Effect and cerebral mechanism of moxibustion at heat-sensitized Yaoyangguan (GV3) in patients with lumbar disc herniation and myofascial pain syndrome by resting-state functionality magnetic resonance imaging: protocol for an observational study

YANG Jun,XIONG Jun,XU Shaozhong,XIE Hongwu,XIANG Jie

YANG Jun,XIONG Jun,XIANG Jie,Jiangxi University of Chinese Medicine,Nanchang 330004,China

XIONG Jun,Department of acupuncture and moxibustion,the Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China

XU Shaozhong,XIE Hongwu,Department of Radiology,the Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China

Abstract We want to explore the analgesic brain effect of the moxibustion at heat-sensitized Yaoyangguan (GV3) in patients with lumbar disc herniation (LDH) and myofascial pain syndrome (MPS).In an assessor-blinded observational study,we will include 15 LDH and 15 MPS.They will accept same treatment of heat-sensitive moxibustion at Yaoyangguan (GV3).The resting-state functionality magnetic resonance imaging image data of brain activities before and after treatment will be analyzed by mean fractional amplitude of low-frequency fluctuation,regional homogeneity analysis and brain functional connection.We select seed of first sensory cortex,second sensory cortex,insula cortex,periaqueductal gray and anterior cingulate cortex as the regions of interest to analyse the relationship between brain functional connectivity of pain-related networks and clinical data.Our study could disclose key brain targets and central response characteristics of the analgesic brain effect and the brain functional connection of heat-sensitive moxibustion.

Keywords: lumbar disc herniation;myofascial pain syndrome;point GV3 (Yaoyangguan);resting-state functionality magnetic resonance imaging;heat-sensitive moxibustion;mean fractional amplitude of low-frequency fluctuation;regional homogeneity;pilot projects

1.INTRODUCTION

Acupoint,designated locations along nerves or organ meridians,is the response point of disease as well as the operative site.It is closely related with meridians,viscera,Qiand blood.Researches have put forward that the function of acupoint is dynamic,it would regulate its nature and strength with the different functional status of its target organs.1Under pathological conditions,the site of the acupoint from resting to active.2Head first observed that when visceral lesions happened,some areas of the skin become more sensitive and even referred pain.3Team of professor Chen Rixin are dedicated to the research of heat-sensitive moxibustion(HSM).They have put forward that there are six HSM sensations,it appears alone or combination in clinically,10% in healthy and 70% in sick people,with the disease recovery those responses gradually disappear.4

Several systematic reviews and Meta-analysis have reported that HSM,as a safe method,is effective in relieving the pain of the patient,especially in lumbar disc herniation (LDH) and myofascial pain syndrome(MPS).5.6Although number of clinical trials have proved the effectiveness of HSM in relieving pain,few researches focus on the analgesia mechanism of heatsensitive.“Acupoints-brain relation hypothesis”suggested that the biological basis of acupoints must depend on the brain regulation effect.7By means of the image technology,it has demonstrated that nociceptive stimuli could activate several cortical network activities,those areas named as “pain matrix” participate the pain perception and process.8

It is believed that pain stimulation can specifically activate the brain regions matrix region,which includes the first sensory cortex (S1),second sensory cortex (S2),insula cortex (IC),anterior cingulate cortex (ACC) and periaqueductal gray (PAG).9S1 and S2 could regulate the sensory part of pain from stimulus localization,intensity and quality discrimination,which are the significant part of lateral pain system.The latter regions of IC and ACC could effectively regulate the emotional part of pain from emotional reactions and stimulus related selective attention,which are consist of the medial pain system.10,11Several studies showed that patients of chronic low back pain with structure and function variation in ACC.12,13PAG is an important center of the pain regulating nerve axis,which could regulate the nociceptive input from spinal dorsal horn and affect the sensory of the pain feeling.14

Recently a variety of functional neuroimaging technology have emerged to explore brain function,while functionality magnetic resonance imaging (fMRI)is widely used in the research of acupoint for its noninvasive,radiation-free and highly sensitive spatial discrimination.Task-state fMRI can reveal structural changes in the brain involved in specific stimuli or instructions;resting-state fMRI (rfMRI) can present the spontaneous activity of each brain area and analyze the data to reflect the changes of brain motor function network connection.15,16Study have showed that moxibustion at heat-sensitized Dubi (ST35) in patients with knee osteoarthritis (KOA) by rfMRI,the change of fractional amplitude of low-frequency fluctuation(fALFF) values and regional homogeneity (ReHo)values were statistically significant when compared with conventional moxibustion.17

Previous studies have proved the significance of the heatsensitized acupoint in patients with pain disease.We explore the central effect mechanism further,when patients with different pain diseases receive moxibustion at same heat-sensitized acupoint.We use rfMRI systematically to investigate the central brain effect and cerebral mechanism of the moxibustion at heatsensitized Yaoyangguan (GV3) in patients with LDH and MPS.

2.METHODS

2.1.Objectives

(a) Whether there is any difference in the central effect mechanism,when patients with different pain diseases receive moxibustion at same heat-sensitized acupoint.

(b) Identify the key brain targets and central response characteristics of the analgesic brain effect when people receive the HSM.

2.2.Study design and setting

The observational study will be conducted at the Affiliated Hospital of Jiangxi University of TCM in Nanchang,with inpatient from acupuncture and moxibustion department and orthopedics department.The study will be sequentially performed in five period,the flowchart presented in Figure 1.

