USA), where the EEG was recording. To prevent wire entanglement, the commutators were used to allow the rats to move freely.
Animal model establishment
The PCPA (4-chloro-DL-phenylalanine, 98+%, CAS: 7424-00-2) was given to rats in the model, taVNS, and stnVNS groups. It was mixed with saline (pH 7.0–8.0) solution (45 mg/100 g, i.p.). To improve PCPA dissolution, Tween-80 was added.[35] The PCPA suspension was injected i.p. in each rat (1 mL/100 g) once a day for 2 consecutive days. The control group was injected with the same volume of saline for 2 days.
Transcutaneous auricular vagus nerve stimulation and sham transcutaneous auricular vagus nerve stimulation intervention
A homemade metal ear clip with plates was used for electrical stimulation [Figure 2]. The taVNS and stnVNS groups were anesthetized with 2% isoflurane inhalant (Hebei Nine Sent Pharmaceutical Co., Ltd., Hebei, China). Two opposite magnetic electrodes (+/−) were placed over both the inner and outer ear to form an electronic circle [Figure 2]. | Figure 2: Administration of transcutaneous auricular vagus nerve stimulation and sham transcutaneous auricular vagus nerve stimulation. The transcutaneous auricular vagus nerve stimulation positions are located in the area of auricular concha with auricular vagus nerve distribution. The sham transcutaneous auricular vagus nerve stimulation positions are located in the auricular margin with no auricular vagus nerve stimulation
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Stimulation was applied with an electroacupuncture apparatus (HANS-100, Nanjing Jisheng Medical Technology Co. Ltd.). The stimulus parameters were as follows: (1) stimulus frequency: 2/15 Hz (2 and 15 Hz, switched every second); (2) stimulus intensity: 2 mA; and (3) stimulus duration: 30 min. The stimulation was applied every morning between 08:00 and 09:00 for 7 consecutive days. These parameters have been proven effective by a previous study.[36]
Sleep electroencephalography acquisition and power spectral analysis
EEG data were collected using Neural Signal Processor (Neurotechnology Systems, Inc., Salt Lake City, UT, USA) between 09:00 and 16:00 for the 7 consecutive days. EEG data collection in taVNS and stnVNS groups started immediately after interventions every day in a shielding room that blocked unwanted electromagnetic signals from the outside.
EEG data were applied with MATLAB (2012b, MathWorks, Inc., American) for computing fast Fourier transforms.[37] When the rats entered the EEG acquisition room, they tended to be more active and interfere more with EEG signal collection. We thus removed the first 3 h of EEG signals. Then, the 100-s stable EEG segments (after removal of ocular artifacts) from the remaining 4 h were used for data analysis [Table 1]. Specifically, four 100-s stable EEG segments separated by about 1 h were selected [Table 2]. | Table 1: Electroencephalography data included in the power spectral analyses
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EEG data from 0.5 to 30 Hz were digitized at 128 samples/s and a sampling period of 100 s. Four frequency bands were then divided into the following frequency bands: delta (0.5–3.5 Hz), theta (3.5–7.5 Hz), alpha (7.5–13 Hz), and beta (13–30 Hz).
Open-field test
The open-field test (OFT)[38] on all rats were conducted the day after postoperative recovery (8 days after surgery), after model establishment (11 days after surgery), and posttreatment (8 days after treatment) [Table 2].
The open-field apparatus was constructed out of black plywood and measured 80 cm × 80 cm with 40-cm high walls. The lines divided the floor into 25 16 cm × 16 cm squares. A central square (16 cm × 16 cm) was drawn in the middle of the open field. A video camera was turned on for video recording from the top of the open-field apparatus, and the activity of the rats was observed for 3 min. Rats were placed on the central square. The squares crossing the bottom of the box were used to score horizontal movement, and the number of vertical movement (stand up) was used to score vertical movement. After the rats were taken out, the box was thoroughly sanitized with low concentration ethanol to avoid leaving a smell and disturbing the observation results of the next rat.
