Synergistic effects of scalp acupuncture and repetitive transcranial magnetic stimulation on cerebral infarction: a randomized controlled trial

Background Presently, there is a need for stroke treatment strategies that combine multiple disciplines, such as neurology, rehabilitation medicine, and traditional medicine. This study investigated the synergistic effects of scalp acupuncture (SA) and repetitive transcranial magnetic stimulation (rTMS) known to be effective for cerebral infarction. Methods This outcome assessor-blinded randomized controlled clinical trial included a per-protocol analysis to compare the efficacy of SA and electromagnetic convergence stimulation (SAEM-CS), which comprises the simultaneous application of low-frequency rTMS, SA, rTMS, and conventional stroke rehabilitation therapy (CSRT) for cerebral infarction patients. The trial was completed by 42 cerebral infarction patients (control group, 12; SA group, 11; rTMS group, 8; SAEM-CS group, 11). All patient groups underwent two sessions of CSRT per day. SA, rTMS, and SAEM-CS were conducted once per day, 5 days per week, for 3 weeks. The primary outcome (motor function recovery) was evaluated using the Fugl‐Mayer assessment (FMA). Other scales were used to assess cognitive function, activities of daily living, walking, quality of life, and stroke severity, which were secondary outcomes. Results There were significant changes (week 7–week 0) between groups in the FMA upper extremity (FMAUE), FMA total (FMAT), modified Barthel index (MBI), and functional independent measurement (FIM) scores. There were no significant changes in the scores of other outcome measures. FMAUE (p=0.015), FMAT (p=0.023), MBI (p=0.002), and FIM (p<0.001) scores significantly increased in the rTMS group compared with the control group and FMAUE (p=0.016), FMAT (p=0.012), MBI (p=0.026), and FIM (p=0.012) scores significantly increased in the rTMS group compared with the SAEM-CS group. However, there were no significant changes in the SA or SAEM-CS group. Conclusions Low-frequency rTMS in the contralesional hemisphere had long-term therapeutic

activities of daily living. SAEM-CS had no positive synergistic effects of SA and rTMS on motor function recovery, cognitive function, activities of daily living, walking, quality of life, and stroke severity. Trial Registration URL: cris.nih.go.kr. Unique identifier: KCT0001768, retrospectively registered (registration date: January 14, 2016).

Background
Stroke is the second most common cause of death and the leading cause of adult disability worldwide [1]. Cerebral infarction (CI) is a common disease with high mortality, recurrence, and disability rates, which accounts for approximately 70% of strokes [2].
Conventional treatment of stroke patients includes pharmacological treatments, surgery, and multiprofessional rehabilitation. These treatments can promote recovery to some extent; however, no single intervention clearly and definitively contributes to stroke recovery. Therefore, stroke treatment strategies should combine multiple disciplines such as neurology, rehabilitation medicine, and traditional medicine [3,4].
Scalp acupuncture (SA) is a specialized acupuncture technique in which a filiform needle is used to penetrate specific stimulation areas on the scalp [5]. Baihui (GV20)-based SA could improve infarct volume and neurological function scores and exhibit potential neuroprotective roles in experimental ischemic stroke [6]. SA is commonly used during the acute, recovery, and sequelae stages of ischemic and hemorrhagic strokes [7][8][9][10].
Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive method that can change the excitability of the brain cortex for at least several minutes. The nature of the after-effect depends on the frequency, intensity, and pattern of stimulation [11].
Neural plasticity is the ability of the brain to develop new neuronal connections, acquire new functions, and compensate for impairments. These processes are crucial for motor recovery after stroke [20][21][22]

Methods
This study followed the standard protocol items of the Recommendations for Interventional Trials (SPIRIT) and CONSORT statement. Detailed methods of this study have been reported previously [24].

