This is a randomized controlled neuroimaging trial which will be performed at the First Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (CDUTCM). A total of 48 patients with persistent asthma will be recruited and randomly allocated in a 1:1 ration to two acupuncture groups: group A (puncturing at acupoints on the Lung Meridian) and group B (puncturing at acupoints on the Heart Meridian). The treatment period will last for 4 weeks. Clinical outcome measurements and fMRI scans will be assessed at baseline and the end of treatment. The details of the study design are shown in Figure 1.
The study protocol conforms to the SPIRIT 2013 statement (Standard Protocol Items: Recommendations for Interventional Trials) [18]. The study has been approved by the Institutional Review Board (IRB) the First Affiliated Hospital of CDUTCM (approved number. 2019KL-045) and registered at Clinical Trial Registry (registration number: ChiCTR1900027478). All participants will provide voluntary written informed consent after a full discussion about the potential benefits and risks before participation. On the consent form, participants will be asked if they agree to use of their data should they choose to withdraw from the trial. Participants will also be asked for permission for the research team to share relevant data with people from the Universities taking part in the research or from regulatory authorities, where relevant. This trial does not involve collecting biological specimens for storage. YSY and XHD will recruiting the patient and gaining informed consent before participation.
Participants
Recruitment strategies
Asthma patients will be recruited mainly from the outpatient clinic in the respiratory department of the First Affiliated Hospital of CDUTCM. Potential patients will also be recruited via advertisement, leaflets and social media. All potential asthma patients will take a physical examination and laboratory tests, such as lung function test, x-ray plate, routine blood test, and blood immune test (immunoglobulin E, IgE). A respiratory physician will make the final diagnosis of potential patients. The diagnosis of asthma according to Western Medicine definition, not use with a specific TCM pattern.
Inclusion criteria
Eligible participants should fulfill all of the following items: (1) belonging to mild to moderate, persist asthma, according to the Chinese guideline for the prevention and management of bronchial asthma (version 2016) [19]; (2) aging from 18 to 65 years; (3) right-handed; (4) not participating in any other clinical trials in the past one month; (5) having assigned informed consent.
Exclusion criteria
Patients matching any of the following items will be excluded: (1) being diagnosed as other lung diseases, such as bronchiectasis, tuberculosis, lung abscess, cystic fibrosis, α-1 antitrypsin deficiency, and restrictive lung disease, etc.; (2) having aggravating malignant tumors or other clinically significant diseases that would jeopardize patient safety, such as uncontrolled heart-failure, severe hypertension, uncontrolled arrhythmias, etc.; (3) occurring only when accidentally exposed to allergen or chemical sensitizer; (4) being pregnant, lactating or those with childbearing requirements for nearly half a year; (5) receiving acupuncture for asthma within the last four weeks; (6) having MRI contraindications, such as having a heart pacemaker, having metallic foreign bodies, having severe claustrophobia, etc.
Sample size
The sample size calculation of neuroimaging study is different from that of classic randomized controlled clinical trials. Power analyses for neuroimaging studies rely upon assumptions about blood oxygenation level-dependent (BOLD) signal amplitude, smoothness, brain location, and other factors that render principled a priori designations difficult. It has been suggested that for fMRI studies, a minimum sample size (n=20) participants should be used in order to obtain 80% power with an error threshold of 0.002 at a single voxel level [20]. Considering a 20% dropout rate, the sample size in this study was increased to 24 per group, making a total of 48.
Randomization
After the baseline assessment, the eligible participants will be randomly allocated with an equal rate to their respective groups. To avoid bias on researchers ‘subjective factors, randomization will be implemented by a clinical information management system (Beijing Bioknow Information Science & Technology Co. Ltd., China). When participant-recruiting staffs decide to recruit an eligible asthma patient, they will send the patient’s name, gender, age, and telephone numbers to this system online. And then, the randomized result will be delivered to the acupuncturists.
Blinding
Because of the different acupoints in two groups, the acupuncturists will not be blinded from the group allocation. Patients in two groups will be separated into cubicles to refrain from communication. Outcome assessors and statistical analysts will be blind to the procedure and results of randomization, group allocation, and intervention.