Statistical analysis
Statistical analysis was conducted by SPSS (Statistical Package for the Social Science) 20.0 statistical software package developed by IBM (International Business Machines Corporation). Results are shown as mean ± standard error. Statistical difference was measured using the repeated measures ANOVA (for consecutive days' comparison) with Sidak correction for post hoc analysis. The values were considered significantly different when P < 0.05.
| Results | | |
Postmodeling state of insomnia rats
28–30 h after the first i.p. injection, the PCPA rats showed symptoms such as tiredness, lack of energy, and unkempt fur without burnishing. They were also more vigilant and less sensitive to sound, light, and other stimuli, but more susceptible to irritability and aggression during the day compared with the control group. It is basically consistent with previous literature studies,[39],[40] which indicated that the models were successfully established.[41]
Open-field test
Repeated measures ANOVA on horizontal movement data showed no significant difference between control and model group (P = 0.06 > 0.05) but did reveal a significant difference between model and taVNS group (P = 0.02 < 0.05) and between model and stnVNS group (P = 0.03 < 0.05). There was no significant difference between taVNS and stnVNS group (P = 0.59 > 0.05).
Repeated measures ANOVA on vertical motion data showed similar results: control versus model group (P = 0.582 > 0.05), model versus taVNS group (P = 0.04 < 0.05), model versus stnVNS group (P = 0.048 < 0.05), and taVNS versus stnVNS group (P = 0.167 > 0.05)
Power spectral analysis of sleep electroencephalography
Percentage variation of power spectrum of frequency bands in control group and model group
We found that compared with the control group, the proportion of delta frequency bands in model rats significantly declined (delta: P < 0.001), while theta, alpha, and beta frequency bands significantly increased (theta: P < 0.001, alpha: P < 0.05, and beta: P < 0.001). These results suggest that the EEG power of the delta and theta frequency bands, which represent the cortex alert increased, thus indicating the success of the insomnia model.[31]
Delta power
The results of test of normality showed that P > 0.05, the data were normally distributed. Repeated measures ANOVA showed that compared with both the model (P = 0.009 < 0.05) and stnVNS (P = 0.009 < 0.05) group, the delta frequency band in the taVNS group increased significantly and tended to be stable from the 1st day to the 7th day. [Figure 3] shows that the increase began on day 2 and reached its peak on day 3. There is no significant difference between the model group and stnVNS group (P = 0.11 > 0.05) [Figure 3]. | Figure 3: Percentage changes of delta frequency band in groups. CG: Control group, MG: Model group, taVNS: Transcutaneous auricular vagus nerve stimulation group, stnVNS: Sham transcutaneous nonvagus nerve stimulation group
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Theta power
The results of test of normality showed that P > 0.05, the data were normally distributed. Repeated measures ANOVA showed a significant difference between the taVNS and model group (P = 0.022 < 0.05) but no significant difference between the stnVNS and model group (P = 0.916 > 0.05). [Figure 4] shows that compared to the model group, the theta power decrease in the taVNS group began on day 2 and reached its bottom on day 3, but there was no significant difference between the taVNS and stnVNS group (P = 0.18 > 0.05). | Figure 4: Percentage changes of theta frequency band in groups. CG: Control group, MG: Model group, taVNS: Transcutaneous auricular vagus nerve stimulation group, stnVNS: Sham transcutaneous nonvagus nerve stimulation group
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Alpha power
The results of test of normality showed that P > 0.05, the data were normally distributed. Repeated measures ANOVA showed no significant difference among the taVNS, stnVNS, and model group (P = 0.312 > 0.05) for taVNS versus model group (P = 0.728 > 0.05), for stnVNS versus model group (P = 0.267 > 0.05), and for taVNS versus stnVNS group (P = 0.156 > 0.05) [Figure 5]. | Figure 5: Percentage changes of alpha frequency band in groups. CG: Control group, MG: Model group, taVNS: Transcutaneous auricular vagus nerve stimulation group, stnVNS: Sham transcutaneous nonvagus nerve stimulation group
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Beta power
The results of test of normality showed that P > 0.05, the data were normally distributed. Repeated measures ANOVA showed no significant difference between the taVNS and model group (P = 0.124 > 0.05) or between stnVNS and model group (P = 0.832 > 0.05), but there was a trend between the taVNS and stnVNS group (P = 0.077 > 0.05) [Figure 6]. | Figure 6: Percentage changes of beta frequency band in groups. CG: Control group, MG: Model group. taVNS: Transcutaneous auricular vagus nerve stimulation group, stnVNS: Sham transcutaneous nonvagus nerve stimulation group
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| Discussion | | |
In this study, we investigated the regulation effect of taVNS with power spectrum analysis on EEG data collected after interventions. We found that taVNS can increase the proportion of the delta frequency band in insomnia rats, thereby demonstrating the potential of taVNS in the treatment of insomnia.
