The effect of moxibustion on brain functional connectivity and effective brain networks in patients with amnestic mild cognitive impairment: study protocol for a randomized controlled trial and preliminary results
Xin-Yan Jia, Xu Yuan, Xiao-Mei Zhou, Rui Jiao, Hong-Liang Xie, Dan Wang, Liang Yin, Ting-Ting Tan, Qi-Qi Liu, Shang-Jie Chen MD
Shenzhen Baoan People’s Hospital, Shenzhen, Guangdong Province, China
|Date of Web Publication||27-Aug-2018|
Shenzhen Baoan People’s Hospital, Shenzhen, Guangdong Province
Source of Support: This study was supported by the Science and Technology Research Project of Shenzhen, No. JCYJ20150402152005642 and the Science and Technology Research Project of Guangdong Province, No. 2016A020215202., Conflict of Interest: None
Background and objectives: Mild cognitive impairment (MCI) is an intermediate state between normal aging and dementia, and can be divided into amnestic and non-amnestic types. Patients with amnestic MCI present with memory impairments that are often considered as the early manifestation of Alzheimer’s disease. Patients with amnestic MCI are more likely to progress to Alzheimer’s disease than patients with non-amnestic MCI. The U.S. Food and Drug Administration has not yet approved any drug that can treat amnestic MCI. Moxibustion is a common noninvasive traditional oriental intervention, which uses mainly the heat generated by burning herbal preparations containing moxa and mugwort (Artemisia vulgaris) to simulate acupoints for alleviating the symptoms. To date, many clinical studies have investigated the clinical use of moxibustion to improve memory impairments of Alzheimer’s disease, but these have failed to make a distinction between amnestic and non-amnestic MCI. Therefore, this trial has been designed to assess the effectiveness of moxibustion on amnestic MCI using the Montreal Cognitive Assessment Scale. We will also assess the safety of moxibustion in healthy controls, and analyze the variation of brain functional connectivity and effective brain networks in patients with amnestic MCI undergoing moxibustion using function magnetic resonance imaging.
Design: This is a prospective, single-center, randomized controlled clinical trial.
Methods: This study will enroll 64 patients with amnestic MCI and 48 healthy controls at Baoan People’s Hospital, Shenzhen, China. The first 64 recruited patients with amnestic MCI will be randomly divided into moxibustion, placebo moxibustion, drug, and control groups (n = 16 per group). In the moxibustion group, patients will be given moxa-wool moxibustion for 12 consecutive weeks. The placebo moxibustion group will receive placebo moxibustion on the same acupoints. Patients in the drug group will be given oral administration of donepezil hydrochloride tablets, 5 mg daily, for 12 consecutive weeks. The control group will receive no intervention. Forty-eight healthy controls will also be randomly assigned into moxibustion, placebo moxibustion, and control groups (n = 16 per group). Interventions will be the same as those received by the patients with amnestic MCI. Evaluators will be blind to group allocation.
Outcome measures and preliminary results: The primary outcome measure will be the improvement in cognitive function 12 months after treatment. Secondary outcome measures will be the scores on the Montreal Cognitive Assessment Scale, Clinical Dementia Rating Scale, Mini-Mental State Examination Scale, and Activity of Daily Living Scale before treatment, after 12 weeks of treatment, and 6 months after the end of treatment, as well as brain function analysis before treatment and after 12 weeks of treatment and adverse events during treatment and follow-up. A correlation analysis between cognitive function scores and brain function results will be performed. Results of our preliminary study involving 60 patients with amnestic MCI who experienced moxibustion or received no treatment showed that moxibustion on acupoints significantly improved cognitive ability and quality of sleep in patients relative to the baseline and compared with the control group. Moreover, the scores on attention and delayed recall in the moxibustion group after treatment were significantly higher than those at base line. In the control group the scores on visual space, execution, and delayed recall were significantly lower than those at baseline. These findings indicate that moxibustion improves patient’s attention and delayed recall. If not, visual space, execution, and delayed recall in MCI patients tend to be declined over time. No obvious adverse responses to moxibustion treatment occurred in the preliminary study.
