Modulatory effects of acupuncture on raphe nucleus‐related brain circuits in patients with chronic neck pain: A randomized neuroimaging trial

Abstract Objective Acupuncture has shown promise in treating neck pain. Clinical trials have shown mixed results, possibly due to heterogeneous methodologies and the lack of knowledge regarding underlying brain circuit mechanism of action. In this study, we investigated the specific contribution of the serotonergic system in treating neck pain, and the specific brain circuits involved. Methods A total of 99 patients with chronic neck pain (CNP) were randomized to receive true acupuncture (TA) or sham acupuncture (SA) 3 times weekly for 4 weeks. Patients with CNP in each group were assessed for primary outcomes by measuring the Visual Analog Scale (VAS) and the duration of each attack; secondary outcomes were measured using the Neck Disability Index (NDI), Northwick Park Neck Pain Questionnaire (NPQ), McGill Pain Questionnaire (MPQ), Self‐rating Anxiety Scale (SAS), Self‐rating Depression Scale (SDS) and the 12‐item Short Form Quality Life Scale (SF‐12); levels of functional circuits connectivity were assessed using resting‐state functional magnetic resonance imaging in the dorsal (DR) and median (MR) raphe nucleus, before and after undergoing acupuncture. Results Patients receiving TA showed more extensive symptom improvement compared with SA. Regarding the primary outcomes, changes observed in the TA group were as follows: VAS = 16.9 mm (p < 0.001) and the duration of each attack = 4.30 h (p < 0.001); changes in the SA group: VAS = 5.41 mm (p = 0.138) and the duration of each attack = 2.06 h (p = 0.058). Regarding the secondary outcomes, changes in the TA group: NDI = 7.99 (p < 0.001), NPQ = 10.82 (p < 0.001), MPQ = 4.23 (p < 0.001), SAS = 5.82 (p < 0.001), SDS = 3.67 (p = 0.003), and SF‐12 = 3.04 (p < 0.001); changes in the SA group: NDI = 2.97 (p = 0.138), NPQ = 5.24 (p = 0.035) and MPQ = 2.90 (p = 0.039), SAS = 1.48 (p = 0.433), SDS = 2.39 (p = 0.244), and SF‐12 = 2.19 (p = 0.038). The modulatory effect of TA exhibited increased functional connectivity (FC) between the DR and thalamus, between the MR and parahippocampal gyrus, amygdala, and insula, with decreased FC between the DR and lingual gyrus and middle frontal gyrus, between the MR and middle frontal gyrus. Furthermore, changes in the DR‐related circuit were specifically associated with the intensity and duration of pain, and the MR‐related circuit was correlated with the quality of life with CNP. Conclusion These results demonstrated the effectiveness of TA in treating neck pain and suggested that it regulates CNP by reconfiguring the function of the raphe nucleus‐related serotonergic system.