Study design
This study was an outcome assessor-blinded single-center randomized controlled pilot clinical trial with a 1:1:1:1 allocation ratio. Participants (n = 60) who fit the inclusion criteria were randomly allocated to the control group (n = 15), SA group (n = 15), rTMS group (n = 15), and SAEM-CS group (n = 15). All groups received CSRT twice per day, five times per week, for a total of 15 times over the course of a 3-week hospitalization period at Chonnam National University Hospital. In addition, the SA group received SA therapy, the rTMS group received rTMS therapy, and the SAEM-CS group received SAEM-CS therapy once per day. Outcome measures were determined at baseline (week 0), 3 weeks after the first intervention (week 3), and 4 weeks after completion of the intervention (week 7). The study design is summarized in Figure 1.

Ethical considerations
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Chonnam National University Hospital (CNUH-2015-114). This trial was registered at cris.nih.go.kr (registration number: KCT0001768). All patients provided written informed consent before participating in this study.

Participant recruitment
To achieve adequate participant enrollment to reach the target sample size, all CI patients who finished treatment for early acute stage CI at the Department of Neurology of Chonnam National University Hospital were screened by physical and rehabilitation medicine doctors. Patients who received an explanation regarding this study from the clinical research coordinator and who voluntarily signed a consent form were transferred to the Department of Physical and Rehabilitation Medicine to participate in this study. The clinical research coordinator continuously monitored the medical conditions of enrolled participants for improved adherence to intervention protocols.

Participation
There were six inclusion criteria: (1) age older than 19 years; (2)  Subjects whose general condition was not fit for SA and rTMS therapies were excluded.
Detailed exclusion criteria were as follows: (1) prior history of brain lesion (e.g., stroke, serious mental illness, loss of consciousness accompanied by head trauma, brain surgery, or seizure disorder); (2) presence of other serious illnesses (e.g., cancer, Alzheimer's disease, epilepsy, head trauma, or cerebral palsy); (3) transient ischemic attack; (4) contraindications to electromagnetic stimulation (e.g., metal implants in the brain, implanted electronic devices in the body such as nondetachable ferromagnetic metals, metal-sensitive implants less than 30 cm away from the brain such as cochlear implants, pacemakers, aneurysm clips or coils, stents, bullet fragments, deep brain stimulation, vagus nerve stimulators, jewelry, or hairpins); (5) continuous convulsion symptoms; (6) previous craniectomy or shunt surgery; (7) increased intracranial pressure symptoms such as headache, vomiting, or nausea; (8) seizure disorder or epilepsy after CI; (9) prior history of stroke accompanied by a clear clinical sign; (10) contraindications to SA (e.g., scalp scarring, inflammation from scalp injury, infection in the treatment region, inability to stop blood flow due to clotting disturbances such as hemophilia, serious unusual response after acupuncture treatment); (11) pregnant or breastfeeding; (12) disagreement with informed consent; and (13) scheduled for surgery within 2 weeks.

Randomization and blinding
After signed informed consent and baseline measurements were obtained, random allocation software (developed by M. Saghaei, MD, Department of Anesthesia, Isfahan University of Medical Sciences, Isfahan, Iran) was used to assign a serial number to the 60 research volunteers and to randomly allocate 15 of them to each group. The serial number codes were inserted in sealed opaque envelopes, kept in a double-locked cabinet, and opened in the presence of the patient and a guardian.
We had no choice but to adopt a single outcome assessor blinding approach because sham treatment was impossible due to the characteristics of SA, which included scalp penetration. During the study, the assessor was blinded to group assignments, and data analysts without conflicts of interest were involved in this study.

Implementation
A clinical research coordinator generated the allocation sequence, enroll participants, and assign participants to interventions.