Discussion: This proposed trial has the potential to uncover that moxibustion will enhance cognitive-related brain function connections and effector brain networks, which is not yet known. If moxibustion is shown to be an effective and safe treatment strategy in patients with amnestic MCI, then this may pave the way for use of this treatment in clinic amnestic MCI.
Ethics and dissemination: This study was approved by the Medical Ethics Committee of the Chinese Clinical Trial Registry (approval No. ChiECRCT-2017018) in October 2016, and registered on April 2017. The study was designed in June 2016. Patient recruitment was initialized in October 2016. Data analysis will be completed in December 2019. Results will be disseminated through publication in a peer-reviewed journal. Protocol version: 1.0.
Trial registration: This trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-POC-17011162).
Keywords: amnestic mild cognitive impairment; moxibustion; Montreal Cognitive Assessment Scale; Clinical Dementia Rating Scale; Mini-Mental State Examination Scale; Activity of Daily Living Scale; donepezil hydrochloride; prospective, randomized controlled clinical trial
|How to cite this article:|
Jia XY, Yuan X, Zhou XM, Jiao R, Xie HL, Wang D, Yin L, Tan TT, Liu QQ, Chen SJ. The effect of moxibustion on brain functional connectivity and effective brain networks in patients with amnestic mild cognitive impairment: study protocol for a randomized controlled trial and preliminary results. Asia Pac J Clin Trials Nerv Syst Dis 2018;3:112-9
|How to cite this URL:|
Jia XY, Yuan X, Zhou XM, Jiao R, Xie HL, Wang D, Yin L, Tan TT, Liu QQ, Chen SJ. The effect of moxibustion on brain functional connectivity and effective brain networks in patients with amnestic mild cognitive impairment: study protocol for a randomized controlled trial and preliminary results. Asia Pac J Clin Trials Nerv Syst Dis [serial online] 2018 [cited 2019 Feb 18];3:112-9. Available from: http://www.actnjournal.com/text.asp?2018/3/3/112/238437
| Introduction|| |
Mild cognitive impairment (MCI) is a transitional state between normal aging and dementia. Patients with MCI present with mild cognitive impairment compared to the normal elderly, but have no deficits in the activities of daily living, MCI can be divided into amnestic and non-amnestic types. Amnestic MCI, mainly manifested by memory decline, is generally progressing to Alzheimer’s disease, while non-amnestic MCI is mostly progressing to vascular dementia, frontotemporal dementia, and Lewy bodies dementia. Therefore, amnestic MCI is often seen as an early stage of Alzheimer’s disease. Patients with amnestic MCI have a predominant memory impairment, which is often regarded as an early stage of Alzheimer’s disease. Existing studies have shown that patients with amnestic MCI tend to develop Alzheimer’s disease at a rate of 10–15% per year (Grundman et al., 2004). As of today, no drug to treat amnestic MCI has been approved by the U.S. Food and Drug Administration (Petersen et al., 2018).
Moxibustion is a traditional Chinese medicine therapy hat involves burning a moxa cone or stick (which is made of dried mugmort) on acupoints on the body. It has been found to reduce memory impairment in animals (Wang et al., 2015a, b; Jiang et al., 2016) and cognitive functioning in patients with Alzheimer’s disease (Liu et al. 2014; Jia et al., 2017), as shown in [Table 1]. Moxibustion is expected to be an effective intervention method for treating amnestic MCI, but there is no relevant evidence of mechanism research at present.
|Table 1: Past studies concerning moxibustion therapy for memory impairments in Alzheimer’s disease in animal experiments and cognitive impairment in clinical practice|
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Previous studies on cognitive impairment have not differentiated between the forms of MCI, investigations into the clinical use of moxibustion in patients with amnestic MCI is therefore warranted.