| INTRODUC TI ON
Neck pain, with an annual prevalence rate exceeding 30%, is the fourth leading cause of disability. 1 Most episodes of acute neck pain will resolve with or without treatment; however, nearly 50% of individuals continue to experience some degree of pain or frequent recurrences. 2Drug therapy, including paracetamol, non-steroidal drugs, anti-inflammatory drugs, and opioids, is a common treatment for acute and chronic neck pain (CNP). 3However, the pharmacological management for low back and neck pain costs over 100 billion dollars in the United States alone 4,5 and often causes non-negligible side effects including fatigue, nausea, headache, and increased pain. 6reover, long-term use of some medications can significantly increase the risk of drug dependence. 7Because of these limitations create an imperative for evidence-based therapies with elucidated mechanisms a priority.
In addition to medication, diverse treatments exist for neck pain including physical therapy and complementary and alternative medicine therapies.Although acupuncture is widely used, its effectiveness remains controversial.Several trials with small sample sizes have shown that true acupuncture (TA) is more effective than sham acupuncture (SA; shallow puncture or needling at non-acupoint locations) for relieving neck pain by modifying the perception of pain or altering physiological functions, that is, pain control for treating certain diseases or dysfunctions. 8However, other trials have shown that acupuncture reduces neck pain and produces a statistically, but not clinically, significant effect compared with that of placebo. 8These beneficial effects may be due to non-specific or specific. 7Although many systematic reviews and meta-analyses of CNP have been conducted, most of them have been inconclusive, and this has led to confusion in clinical policy and practice. 9The inconsistency in these findings may have resulted from variations in the design characteristics (such as the choice of SA) and the lack of clear longitudinal neuroimaging evidence.
Many studies have reported that acupuncture stimulation can relieve chronic pain by modulating the trigeminal spino-thalamocortical circuit, [10][11][12][13] which is a key network in the regulation of pain, 10 and has a high degree of congruence with the serotoninacting regions. 14,157][18][19] Recent studies have focused on acupuncture for treating chronic pain via this descending pain regulation loop.Gao et al 20 found that acupuncture improves pain intensity by enhancing the functional connectivity (FC) of the dorsal raphe (DR) nucleus in knee osteoarthritis with chronic pain.Liang et al 11 reported that acupuncture relieves knee osteoarthritis knee pain by modulating the FC between the ventrolateral periaqueductal gray and thalamus.However, little is known about whether and how acupuncture modulates CNP via this circuit.
2][23] Han et al 24 used the raphe nucleus as seeds and found that the dysconnectivity between it and the subcortical serotoninrelated regions contributes to altered salience network in schizophrenia.Similarly, increased FC between the raphe nucleus and periaqueductal gray has been reported in chronic migraine. 25Using resting-state FC, Li et al. reported the abnormal dynamic activity of the striatal-sensorimotor circuit in patients with benign epilepsy. 26erefore, fMRI is suitable for exploring the raphe nucleus-related serotonin circuit in CNP.
In this trial, we compared the clinical outcomes of TA with those of SA, and investigated how acupuncture improves the clinical outcomes of patients with CNP by regulating the raphe nucleus-related serotonergic system.We hypothesized that TA could better reduce pain intensity, duration and pain-related disability and improve the quality of life of patients with CNP.Moreover, we proposed a new perspective, in which TA was effective by reconfiguring the function of the serotonin system in CNP.

| ME THODS
We collected the clinical and fMRI data of patients with CNP from the Hospital of Chengdu University of Traditional Chinese the DR and thalamus, between the MR and parahippocampal gyrus, amygdala, and insula, with decreased FC between the DR and lingual gyrus and middle frontal gyrus, between the MR and middle frontal gyrus.Furthermore, changes in the DR-related circuit were specifically associated with the intensity and duration of pain, and the MR-related circuit was correlated with the quality of life with CNP.

| Participants
We included patients with CNP, whose criteria were based on the guidelines developed by the Orthopedic Section of the American Physical Therapy Association. 27The inclusion criteria were as follows: ( 1

| Study design
In this trial, the patients experiencing CNP were observed for 6 weeks, of which the first 2 weeks of the run-in period checked whether patients with CNP were eligible and willing to participate.
A total of 99 patients with CNP were randomized to receive TA or SA with a 2:1 ratio [28][29][30][31] (TA = 66; SA = 33) by using a random number table (Figure 1).An independent staff generated the random number and grouping scheme using the SPSS software (version 20.0;IBM Corp.) to set the block length and number of segments to nine according to the block and the number of segments to 11 according to the block randomization principle.The TA and SA groups were blinded to their group assignments and were treated in a closed unit.Acupuncturists could not be blinded to the treatment assignments because of the nature of the interventions; thus, they were not engaged in the evaluation of outcomes or data analysis.Other researchers, including outcome assessors, data collectors, and statisticians, were blinded to the treatment allocation.Jianjing (GB21), Jianzhongshu (SI15), Wangu (GB12), Fengchi (GB20), Tianzhu (BL10), Dazhui (DU14), Dazhu (BL11), Jianwaishu (SI14), Tianliao (SJ15), Jugu (LI16), Tianzong (SI11), Shousanli (LI10), Lieque (LU7), Zhongzhu (SJ3), and Houxi (SI3). 32,33[36] We have previously published a clinical study protocol related to the acupoint selection and treatment. 36l acupoints were punctured using single-use stainless steel fili- with a depth of 2 mm was performed at the five non-acupoints, without manual stimulation to yield the Deqi sensation. 37Both groups received acupuncture treatment 3 times weekly for 4 weeks with a total of 12 sessions.Each session lasted for 30 min.None of the patients were allowed to receive any sustained-release or prophylactic analgesics.For intolerable neck pain, patients were allowed to receive analgesic medications (such as non-steroidal anti-inflammatory drugs) or effective analgesic medications, which they considered as rescue medications.The details are documented in a Case Report Form.