Interventions
Subjects receive regular medical therapy (Symbicort or Seretide) as usual while participating in this study, except that it is not allowed to change the asthma control medication during this study. Based on the regular therapy, asthmatic patients randomly allocated will receive two different acupuncture treatments: puncturing at acupoints on the Lung Meridian or acupoints on the Heart Meridian.
Acupuncture intervention
According to records in the ancient Traditional Chinese Medicine work Huangdi Neijing, acupuncture points belonging to Lung Meridian have a noticeable effect on Lung disease. Therefore, we chose the following acupuncture points: patients in group A will receive manual acupuncture at six acupoints belong to Lung Meredian (bilateral Taiyuan (LU9), Lieque (LU7), Chize (LU5)) with disposable sterile filiform needles (0.25 × 25mm,0.35 × 40mm, Huatuo Medical Instrument Co., Ltd., China). Patients in group B is the reference group, will receive manual acupuncture at six acupoints belong to Heart Meredian (bilateral Shenmen (HT7), Yinxi (HT6), Shaohai (HT3). The manipulations are as follows: needles will be inserted into acupoints at a depth of 20-30 mm after skin disinfection using alcohol; acupuncturists will then bi-directionally twist needles by 90°–180°, lifting and thrusting needles with the amplitude of 3–5 mm for 1–1.5 Hz to induce deqi sensation. After the deqi sensation is attained, needles will be retained at the acupoints for 30 min. During the 30 min, the above procedures will be manipulated intermittently to maintain the deqi sensation.
Patients both in the two groups will receive a total of 12 sessions of acupuncture in four weeks with three sessions per week. All the acupuncture manipulation will be performed by two licensed acupuncturists with at least three years of clinical experience.
The acupuncture treatment will be conducted by doctors of department of Acupuncture and Tuina School of CDUTCM, with more than 6 years of Traditional Chinese medicine college education and at least 4 years of clinical experience.
Regular medical therapy
During the study period, all participants will receive regular medical therapies according to the Chinese guideline for the prevention and management of bronchial asthma. Asthma medications, such as sodium cromolyn, nedocromil sodium, leukotriene modifiers, and theophylline, require a stable dose for 3 months before enrollment; Nasal glucocorticoids and antihistamines require a stable dose for 2 months before enrollment; A short course of antihistamines or nasal corticosteroids is allowed to treat pollinosis; Desensitization treatment in the maintenance period requires a stable dose for at least 1 month before enrollment. During the study, short-term (<10 days) use of systemic corticosteroids and temporary use of aerosolized therapy (including beta 2-agonists, anticholinergics, and steroids) and antibiotic therapy were allowed for acute asthma attacks. All medications used need to be maintained at a stable dose throughout the study period. Furthermore, a salbutamol metered-dose inhaler (100 mg per puff) will be provided as rescue medication throughout the trial.
MRI data acquisition
MRI data will be acquired with a 3.0-T MR scanner (Siemens AG, Germany) at Huaxi Magnetic Resonance Research Center, West China Hospital of Sichuan University, Chengdu, China. All patients will be asked to stay awake, keep their eyes open, and remain still at the stage of the fMRI can. The participant’ head should be placed in the head mask and a sponge will be inserted to strengthen the fixation of the head. The scanning procedure contains a localizer, a high-resolution three-dimensional T1-weighted imaging (3D-T1WI), a blood oxygenation level-dependent fMRI (BOLD-fMRI) and a diffusion tensor imaging (DTI) sequence. The 3D-T1WI scanning parameters will be as follows: repetition time (TR)/echo time (TE) = 1900/2.26ms; slice thickness = 1 mm; slice number = 30; matrix size= 128 × 128; and field of view (FOV) = 256 × 256 mm. The BOLD-fMRI scanning parameters will be as follows: TR/TE = 2000/30 ms; flip angle = 90°; slice number = 30; matrix size = 128 × 128; FOV = 240 × 240 mm; slice thickness = 5 mm; and total volume = 240. The DTI data will be acquired with the following parameters: FOV = 240 ×240 mm; TR/TE = 6800/93 ms; matrix size = 128 × 128; and slice thickness = 3 mm with no gap. Two diffusion-weighted sequences were acquired using gradient values b= 1000 s/mm2 and b = 0 with the diffusion-sensitizing gradients applied in 64 non-collinear directions. All images will be checked by a consultant radiologist at West China Hospital of Sichuan University to exclude unexpected brain lesions in recruits.