This trial will evaluate the effectiveness and safety of moxibustion to treat deficits associated with amnestic MCI and will provide objective evidence of brain function mechanism. We will use several cognitive assessment scales, including the Montreal Cognitive Assessment Scale (MoCA), and will analyze changes in brain functional connectivity and effective brain networks in patients with amnestic MCI using functional magnetic resonance imaging (fMRI).
| Methods/Design|| |
This is a prospective, single-center, randomized, controlled clinical trial.
The first 64 recruited patients with amnestic MCI will be randomly divided into moxibustion, placebo moxibustion, drug, and control groups (n = 16/group). In the moxibustion group, patients will be given moxa-wool moxibustion on the Baihui (DU20), Guanyuan (CV4), Zusanli (ST36), Xuanzhong (GB39), and Taixi (KI3) acupoints for 30 minutes at a time, five times a week, for 12 consecutive weeks. The placebo moxibustion group will receive placebo moxibustion on the same acupoints. Patients in the drug group will be administered with oral donepezil hydrochloride tablets, 5 mg daily, for 12 consecutive weeks. The control group will receive no intervention.
Forty-eight healthy controls will also be recruited, and be randomly assigned into moxibustion, placebo moxibustion, and control groups (n = 16/group). Interventions will be the same as those received by the corresponding groups of patients with amnestic MCI.
The primary outcome measure will be the improvement in cognitive function at 12 months after treatment as determined by the percentage of patients with an MoCA score ≥ 26 before treatment vs. 12 months after treatment. Secondary outcome measures will be scores on the MoCA, Clinical Dementia Rating Scale (CDR), Mini-Mental State Examination Scale (MMSE), and Activity of Daily Living Scale (ADL) before treatment, after 12 weeks of treatment, and 6 months after the end of treatment, as well as brain function analysis before treatment and after 12 weeks of treatment and adverse events during treatment. A correlation analysis between cognitive function scores and brain function results will be performed.
Flow chart of the trial is shown in [Figure 1] and timing for outcome measurement is shown in [Table 2].
Paper advertisement for recruitment information will be released through the bulletin board of the Shenzhen Baoan People’s Hospital, Shenzhen, China. On-site lectures and recruitment in local community will be conducted. Interested patients or healthy volunteers can contact the first author through their attending doctor or medical examiner, or via telephone, e-mail, or WeChat. Written informed consent will be obtained from all patients, participants, and participants who meet the inclusion criteria and do not meet the expulsion criteria will be selected.
Patients with amnestic MCI
We will enroll patients with amnestic MCI who receive treatment at the Department of Memory Disorder and Cognitive Rehabilitation Center at Shenzhen Baoan People’s Hospital.
Patients with amnestic MCI who meet all of the following criteria will be considered for study inclusion:
- Loss of subjective memory
- Scores on the Delayed Story Recall (DSR; Reger et al., 2008): Illiteracy ≤ 2 points, primary school education ≤ 4 points, junior high school education and above ≤ 8 points
- MoCA (Nasreddine et al., 2005) score < 26 points; MMSE (Forman et al., 1995) scores: Primary school education ≥ 21 points, junior high school education and above ≥ 24 points
- A CDR (Morris, 1993) score of 0.5 or a Global Deterioration Scale (GDS; Reisberg et al., 1988) score 2 or 3 points
- Unimpaired activities of daily living, as assessed by the ADL (Lawton and Brody, 1969)
- A Hamilton Anxiety Rating Scale (HAMA; Hamilton, 1959) score < 6 points
- A Hamilton Depression Rating Scale (HAMD) 21-Item scale (Hamilton, 1960) score < 8 points
- A Hachinski Ischemic Score (HIS; Hachinski et al., 1975) ≤ 4 points
- Aged 55–75 years, irrespective of sex
- Provision of written informed consent by patients and their families
Patients with amnestic MCI presenting with one or more of the following conditions will be excluded from the study:
- Any form of dementia
- Cognitive impairments caused by depression, frontal degeneration, epilepsy, encephalitis, Parkinson’s disease, or other neurological diseases
- Mental illness
- A history of medication that affects cognitive function
- Inability to complete the trial because of serious misrecognition, aphasia, apraxia, and visual, auditory, and consciousness disorders
- Incapable of completing or cooperating with neuropsychological testing
- Multiple stroke
Patients with amnestic MCI will be withdrawn from the study if they fulfill any of the following criteria:
- Patients who do not stick to their treatment or medication guide
- Incomplete data that affect the assessment of efficacy or safety
- During the trial, the development of comorbidities or complications that affect the assessment outcomes
- Patients who accept other therapies or medications without permission
Healthy controls that receive health examinations at Shenzhen Baoan People’s Hospital will be enrolled.