| Clinical assessments
Changes in the VAS score (with 0 representing no pain and 10 representing the worst pain imaginable) and the duration of each attack from baseline to 4 weeks of treatment were set as the primary outcomes to assess the severity of the chronic pain.Secondary

| Clinical data analysis
The sample size, which was calculated using the NQuery Advisor software (version 4.0; Statistical Solutions) with a 2-sided significance level of 5% and a power of 90%, included 80 participants. 38e estimated lost-to-follow-up rate was 15%. 39Thus, we enrolled 99 participants in the two groups (66 in the TA group and 33 in the SA group).
All statistical evaluations were performed using SPSS 22.0 (SPSS Inc.).The normality distribution of the data was assessed using the visual inspection of histograms and the Shapiro-Wilk test.Data that did not follow a normal/Gaussian distribution were analyzed using a non-parametric test.We used the following statistical tests: the chi-squared test, Mann-Whitney U test, and Kruskal-Wallis H test.
Statistical analysis was performed using a two-tailed test, and the significance level was set at 5%.

| Raphe nucleus seed-based FC analysis
Resting-state fMRI images were preprocessed using the data processing and analysis of brain imaging toolbox.The first 5 volumes were discarded to avoid the non-equilibrium effects of magnetization, and slice timing and realignment correction were performed for the remaining images.Any participant with maximum head movement >2.0 mm translation or >2.0° rotation was not included.In this step, two patients in the TA group and three patients in the SA group were excluded from the following analyses.Data were further normalized to the EPI template (resampled voxel size: 3 × 3 × 3 mm 3 ).
Several covariates including the Fristion 24 motion parameters, cerebrospinal fluid, and white matter signals were regressed as nuisance variables to reduce spurious variance.No global signal regression was performed to avoid introducing distortion into the time series data. 40Detrending and band-pass filtering (0.01-0.08 Hz) were then conducted.Finally, since the resting-state activity is sensitive to minor head movements, we calculated the mean frame-wise displacement (FD) to further determine the comparability of head movement among groups."Bad" time points (FD > 0.5 mm), as well as their one-back and two-forward time points, were scrubbed and interpolated by spline interpolation. 41 described in previous positron emission tomography (PET)   and fMRI studies, the raphe nucleus is subdivided into the DR and median raphe (MR) nucleus. 42,43The central coordinates of the seeds were obtained with a spherical radius of 4 mm.Accordingly, the ROIs were defined as follows: DR (0, −27, −9) and MR (0, −31, −21) 27 (Figure 4).The voxel-wise FC between the seeds and each voxel in the brain was then calculated using the Pearson's correlation and the correlation coefficients were converted to Z-values using the Fisher's r-to-z transformation.
ancova analysis was used to assess the reconfiguration of the TA on the raphe nucleus-related circuits with age and gender as covariates in CNP.Gaussian random field corrections (with voxel p < 0.005, cluster p < 0.05) were conducted for the multiple comparison correction. 29Correlation analysis was performed between the altered FC between the seed regions to the ROIs and the improvements in clinical symptoms in patients with CNP.

| RE SULTS
After 243 patients were screened, 99 with CNP aged 18-65 years were randomized, of whom 63 (64.9%) were women.Two patients in the TA group discontinued the study and three patients in the SA group discontinued the neuroimaging analyses because of a maximum head movement >2 mm translation or >2° rotation.Two patients in the SA group discontinued the clinical assessment because of unsatisfactory outcomes.Finally, 97 patients (66 in the TA group and 31 in the SA group) were included in the final clinical analysis, and 92 patients (64 in the TA group and 28 in the SA group) were included in the neuroimaging analysis (Figure 1).No significant difference in head movement was observed among the groups (TAbefore treatment: 0.07 ± 0.03; TA-after treatment: 0.11 ± 0.06; SA-before treatment: 0.13 ± 0.04; SA-after treatment: 0.09 ± 0.07; FD mean ± SD, p = 0.37).