Outcomes
The primary outcome measurements are the Asthma Quality of Life Questionnaire (AQLQ) [21], for assessing health-related impairment of quality of life in adult asthmatic patients [22]. AQLQ contains 32 items which including four domains: activity limitation, symptoms, mental health, environmental stimuli. It will take 5-10 minutes to the assessor.
The secondary outcome measurements including the Asthma Control Test (ACT), the Rate of Peak Expiratory Flow (PEFR), Forced Expiratory Volume in 1 second (FEV1), Montreal Cognitive Assessment (MoCA), Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). The ACT is a questionnaire consisting of five questions with a 5-point scale for each, which is validated for assessing asthma control [23]. PEFR and FEV1 are the main and objective monitors for asthma therapy. Furthermore, this trial also selects SAS, SDS, and MoCA as the secondary clinical outcomes to assess the emotional state and the general cognitive function, for psychological cognition importance in asthma patient’s pathogenesis.
All outcomes will be assessed at baseline and the end of treatment. The overview of the outcome measurement at different time points is shown in Table 1.
Patient safety
Safety monitoring will be conducted throughout the trial with reporting of adverse events (AEs) and serious adverse events (SAEs) in each participant’s case report form (CRF). All AEs/SAEs will be reported immediately to the study principal investigator and attending physician. Each AE will be recorded separately.
Data management
Clinical data will be managed with printed and electronic CRFs. Only outcome assessors have access to CRFs and will perform double-data entry. The researchers will be required to follow the requirements of the case report form and fill in the relevant information in a timely and accurate manner. The Evidence-based Medicine Center of the CDUTCM will be responsible for monitoring the study and data every three months and will make the final decision to terminate the trial.
Data analysis
The primary analysis will be conducted on a per-protocol basis. The data analysis will be completed by statisticians who are independent of the research and blind to the group mark. For non-imaging data, statistical analyses will be conducted with SPSS 23.0 software (IBM Corporation, Armonk, NY, USA). For clinical information, data will be presented as means with SDs. Student T-test and Chi-Square test will be used to compare group differences at baseline. Paired t-test will be used to compare the clinical outcomes within the group. Analysis of variance and Kruskal–Wallis Test will be used to compare the clinical feature change between groups. The significance will be set at 5% level with two-side test. The clinical and neuropsychological data including AQLQ, ACT, MoCA, SAS, and SDS will be converted to domain z scores for correlational analysis with imaging data.
The MRI data will be preprocessed and analyzed by SPM12 (http://www.fil.ion.ucl.ac.uk/spm/) and CONN toolbox 18b (https://web.conn-toolbox.org/) performed on MATLAB 2014b (MathWorks, Inc., Natick, MA, USA). The structural MRI data will analyze using VBM toolbox within SPM12. The steps including checking for artifacts, structural abnormalities, and pathologies; image segment; normalizing to standard template; spatial smoothing. The preprocessing steps of fMRI data including slice timing correction; head motion correction; skull-stripping using BET; co-registration of the anatomical image to the mean functional image; segmentation of the anatomical gray matter, white matter, and CSF; normalization to the MNI152 standard template; smoothing; Band-pass filtering. After preprocessing, a series of brain activity information including amplitude of low-frequency fluctuation (ALFF), group Independent Component Analyses (ICA), seed-based functional connectivity, complex-network analyses, and dynamic connectivity analyses, will be calculated to investigate the brain response for different treatment. Finally, the Pearson correlation analysis will be used to assess the association between the changes of cerebral activity features and the improvement of clinical outcomes in each group.