Participants who meet all of the following criteria will be considered for study inclusion: (1) Healthy, with no mental illness or neurological disease; (2) aged 55–75 years old, irrespective of sex; (3) provision of written informed consent by participants and their families.
Participants who fulfill any of the following criteria will be excluded from the trial: (1) Incapable of completing or cooperating with neuropsychological testing; (2) a history of medication that affects cognitive function.
Randomization and blinding
Forty-six patients with amnestic MCI will be randomized into moxibustion group, placebo moxibustion group, drug group, and control group. A random number will be selected from the random data table. Recruited patients will be then numbered randomly in the same direction beginning with this random number. Any random number given to the patients will be divided by 4, and the remainder is 0 for the moxibustion group, 1 for the placebo moxibustion group, 2 for the drug group, and 3 for the control group. The treatment regimen for each patient will be placed in a sealed, opaque envelope. When an eligible patient agrees to participate in the trial, the envelope will be opened and this patient will be given the corresponding treatment. The randomization allocation will be repeated in the healthy controls, who will be also randomly assigned into moxibustion, placebo moxibustion, and control groups (n = 16/group). Evaluators will be blind to grouping information.
Patients with amnestic MCI
In the moxibustion groups, participants will be given moxa-wool moxibustion on Baihui (DU20, located at the intersection between the midline of the head and the line between the tips of the two ears), Guanyuan (CV4, located at the anterior midline of the lower abdomen, 3 cun (1 cun = 1.31 inch) below the umbilicus), Zusanli (ST36, located at 3 cun below the knee joint), Xuanzhong (GB39, located at the lateral lower leg, 3 cun above the tip of lateral malleolus, and the anterior edge of tibia), and Taixi (KI3, located at the medial part of the foot, behind the medial malleolus, and at the recess formed by the medial malleolus and the Achilles tendon). The device for moxibustion is produced by Shenzhen Dr. Moxibustion Medical Science & Technology Co., Ltd., Shenzhen in China with license No. 20122179. Moxibustion will be performed for 30 minutes each time, five times a week. Specific operations are as follows: A participant will be asked to be in the supine position, relax, and uniformity of breathing and his/her abdomen skin and skin below the knees will be exposed. Moxibustion probes will be fixed at the Baihui, bilateral Guanyuan, bilateral Zusanli, bilateral Xuanzhong, and bilateral Taixi acupoints, respectively, and the moxibustion device will be started up. The temperature of each probe will be set to be moderate, and the time for moxibustion will be set to 30 minutes. After moxibustion, the probes will be removed [Figure 2].
|Figure 2: Moxibustion acupoints in the treatment of amnestic mild cognitive impairment.|
Note: (A–E) Baihui (DU20), Guanyuan (CV4), Zusanli (ST36), Xuanzhong (GB39), and Taixi (KI3).
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In the placebo moxibustion groups, placebo moxibustion on the same acupoints will be performed for 12 consecutive weeks. The device for placebo moxibustion is produced by Shenzhen Dr. Moxibustion Medical Science & Technology Co., Ltd. Special operations are same as those in the moxibustion group, except that the placebo moxibustion probe is at around room temperature.