Sleep is characterized by synchronized oscillation in billions of synaptically coupled neurons in thalamocortical systems.[41] EEG activity can be quantified by calculating the power spectrum density at various frequencies and frequency bands (e.g., delta, theta, alpha, and beta) directly from the EEG signal.[42] Using power spectrum analysis, the percentage changes of each frequency band in EEG can also be obtained. Different frequency bands represent different pathophysiological significance, as delta spectrum increase is associated with the depth of sleep.[43],[44],[45],[46],[47],[48],[49] Theta, alpha, and beta frequency bands imply a heightened state of alertness, and activities of these frequency bands may serve as a marker of cortical arousal.[49] Thus, EEG spectrums may be a valuable tool in evaluating the changes of cortical hyperarousal for insomnia. It is generally believed that the delta frequency bands may represent relatively low cortical arousal (such as that which occurs during sleep), and high-frequency bands such as alpha and beta frequency band represent elevated cortical arousal.[49]
Compared with the control group, the power spectrum percentage of the delta frequency band decreased while percentage of theta, alpha, and beta frequency bands increased in the model group. These findings are consistent with other studies in which the authors found weakened delta and enhanced theta, alpha, and beta power in insomnia.[37],[43],[44],[50] These results indicate that the rats in the model group achieved an excited state in 7 days and may suggest that the insomnia models were built successfully.
Compared with the stnVNS group, taVNS can significantly increase the power spectrum percentage of the delta frequency band. In addition, results showed that taVNS achieved a better effect after 3 days of treatment (rats in the taVNS group were able to recover to 91.8% of the delta frequency band as indicated by control group level). Although the effect decreased from the 4th day to the 7th day, it stabilized at about 85% of the normal level on the 7th day. This indicates an improvement in sleep of the insomnia rats since the delta frequency band is associated with depth of sleep,[49] thus suggesting that taVNS can enhance the deep sleep in rats.
Our results are consistent with previous studies, which found that improved sleep quality can lead to changes in major brain waves, such as delta waves. For instance, Baoci et al.[47] tried to apply a weak current (4 Hz) to stimulate insomniacs on the Anming acupoint (an acupuncture point on the neck) and found that electrical stimulation could increase delta waves, and the deeper the sleep, the higher the proportion of the delta frequency band. Steriade[51] also found that the delta brain waves are the common nonrapid eye movement signs. Li and Liu[52] found that the delta wave of the insomnia rats was significantly increased and the beta wave was significantly decreased when stimulate on the Shenmen acupoint (HT7). Hallbook et al.[31] tracked insomnia patients treated with VNS and, at a 9-month follow-up, found that daytime sleepiness was improved, the incubation period of sleep was shortened, and the proportion of delta waves increased during slow-wave sleep and phase 2 sleep. Our findings are also consistent with previous clinical studies suggesting that taVNS can relieve symptoms of insomnia. For instance, Aihua et al.[17] found that taVNS can improve sleep quality at night, alleviate daytime sleepiness, and improve mood and quality of life in patients with epilepsy. These effects are all independent of the antiepileptic and antidepressant effects of taVNS, suggesting that the intervention might provide beneficial effects in the treatment of insomnia. In another study,[53] we also found that taVNS can safely and effectively reduce symptoms of insomnia patients.