Intervention
All participants underwent conventional stroke rehabilitation therapy (CSRT), which focused on practicing fine and gross motor movements, activities of daily living, taskoriented therapeutic exercises, and muscular electrical stimulation therapy as needed.
Training for swallowing and improving language was also performed for dysarthria. These sessions were conducted for 30 minutes (excluding Saturdays and Sundays) twice daily for 3 weeks for a total 15 times. SA, rTMS, and SAEM-CS therapies were conducted once daily for 20 minutes (excluding Saturdays and Sundays) for 3 weeks for a total of 15 times.
SA was conducted as follows: one or two needles were horizontally inserted approximately 3 cm into the lesion site and upper limb regions of MS6 (line connecting GV21 and GB6) and MS7 (line connecting GV20 and GB7) in the directions from GV21 to GB6 and from GV20 to GB7 [10]. Manual stimulation and electroacupuncture were not applied, and the needles (KOS 92 nonmagnetic steel acupuncture needles, size 0.25 mm × 30 mm, product no. A84010.02; Dongbang Acupuncture, Inc., Boryeong, Republic of Korea) were left in position for 20 minutes ( Table 1).
The rTMS was conducted as follows: a 70-mm figure-8 coil and a Magstim Rapid stimulator (Magstim Co., Dyfed, UK) were used to deliver 1 Hz of rTMS to the skull of the contralesional hemisphere at the site that elicited the largest motor-evoked potentials (MEPs) in the first dorsal interosseous (FDI) muscle of the unaffected upper limb. One LF-rTMS session consisted of 1200 pulses and lasted for 20 minutes. Stimulation intensity was set to 80% of the motor threshold of the FDI muscle, which was defined as the lowest intensity of stimulation that provokes MEPs. All patients sat in a reclining wheelchair and were asked to relax as much as possible with their heads strapped to a headrest [25].
The SAEM-CS was conducted as follows: the aforementioned SA and LF-rTMS therapies were performed simultaneously. After SA treatment of MS6 and MS7 on the lesion side, LF-rTMS stimulation was conducted on the contralateral hemisphere for 20 minutes.

Outcome measurements
The primary outcome was motor function, and the secondary outcomes were cognitive function, activities of daily living, walking, quality of life, and stroke severity. Primary and secondary outcome assessments were conducted at baseline (before intervention), 3 weeks after the first intervention, and 4 weeks after completion of intervention (except

Statistical analyses
Sample size calculation was detailed in our study protocol [24]. We performed perprotocol analysis for the assessment of efficacy and a supplementary full analysis set.
Analysis was performed by blinded biostatisticians using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA) using two-sided significance tests with a 5% significance level.
Continuous variables were presented as means and standard deviations (SD), and categorical variables were presented as count frequencies and percentages.
Baseline data were collected and compared by first using the independent k-sample Kruskal-Wallis test, nonparametric tests, and χ 2 test. Differences between all outcome value changes (week 0 vs. week 7) in the four groups were compared by the two related samples test and the Wilcoxon signed-rank test (nonparametric tests). Values of FMAUE, FMA lower extremity (FMALE), FMA total, NIHSS, MBI, FIM, 9HPT, AHSA-NOMS, FAC, mRS, EQ-5D, K-MMSE, MAS elbow, and MAS ankle were compared by repeated-measures analysis of variance (ANOVA) across two to three testing time points (week 0, week 3, week 7). An F test was conducted to detect differences between therapies, and the Scheffé post hoc test was conducted to identify groups. Differences between two groups of outcome value changes (week 0 vs. week 7 and week 0 vs. week 3; significant changes were observed in the ANOVA and the Scheffé post hoc test) were compared by the two independent samples test and the Mann-Whitney U test (nonparametric test).

Participants
We recruited participants between July 31, 2015, and December 31, 2017. During the study period, 2200 patients were assessed for eligibility and 2140 were excluded due to nonconformity to the inclusion criteria, conformity to the exclusion criteria, or refusal to participate. Sixty patients were included in this study and were randomly assigned to four groups: control group, 15; SA group, 15; rTMS group, 15; and SAEM-CS group, 15. Three did not complete treatment in the control group. Two did not complete treatment and two were lost to follow-up in the SA group. One exited the study due to orthopedic surgery, four did not complete treatment, and two were lost to follow-up in the rTMS group. One exited the study due to orthopedic surgery, two did not complete treatment, and one was lost to follow-up in the SAEM-CS group (Fig. 2). The results of the per-protocol analysis for the assessment of efficacy were not different from those of the full analysis set. Data for 42 CI patients were used in the final analysis.