In the drug group (patient group only), patients will be given oral donepezil hydrochloride tablets (SFDA approval No. H20050978), 5 mg daily, for 12 consecutive weeks. Oral administration will be stopped at 1 day prior to the MRI scanning. In the control groups, no intervention will be conducted.
Interventions of healthy controls will be the same as the corresponding groups of amnestic MCI patients.
Primary outcome measure
The primary outcome measure will be the improvement in cognitive function at 12 weeks after treatment, as determined by the percentage of patients with MoCA score ≥ 26 at 12 weeks after treatment. The MoCA scale assesses 8 cognitive domains, including attention and concentration, executive function, memory, language, visuospatial structure, abstract thinking, calculation, and orientation, with 11 testing items. MoCA scores range from 0 to 30, and a lower score indicates worse cognitive function (Nasreddine et al., 2005).
Secondary outcome measures
- The rate of improvement in cognitive function at 12 weeks of treatment vs. baseline and 6 months after treatment vs. baseline.
- The CDR scores at 12 weeks of treatment and 6 months after treatment.
- The MMSE scores at 12 weeks of treatment and 6 months after treatment.
- The ADL scores at 12 weeks of treatment and 6 months after treatment.
- Brain function analysis: functional connectivity and effective network analysis will be performed based on the MRI scanning results. Functional connectivity analysis can indicate the intensity of functional connectivity between brain regions, but cannot reflect the direction of information flow in brain regions. Effective network analysis cannot only reflect the intensity of brain connectivity but also the direction of information flow. The combination of these two methods can reflect the brain function mechanism comprehensively and truly. Brain function images will be acquired using a 3.0T MRI apparatus (Siemens, USA). Conformation before treatment and at 12 weeks of treatment: 3D BRAVO sequence will be used with the repetition time (TR) = 8.216 seconds, echo time (TE) = 3.22 ms, Flip-angle = 12°, field of view (FOV) =24 cm × 24 cm, 120 slices. 6-minute resting state head scan with gradient-recalled echo planar imaging (EPI) sequence: TR = 2 seconds, TE=30 ms, Flip-angle = 85°, FOV = 25.6 cm × 25.6 cm, Matrix = 64 × 64, Slice Thin = 6 mm, Slice Space = 6 mm, 20 slices.
- Adverse events: Any adverse events during the treatment, including burns, allergies, convulsions, nausea, vomiting, and diarrhea, will be recorded. After treatment, each patient will receive a physical examination and electrocardiogram. Adverse events will be discovered through spontaneous patient reports or direct observations by physicians. Each patient will be asked non-inductively about adverse events at each visit or when they are tested. During the trial, the adverse event will be accurately recorded in the adverse event list of a case report form Case Report Form including the occurrence time, severity, duration, measures taken and outcomes. Any adverse events that occur during the trial must be reported immediately to the Shenzhen Baoan People’s Hospitals well as to the Guangdong Provincial Drug Administration and the Department of Food Supervision of China Food and Drug Administration within 24 hours.
Based on the findings of Jia et al. (2017), the effectiveness of moxibustion and placebo moxibustion was estimated to be 70% and 50%, respectively. Taking a statistically significant level of α = 0.05 (two-sided) and a power of 1 – β = 0.90, the calculated sample size was 124 cases per group using PASS 11.0. Assuming a 20% dropout rate, the sample size was finally set as 149 cases. Sixty-four amnestic MCI cases (n = 16 per group) as the first batch will be included. Meanwhile, 48 healthy controls (n = 16 per group) will be recruited.