| Patient characteristics
The baseline demographic characteristics of all patients are summarized in Table 1.These values were comparable between the TA and SA groups (p > 0.05).Two patients (2.1%) in the SA group did not undergo follow-up because of unsatisfactory outcomes.

| Clinical outcomes
The clinical outcomes conformed to a normal distribution and are presented in both the scatter and distribution plots shown in   3 show the clinical outcomes of the TA and SA groups.The results were analyzed to determine the changes that occurred from baseline to the end of the 4-week treatment period.

| Primary outcomes
Regarding the primary outcomes, the VAS scores significantly decreased in the TA group (16.9 mm, 95% CI: 12.15-21.61mm, p < 0.001) and did not in the SA group (5.41 mm, 95% CI: 1.85-12.68mm, p = 0.138), and the duration of each attack was significantly reduced in the TA group (4.30 h, 95% CI: 2.70-5.90h, p < 0.001) and was not in the SA group (2.06 h, 95% CI: 0.07-4.19h, p = 0.058) after 4 weeks of treatment, with a significant difference between the two groups (p < 0.05).

| Neuroimaging outcomes
Considering the impact of head movement on imaging results, the neuroimaging analyses included 92 patients with CNP (TA = 64; SA = 28) who did not differ in gender, age, or years of education.
As shown in Figure 4 and Table 3, with the DR as the seed, ancova analyses showed a significant interaction effect (time and group factors), mainly located in the left lingual gyrus, right thalamus, and left middle frontal gyrus (p < 0.05).With the MR as the seed, ancova analyses showed a significant interaction effect (time and group factors), mainly located in the right amygdala, bilateral insula, left parahippocampal gyrus, and right middle frontal gyrus (p < 0.05).A significant main effect of group factors was mainly located in the left lingual gyrus, right thalamus, and left middle frontal gyrus with the DR as the seed, and in the right amygdala with the MR as the seed.
The main effects of time were mainly located in the right thalamus, bilateral insula, and left parahippocampal gyrus.
Post hoc analyses revealed reconfiguration of the TA in the FC of the raphe nucleus subdivision in CNP (Figure 5).

| Correlation with the clinical improvements in the TA group
As shown in Figure 6, changes in FC between the DR and left middle frontal gyrus were positively correlated with the NDI improvements TA B L E 1 Participant demographic and baseline characteristics.

| Medication uses
No patient reported pain medication usage during the 4-week treatment period in this study.However, four patients used a sticking plaster on the neck for pain relief (two in the TA group and two in the SA group).

| Patients safety
All 99 patients were monitored for safety and tolerability.Four patients in TA and three patients in the SA group developed mild bruising around the acupoints/non-acupoints, all of which were back to normal after 1 week of follow-up.