Baseline characteristics
Participants were divided into the control group (n=12), SA group (n=11), rTMS group (n=8), and SAEM-CS group (n=11). Baseline demographic characteristics of the 42 CI patients in the four groups, including sex, age, lesion site, and all variables, are presented in Table 3. No significant differences in the baseline demographic characteristics were detected among the four groups (P<0.05; Table 2).

Efficacy of primary and secondary outcomes
All variables at each testing time point are detailed in Table 4. All variables except ASHA-NOMS, K-MMSE, and FAC were estimated at week 0 (baseline), week 3 (end of intervention), and week 7 (follow-up). ASHA-NOMS, K-MMSE, and FAC were estimated at week 0 (baseline) and week 3 (end of intervention; Table 3).  Changes in the FMAUE scores (week 0 vs. week 3) of the rTMS group were significantly larger than those of SAEM-CS group, and the changes in the FMAUE scores (week 0 vs. week 7) of rTMS group were significantly larger than those of control and SAEM-CS group according to Scheffé post hoc test (Table 6). Changes in the FMAT scores (week 0 vs. week 7) of the rTMS group were significantly larger than those of control and SAEM-CS group according to Scheffé post hoc test (Table 6).
Changes in the MBI scores (week 0 vs. week 3) of the rTMS group were significantly larger than those of control group, and the changes in the MBI scores (week 0 vs. week 7) of the rTMS group were significantly larger than those of the control and SA groups according to Scheffé post hoc test (Table 6).Changes in the FIM scores (week 0 vs. week 7) of the rTMS group were significantly larger than those of the control and SA groups according to Scheffé post hoc test (Table 6).
We conducted multiple comparisons of FMAUE, FMAT, MBI, and FIM, and significant score changes (week 0 vs. week 3 and week 0 vs. week 7) were observed in the ANOVA and Scheffé post hoc test for the four groups to investigate the synergistic effects of SA and rTMS.
Changes in the MBI (p=0.005) and FIM (p=0.03) (week 0 vs. week 3) scores of the rTMS group were significantly larger than those of control group. Changes in FMAUE (p=0.026) and MBI (p=0.043) (week 0 vs. week 3) scores of the rTMS group were significantly larger than those of SAEM-CS group (Table 7).

Safety evaluation
Adverse events that occurred in this study were recorded on a case report form after evaluating their relationships with the intervention. Fortunately, no adverse events that were related to the intervention occurred in this study.

Discussion
To our knowledge, this is the first randomized controlled study to investigate the synergistic effects of SA and rTMS on motor-function recovery, stroke severity, activities of daily living, cognitive function, dysphagia, walking ability, quality of life, and spasticity of CI patients by comparing the effects of simultaneous application of LF-rTMS and SA with the effects of SA, LF-rTMS, CSRT. There were several main findings. First, rTMS combined with CSRT led to better improvements in FMA, MBI, and FIM than CSRT alone and SAEM-CS combined with CSRT. Second, SA combined with CSRT and SAEM-CS combined with CSRT did not lead to significant differences compared with CSRT alone. Third, SAEM-CS did not show the positive synergistic effects of SA and rTMS on motor-function recovery, stroke severity, activities of daily living, cognitive function, dysphagia, walking ability, quality of life, and spasticity of CI patients.

Effect of CSRT on patients with CI
CSRT itself has some beneficial effects on motor recovery, activities of daily life, and stroke severity. These results may be related to the therapeutic effects of physical therapy, occupational therapy, and functional electrical stimulation. Two systematic reviews have suggested that the intensity of stroke rehabilitation is an important factor associated with better and faster improvement [38,39]. Intensive occupational therapy was confirmed to be significantly beneficial in a study involving a large number of patients with upper limb hemiparesis after stroke [40].