SPSS 23.0 (IBM, Armork, NY, USA) will be used for statistical analyses. Normally distributed measurement data will be expressed as the mean ± SD, while non-normally distributed data will be expressed as the lower quartile (q1), median, and upper quartile (q3). Enumeration data will be presented as a percentage. Repeated measurement analysis of variance will be use to compare scores on different cognitive scales and brain function results among groups. Intergroup comparison at the same visit time will be performed using a single factor analysis of variance and least significant difference test. The incidence of adverse event at the same visit time among the three groups will be compared using the Pearson’s X2 test or the Fisher’s exact test. Test level bilateral α = 0.05. A value of bilateral α = 0.05 will be set.
Full analysis set: The analysis will be used in all patients/healthy controls randomized according to the intention-to-treat principle.
Per protocol set: The analysis will be restricted to the subjects randomized except for those who discontinue the treatment or seriously violate the protocol at least once.
Intention-to-treat analysis will be performed in the trial.
Data collection and management
All data will be recorded by trained clinical researchers in a case report form (CRF). To ensure the accuracy and reliability of the data, supervisors will verify and cross-check the CRF data based on the original records. Missing data or specific errors in the CRF will be detected and sent to the investigator for resolution. All electronic data will be stored confidentially and only be accessed through a password. Anonymized trial data will be publicly available at www.figshare.com.
During the trial, regular audits by SJC and RJ responsible for quality control will be performed to ensure that the trial follows the protocol and complies with the relevant regulations, as well as that the information is recorded correctly. The researchers involved in the trial will carefully implement the standard operating procedures of clinical trials before, during, and after the trial. During the trial, inspectors responsible for quality control, assigned by the principal investigator, will audit the trial records for the correctness and completeness of the CRF data. These researchers must receive professional training and follow uniform data recording methods and evaluation standards. The CRF data will be recorded truthfully, detailedly and carefully to ensure the reality and reliability of the CRF. Patients included in this trial will receive a transportation allowance and have their appointment and follow-up examination fees waived. The researchers will provide insurance coverage for all subjects, cover the costs of treatment, and provide financial compensation for subjects suffering damage or death associated with the trial.
Ethics and dissemination
This trial was approved by the Medical Ethics Committee of the Chinese Clinical Trial Registry (approval No. ChiECRCT-2017018). The implementation of the trial will follow the relevant laws and regulations of the Declaration of Helsinki, as well as the relevant ethical medical principles in China. The entire trial process will be supervised by the ethics committee. All subjects will voluntarily participate in the trial and provide written informed content prior to its commencement. This manuscript was prepared and modified according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines (Additional file 1 [Additional file 1]). Dissemination plans include presentations at scientific conferences and peer-reviewed journals. Anonymized trial data will be published at www.figshare.com.
| Results|| |
Grouping and treatments
Sixty patients with MCI enrolled in the preliminary study from October 2015 to April 2017, and were randomly divided into a moxibustion group and a control group (n = 30 per group). Patients in the two groups were comparable as there were no significant differences in sex, age, or education between the two groups [Table 3].
|Table 3: Information about patients in the preliminary study (Jia et al., 2017)|
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Post-treatment MoCA scores
After treatment, the MoCA scores in the moxibustion group were significantly increased compared to baseline, and significantly lower than those of the control group. The scores on attention and delayed story recall in the moxibustion group were significantly higher than those at baseline (P < 0.05). After treatment, the scores on visual space, execution, and delayed recall in the control group were significantly lower than those at baseline (P < 0.05), and were also significantly lower than those in the moxibustion group (P < 0.05; [Table 4].
|Table 4: Cognitive function of patients with amnestic mild cognitive impairment before and after treatment|
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Post-treatment assessment of sleep quality
After two treatment sessions, the moxibustion group had significantly lower scores on the Epworth Sleepiness Scale (ESS), Insomnia Severity Index scale (ISI), and Pittsburgh Sleep Quality Index (PSQI) relative to the baseline (P < 0.05) and compared with the control group (P < 0.05; [Table 5].