| DISCUSS ION
TA has demonstrated significant and clinically relevant benefits for CNP, as it reduces the pain intensity and duration of each attack compared with SA.Moreover, improvements in pain-related emotional impairment and quality of life were observed.Furthermore, SA had a weak statistically significant effect in controlling pain-related disability measures, including the NPQ and MPQ, after 4 weeks of treatment.Thus, acupuncture should be considered a treatment option for neck pain.Moreover, we found that the modulation of the TA mainly included the DR-thalamus-frontal gyrus and MR-limbicfrontal gyrus circuits.The former was specifically associated with the primary outcomes (intensity and duration of pain), whereas the latter was associated with the secondary outcomes.To the best of our knowledge, this study is the first randomized neuroimaging trial to combine clinical and neuroimaging analyses to test the efficacy of TA and explore its underlying neuroimaging mechanisms in CNP.
Rigorous paired control groups were used to assess both the clinical and neuroimaging outcomes, providing valuable insights into the role of raphe nucleus-related circuits as neuroimaging markers in acupuncture treatment for CNP.
First, we found that TA was effective in reducing the intensity of neck pain and duration of each attack, and it was more effective than SA in reducing the VAS scores in CNP.These findings are congruent with those of previous clinical trials 44 and demonstrated the effectiveness of acupoint TA in relieving chronic pain. 45In addition, TA showed effectiveness in improving secondary clinical outcomes including the NDI, NPQ, MPQ, SAS, and SDS, as well as, in increasing the SF-12 scores.Some studies have shown that acupuncture can effectively reduce pain and improve function in patients with CNP, supporting its appropriateness and effectiveness in treating chronic pain. 46,47However, a study using SA found that although the VAS scores in both groups were significantly reduced after therapy, there is no significant difference between groups was detected. 48milar to our findings, most clinical outcomes were significantly improved in the TA group compared with those in the SA group, except for the VAS and SAS scores; this may be attributed to the placebo effect of SA.The control intervention method we selected was non-acupoint shallow acupuncture, to explore the specificity of acupoints.Previous studies have suggested that acupuncture analgesia may work through diffuse harmful inhibitory controls, which are responses to a painful stimulus that suppresses pain from another stimulus (pain-inhibiting-pain effect). 49However, another study showed that the effect could be explained by improving tactile sensitivity due to primary somatosensory cortical neuroplasticity in specific body structures, which could be induced by penetrating the sham control. 50Moreover, needling can enhance patients' confidence in receiving acupuncture, indicating that non-acupoint acupuncture has psychological analgesic effects similar to those of TA.Moreover, the study demonstrated that the placebo effect of acupuncture was definite and showed high variability among different types of sham controls and that the placebo effects were influenced by the depth of needle insertion and puncture location. 8Therefore, the placebo effect can be avoided to a large extent by using a nonacupoint shallow puncture.Nevertheless, currently, there is still no consensus on the selection of control/sham acupuncture interventions, indicating the need for further search into different neuroimaging mechanisms.
To explore the modulation on the serotonergic system via TA in the treatment of CNP, we selected important source nucleus of serotonin in the raphe nucleus, that is, the DR and MR according to previous PET and fMRI studies.We found that TA modulated the functional connection between the DR and thalamocortical networks by reducing the FC between the DR and the lingual gyrus and middle frontal gyrus, but increasing the FC between the DR and thalamus.Consistent results have been reported for other chronic pain, such as chronic migraine 13 and chronic low back pain. 51The DR, thalamus, and middle frontal cortex play central roles in pain processing.The thalamus integrates the pain stimulus and sends it to the high brain processing area (mainly located in the frontal cortex), which further converts the stimulus into a perceptual signal; this perceptual signal is further used to downregulate the pain (including in the subcortex and DR). 524][55] The modulation of these abnormalities by acupuncture contributes to pain processing, such as pain sensation and pain perception. 52,56These findings suggest that acupuncture relieves and treats CNP by regulating endogenous pain processing via the DR-related circuit.
Moreover, we found that TA modulated the connections between the MR to limbic networks and the middle frontal gyrus by increasing the FC between the MR and the amygdala, bilateral insula, and parahippocampal gyrus and reducing the FC between the MR and the middle frontal gyrus.Interestingly, acupuncture enhanced the association between the MR and limbic regions but weakened the association between the MR and frontal cortex.The limbic regions, particularly the insula, are important components of the pain matrix. 57Baliki et al. proved that the insula includes circuits specific to pain perception and is generally linked to encoding the intensity or magnitude of sensory stimuli, such as painful stimuli. 58,59The amygdala plays a key role in emotion processing and the emotion-affective dimension of pain. 60,61Coppieters et al 61 found that the dysfunction of the amygdala could affect the link between cognitive-affective and sensory modulation of pain in CNP.The parahippocampal gyrus, which is located at the junction between the hippocampus and the fusiform cortex, is involved in diverse classes of stimuli, tasks, environments, and pain perception. 62These results suggest that acupuncture may alleviate a wider range of pain dimen-  This study has some limitations.First, it was a single-center trial, which might have introduced potential biases that influenced the generalizability of the results.However, quality is more easily controlled and internal authenticity is better guaranteed in single-center trials than in multicenter trials.Second, acupuncture was performed by experienced acupuncturists who could not be blinded.Whether non-acupuncture practices produce similar results remained unknown.Third, the intervention was an individualized treatment; therefore, the results did not clearly explain the relationship between left-and right-side pain and brain circuits.In the future, we will conduct a subgroup analysis of unilateral and bilateral pain in patients with neck pain to assess the specific relationship between neck pain, acupuncture points, and different brain circuits.