Analysis of the efficacy of SA for patients with CI
SA combined with CSRT has a beneficial effects on motor-function recovery, stroke severity, and activities of daily living of CI patients, however, there were no significant differences in primary and secondary outcome score changes between the SA group and control group. These results may be related to acupoint specificity, acupuncture manipulation, and electrical stimulation.
Acupuncture is a complex intervention involving both specific and nonspecific factors associated with therapeutic benefits. Apart from needle insertion, issues such as needling sensation, psychological factors, acupoint specificity, acupuncture manipulation, and needle duration also have relevant influences on the therapeutic effects of acupuncture When SA was used to treat stroke patients, manipulation or electroacupuncture (acupuncture combined with electrical stimulation) were usually used to reinforce the therapeutic effects of SA. There are some methods of reinforcing-reducing acupuncture manipulations in Traditional Chinese Medicine. In clinical practice, mastering the reinforcing-reducing manipulations of acupuncture will contribute to improvements in therapeutic effects [46]. With various factors of manipulation, including lifting-thrusting, twirling-rotating, and variations in the direction, angle, and depth of needle insertion, it is possible to affect the outcomes of acupuncture treatment [47,48]. In most systematic reviews reporting acupuncture for neurogenesis with experimental ischemic stroke [49], Baihui (GV20)-based SA for experimental ischemic stroke [6], and SA for stroke recovery in randomized controlled trials [10], electroacupuncture or manipulation of twirling (needles should be twirled more than 200 times per minute) was applied.
This study aimed to investigate the synergistic effects of SA and rTMS on stroke. Therefore, the same acupuncture treatment method used for the SAEM-CS approach must be used for SA therapy. Subsequently, we could not use the combination of SA and body acupuncture, manipulation, and electroacupuncture to reinforce the therapeutic effects of SA in the SA group.

Analysis of the efficacy of LF-rTMS for patients with CI
LF-rTMS combined with CSRT has beneficial effects on motor-function recovery, stroke severity, activities of daily living, walking ability, and quality of life of CI patients. The rTMS group showed better effects on motor-function recovery and activities of daily living than the control group and SAEM-CS group at 4 weeks after the intervention, however, there were no significant differences in outcome score changes except for MBI and FIM (week 0 vs. week 3) between the rTMS group and control group. These results are similar to those of a previous study [17] and may be related to the long-term effects of rTMS on stroke.

Limitations
This study had some limitations. First, our trial was a pilot study with a small sample size.
We lost some subjects because of various reasons; therefore, the number of subjects included in the final analysis was small. Second, according to our study design, we did not perform outcome measurements of K-MMSE, ASHA-NOMS, or FAC at week 7. Therefore, we did not explore the long-term additional effects on cognitive function, dysphagia, and walking. Third, our study lacked long-term follow-up and evaluation. Although we conducted 3 weeks of treatment and 4 weeks of follow-up, this was not sufficient to assess the long-term efficacy of SA, rTMS, and SAEM-CS. Fourth, we did not investigate synergistic effects of SA and rTMS through various combination methods. We used only ). This trial was registered at cris.nih.go.kr (registration number: KCT0001768). The purpose and potential risks of this clinical trial will be fully explained to the participants and their families. All patients were asked to provide written informed consent before participating in this study.

Consent for publication
Written informed consent for publication of individual details and accompanying images will be obtained from the trial participants. The consent form is in possession of the authors and available for review by the Editor-in-Chief.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no conflicts of interest concerning this article.

Authors' Contributions
Han JY and Kim JH designed or conceptualized the trial, wrote the initial draft, and analyzed data. Song MK and Park GC designed the trial and conducted the trial. Lee JS is responsible for planning data analysis and interpreting the data resulting from the trial.