|Table 5: Sleep quality of patients with amnestic mild cognitive impairment before and after treatment|
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Adverse events during the treatment
After No obvious adverse responses to moxibustion treatment occurred in the preliminary study.
| Trial Status|| |
This study was designed in June 2016, approved by the Medical Ethics Committee of the Chinese Clinical Trial Registry in October 2016, and registered in April 2017. Patient recruitment was initialized in October 2016. Data analysis will be completed in December 2019. Recruitment is ongoing at the time of submission.
| Discussion|| |
To date, there have been many studies concerning the use of moxibustion to improve memory impairments of Alzheimer’s disease in both animal models (Wang et al., 2015a, b; Jiang et al., 2016) and in clinical practice (Wang et al., 2015a, b; Jiang et al., 2016). However, there is still a lack of clinical studies on the improvement of cognitive function by moxibustion compared to donepezil hydrochloride, which is commonly used to treat mild cognitive impairment. Thus, this trial will use donepezil hydrochloride as a positive control and placebo moxibustion treatment as a negative control to evaluate the efficacy of moxibustion in the treatment of amnestic MCI, as well as to validate the safety of moxibustion in patients with amnestic MCI through a comparative study with healthy controls.
The prevention and treatment of amnestic MCI requires long time persistence, Moxibustion is a kind of green and healthy treatment method and leads to few adverse events. Participants are more likely to use moxibustion for a long period of time and have higher compliance than acupuncture or drugs. Moreover, why can moxibustion improve the cognitive function of amnestic MCI, and what is the mechanism of its effect? Brain function magnetic resonance imaging will allow us to observe what such mechanisms might be.
Limitations of this study
There is a small sample size.
Significance of this study
Findings from this trial will offer objective evidence for the treatment of amnestic MCI with moxibustion.
Additional file 1: SPIRIT checklist.
We wishes to thank the Shenzhen Science and Technology Innovation Committee for the financial support, as well as all the members of the research team, involving recruiters, evaluators, NMR scanners, and therapists for their contributions and dedications.
SJC: Trial design and supervision of trial implementation. XYJ: Data reduction and management. HLX and RJ: Diagnosis and randomization. LY: NMR scanning. XY and QQL: Moxibustion treatment. XMZ and TTT: Recruitment. DW: Evaluation. All authors have read and approved the final version of the manuscript for publication.
Conflicts of interest
Although moxibustion and donepezil hydrochloride will be used as interventions, all the authors declare that the trial will be conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by the Science and Technology Research Project of Shenzhen, No. JCYJ20150402152005642 and the Science and Technology Research Project of Guangdong Province, No. 2016A020215202. Funders had no involvement in the study design; data collection, management, analysis, and interpretation; paper writing; or decision to submit the paper for publication.
Institutional review board statement
The protocol has been approved by the Medical Ethics Committee of the Chinese Clinical Trial Registry (approval No. ChiECRCT-2017018). All the patients and their families will voluntarily participate in the trial, and provide written informed consent with the premise of fully understanding the treatment plan.
Declaration of patient consent
The authors certify that they obtained all appropriate patient or their legal guardian consent forms. In the form the patients or their legal guardians have given their consent for patients’ images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
This study follows the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidance for protocol reporting.
The statistical methods of this study were reviewed by the biostatistician of the Shenzhen Baoan People’s Hospital, China.
Copyright license agreement
The Copyright License Agreement has been signed by all authors before publication.
Data sharing statement
Individual participant data that underlie the results reported in this article after deidentification (text, tables, figures, and appendices) will be shared. Study protocol, informed consent form and clinical study report will be promulgated within 6 months after the completion of the trial. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Anonymized trial data will be available indefinitely at www.figshare.com.
Checked twice by iThenticate.
Externally peer reviewed.
Funding: This study was supported by the Science and Technology Research Project of Shenzhen, No. JCYJ20150402152005642 and the Science and Technology Research Project of Guangdong Province, No. 2016A020215202. 
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]