| CON CLUS ION
In summary, TA showed superior treatment effects in CNP than those of SA.TA resulted in significant reductions in the intensity and duration and improvements in emotional well-being and quality of life, rather than being merely a placebo.After TA treatment, the improvement in primary and secondary clinical outcomes was significantly associated with changes in raphe nucleus-related serotonergic circuits in CNP.These findings demonstrate that acupuncture can be considered a treatment option for CNP and support the hypothesis that acupuncture could treat CNP by regulating the serotonergic circuits of the brain.

:| 3 of 13 WANG
These results demonstrated the effectiveness of TA in treating neck pain and suggested that it regulates CNP by reconfiguring the function of the raphe nucleus-related serotonergic system.K E Y W O R D S fMRI, neck pain, raphe nucleus, serotonergic et al.Medicine (TCM), where the participants were recruited from the acupuncture and orthopedic clinics.The research protocol was submitted to the Chinese Clinical Trial Registry with the identifier (ChiCTR1800017718) and was reviewed by the Sichuan Regional Ethics Review Committee on Traditional Chinese Medicine (ethical approval number: 2018KL-056).The recruitment process started since August 2018 and continued until December 2019.
) 18-65 years old men or women with neck pain and discomfort or limited cervical motion as the main symptoms; (2) right-hand dominance; (3) pain score of ≥4 cm on the Visual Analog Scale (VAS) for 5 of 7 days (range, 0-10 cm); (4) more than 3 months duration of disease; and (5) informed consent signed by patients.Patients with any of the following conditions were excluded: (1) macroscopic T2-visible brain lesions on the MRI scans; (2) accompanied by other serious organic lesions, including malignant neoplasms, tuberculosis, fracture, or osteomyelitis; (3) complications associated with serious primary diseases, including cardiovascular, cerebrovascular, liver, kidney, and hematopoietic systems; (4) mental disorders and other mental disorders that could not be matched with the questionnaire (a score of self-rating anxiety scale [SAS] or self-rating depression scale [SDS] > 72); (5) bleeding tendencies, allergies, and skin diseases; pregnancy, lactation, or fertility issues within the past 6 months; (6) contraindications such as metals in the body; and (7) participation in other simultaneous clinical trials.
Acupuncture therapy was administered to both groups in turn by two specialized acupuncturists with at least 5 years of training and 3 years of experience.The acupoints and non-acupoints used in this study are shown in Figure 2.After detecting individualized tenderness threshold at 15 acupoints, five sensitive acupuncture points with the largest absolute value of changes in the pressure pain threshold were selected as the treatment acupoints in the TA group, including the Tianliao point (SJ15), Jianwaishu point (SI14), Jianzhongshu point (SI15), Dazhu point (BL11), and Jugu point (LI16).The 15 acupoints were determined by data mined from the literature and expert consensus on the treatment of CNP, including

| 5 of 13 WANG
form needles (Hwato Needles, Sino-foreign Joint Venture Suzhou Hua Tuo Medical Instruments Co.), which were 25-40 mm in length and 0.25-0.30mm in diameter.In the TA group, the depths of the inserted needles varied but were approximately 0.5-1.5 cm.Rotating or liftingthrusting manipulation was performed for Deqi sensation (a sensation of soreness, numbness, distention, or radiation that indicated effective needling) after needle insertion.In the SA group, shallow acupuncture F I G U R E 2 Experimental design and acupoint/non-acupoint location.SA, sham acupuncture; TA, true acupuncture.Non-acupoint 1, midpoint between the brachial arch and acromial arch; non-acupoint 2, medial edge of the upper arm, the joint of the deltoid muscle, and biceps brachii muscle; non-acupoint 3, midpoint between the elbow tip and axilla; non-acupoint 4, midpoint between the medial epicondyle of the humerus and the ulnar end of the wrist stripe; and non-acupoint 5, tibial front.et al.
outcomes included the Neck Disability Index (NDI), Northwick Park Neck Pain Questionnaire (NPQ), and McGill Pain Questionnaire (MPQ) for holistic pain measurement.The SDS/SAS and 12-item Short Form Quality Life Scale (SF-12) were further used as secondary outcomes to evaluate the pain-related impairment of emotion and quality of life.The pre-and post-treatment values for each group were compared using paired t tests.