According to the power spectrum change of delta, we assume that maybe the synchronous oscillation of the brain neuron is stimulated by taVNS, which makes the EEG signals of the delta power spectrum distribution density changed. In addition, there are now convincing mechanisms from some different approaches supporting the conclusion that VNS can improve insomnia. Recent studies[54],[55],[56],[57],[58] have shown that insomnia is closely related to the decline of central melatonin function, including the decrease of melatonin secretion and the down-regulation of melatonin receptor expression. There is no feedback regulation of melatonin secretion. The pineal secretion of melatonin is affected by the light or dark cycle and controlled by the autonomic nervous system: When the vagus nerve, which is mainly composed of parasympathetic nerve, is excited, it can promote the secretion of melatonin in the pineal gland. Sympathetic stimulation, however, has the opposite effect.[59],[60],[61],[62] The mutual promotion of parasympathetic vagus nerve excitation and melatonin secretion constitutes the basis of melatonin treatment for insomnia.[55],[57],[63],[64] taVNS can directly stimulate the afferent fibers of vagus nerve on auricular region. Moreover, studies have also shown that VNS or taVNS can increase the vagus nerve upward afferent impulse, the concentration of gamma-aminobutyric acid in the central nervous system, and decrease glutamate concentration, so as to improve the quality of sleep at night, relieve daytime sleepiness, and improve mood.[65]
Regarding the medical imaging, clinical trial[66] pointed out taVNS can improve sleep in poststroke insomnia (PSI) patient by BOLD-fMRI. The results indicated that taVNS has provided a potentially useful and safe new technique for PSI patients. The neural mechanism probably is due to the taVNS regulate the function connection of default mode network, visual cortex, and emotional circuit.
We did not find significant behavioral results between the taVNS and stnVNS in OFT. This may be due to (1) external noise in the experimental environment interfering with the behavior and activity of rats in the open-field box and (2) peritoneal injection of PCPA used in the modeling causing some ulceration in the abdomen of rats and affecting the activity of rats in the open-field box. These need to be improved in future research.
| Conclusion | | |
We found that taVNS can significantly increase the delta frequency band power spectrum percentage in insomnia rats. Our results suggest that taVNS may constitute a low-cost alternative treatment for insomnia.
Limitation
There are several limitations to this study. First, the EEG data were systematically sampled for 7 consecutive hours, and the power spectrum was subsequently analyzed on four 100-s segments. The consciousness of the rats was characterized by the percentage of each frequency band. Given that there was no way to verify that the rats were asleep during the EEG recording, the sleep stage EEG was not performed. Second, the sample size of this study was relatively small; thus, studies with larger sample sizes are needed to validate and expand upon these findings in the future.
Acknowledgment
This work was funded by the National Natural Science Foundation of China (81473780), Beijing Municipal Science and Technology Commission (Z161100002616003), Joint Sino-German Research Project (GZ1236), and Guangdong Province Higher Vocational Colleges and School Pearl River Scholar Funded Scheme (2016-A1-AFD018161Z0146). The authors also would like to thank Dr. Yang Yongsheng for his skilled technical assistance.
Ethical Statement
Permission was obtained to conduct this experiment from the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences. The experimental process and treatment of animal subjects strictly abided by the Guidelines for Ethical Review of Experimental Animal Welfare in Beijing. The certificate number is D2018-07-10-1.
Financial support and sponsorship
This work was funded by the National Natural Science Foundation of China (81473780), Beijing Municipal Science and Technology Commission (Z161100002616003), Joint Sino-German Research Project (GZ1236), and Guangdong Province Higher Vocational Colleges and School Pearl River Scholar Funded Scheme (2016-A1-AFD018161Z0146).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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