2. 5 |
MRI data acquisition fMRI images were acquired using a 3.0 T MRI scanner (GE, Discovery MR750) in the MRI Center of the Hospital of the Chengdu University of TCM.All participants were instructed to rest with their eyes closed, not to think of anything in particular, and not to fall asleep during the scan.The functional images were collected transversely using a gradient-recalled echo-planar imaging pulse sequence with the following settings: TR/TE = 2000/30 ms, flip angle = 90°, 40 slices, slice thickness = 5 mm, 64 × 64 matrix, field of view = 250 × 250 mm 2 , interslice gap = 0 mm, and voxel size = 3.75 × 3.75 × 5 mm 3 .The scan lasted for 7 min for each subject, and 210 functional volumes were obtained.
in the TA group (r = 0.37, p = 0.007), while those between the DR and right thalamus were inversely correlated with the duration of each attack improvement in the TA group (r = −0.32,p = 0.009).Moreover, changes in FC between the MR and right middle frontal gyrus were positively correlated with improvements in the NPQ (r = 0.34, p = 0.006) and SDS (r = 0.32, p = 0.009) scores, while those between the MR and parahippocampal gyrus were inversely correlated with improvements in the NDI (r = −0.31,p = 0.012) and SDS (r = −0.27,p = 0.033) scores in the TA group.

F I G U R E 3
Clinical outcomes in TA and SA groups, respectively.MPQ, McGill Pain Questionnaire; NDI, Neck Disability Index; NPQ, Northwick Park Neck Pain Questionnaire; SA, sham acupuncture; SAS, Self-rating Anxiety Scale; SDS, Self-rating Depression Scale; SF-12, 12-item Short Form Quality Life Scale; TA, true acupuncture; VAS, Visual Analog Scale.
sions, such as pain emotion and perception, in CNP through the FC of the MR.Correlation analysis revealed that the modulation of acupuncture on the FC of the DR was significantly correlated with improvement in the NDI and the duration of each attack.The modulation of the FC of the MR by TA was significantly associated with improvements in the NPQ, SDS, and NDI.The current findings suggest that acupuncture can regulate the DR-related circuit to alleviate and treat CNP, including the sensation and perception of pain.Moreover, acupuncture can reduce pain-related emotion impairment mainly by regulating the MR-related limbic circuit in CNP.Overall, the subdivision of the raphe nucleus may play a unique role in using acupuncture in treating CNP.

F I G U R E 4 F I G U R E 5
ancova analyses revealed the interaction effect of time and group factors.AMYG, amygdala; DR, dorsal raphe nucleus; INS, insula; L, left; LING, lingual gyrus; MFG, middle frontal gyrus; MR, median raphe nucleus; PHG, parahippocampal gyrus; R, right; THA, thalamus.The reconfiguration of TA on the raphe nucleusrelated serotonergic circuits in CNP.The warmer yellow color represents the higher F value in the ancova analyses.The yellow color line denotes TA increased the FC between the regions in CNP.The blue color line denotes TA decreased FC between the regions in CNP.AMYG, amygdala; DR, dorsal raphe nucleus; INS, insula; LING, lingual gyrus; MFG, middle frontal gyrus; MR, median raphe nucleus; PHG, parahippocampal gyrus; THA, thalamus.
TA B L E 2 Neuroimaging outcomes obtained by ancova analyses with time and group as factors.T values for comparison between different time points.p T*G based on Time*Group interaction.p G based on the comparison between different groups.