Figure 1 Flowchart of the participants

2.3.Ethics approval and consent to participate

The trial follows the principal ethical guidelines of Helsinki Declaration and has received approval from the Independence Ethics Committee of the Affiliated Hospital of Jiangxi University of TCM (ethic approval number: JZFYKYLL20200525013).Each enrolled participant will provide written informed consent and be asked to sign for it before participating.Our study has been registered in the Chinese Clinical Trial Registry(ChiCTR2000035027).The trial was approved by the research ethics committee.The results of the study will be published in a peer-reviewed academic journal and will also be disseminated electronically through conference presentations.

2.4.Participants

Eligible participants met diagnostic criteria of MPS(Simons 1990 clinical criteria)18or LDH (1995,Guiding principles of clinical research on the treatment of lumbar intervertebral disc herniation with new Chinese medicine)will be recruited.19

2.5.Eligibility criteria

(a) Patients aged 16-60 years,male or female;(b) Righthanded;(c) With normal consciousness,coordinated behavior,they can correctly express the moxibustion sensation;(d) Acupoint heat-sensitization phenomenon exists in the region of Yaoyangguan (GV3).The location of Yaoyangguan (GV3) is on the lower back which is below the 4th lumbar spine.The following patients sensation will suggest the acupoint heat-sensitization:heat penetration means the heat sensation conducting from the moxa local skin surface into deep tissue,or even into the lumbar vertebra or abdomen;heat expansion means the heat sensation spreading the surrounding little by little around the moxa point;heat transmission means the heat sensation transferring along some pathway or direction,even to the back or thigh;local not or slightly but far heat,surface not or slightly but deep heat and nonheat-sensitive sensations.Some patients may exist more than one heat-sensitive sensation;(e) Modified Japanese Orthopaedic Association low back pain score (M-JOA) ＞10;(f) Sign the informed consent by the patient himself or voluntarily.

2.6.Exclusion criteria

(a) Single nerve palsy or cauda equina paralysis manifested as muscle paralysis or rectal and bladder symptoms;(b) lumbar spinal stenosis and space occupying lesions caused by other reasons,or lumbar tumor,infection,tuberculosis,etc.;(c) combined with serious life-threatening primary diseases referred to cardio cerebrovascular system,liver,kidney and hematopoietic system,as well as psychosis;(d) pregnant or lactating women;(e) fainting during moxibustion or repelling moxibustion;(f) patients who are prohibited by magnetic resonance testing.

2.7.Sample size

It is not applicable to calculate the sample size for lacking of similar research.Professor Thirion and José M.Soares suggested that an optimal sample size of 16-32 could provide adequate reliability for fMRI investigations.20.21So we plan to include 30 participants in our study.

2.8.Allocation

Eligible patients will be allocated into two parallel groups after baseline assessment: LDH group and MPS group.Therapists get professional training to ensure each participants receive the same treatment.Assessors who did not participate in the treatment and who are blinded to the allocation results will perform the outcome assessment.

2.9.Participant timeline

Our study will be carried out sequentially as follows:enrollment period of 6 months to set up the research group and form the operation manual,establish information acquisition database framework to select eligible patients to participate the research;a run-in period of one week prior to allocation,which give the patient enough time to familiar with the process of the research;a treatment period of 14 d (7 sessions per week);assessment period with five phrase (1st day before and after treatment,the 7th day during treatment,the 14th day and 21th after treatment).The schematic diagram is shown in Figure 2.

Figure 2 Study schedule for data collection

2.10.Intervention

(a) Environment: in a quiet,sealing,opacity room and temperature between 24 ℃ to 30 ℃;(b) position: the patient lies on the bed in prone position and fully expose the moxibustion site;(c) material: we choose the moxa stick manufactured by Henan Nanyang Hanfang Aiye Co.,Ltd.,China.The specification parameter is 12 cm(length) × 3 cm (diameter);(d) therapists: those who have received professional training with clinical experience more than 5 years in acupuncture and moxibustion are qualified;(e) method: clinical studies showed that Yaoyangguan (GV3) is high frequency point for MPS and LDH with great therapeutic effect.22Therapist hold the ignite moxasticks over the region of Yaoyangguan(GV3) about distance of 3cm to search acupoint heatsensitive phenomenon.The specific operation should refer to the book of Heat-sensitive moxibustion practical reader.23(f) course: patients receive the treatment once a day for 15 min,a total of 14 sessions.

2.11.rfMRI

rfMRI is performed by 3.0TSigna scanner (Sign HDx 3.0T,General Electric Company,Wauwatosa,WI,USA)at the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine,China.Each participant will receive 5 MRI sessions (one structural image before treatment,4 functional images after treatment on the 1st,7,14,21th).Before scanning,patients should be familiar with the surroundings,and they are asked to wear blinder and earplugs to reduce audio-visual interference.They are introduced to close their eyes and think of nothing without falling asleep.Head motion is minimized using foam pads and a headband.At the beginning of each session,an 8 min 3D-T1 weighted structural image is required to use gradient echo sequence to scan the whole brain,which is acquired with TR/TE/FA=12 ms/4.2 ms/15°,a field of view (FOV) of 240 × 240 mm2with 249 slices with 1.2 mm slice thickness,interval of 1.0 mm,a matrix size of 64 × 64 mm and resolution of 1 mm.The 7 min 10 s functional images are required to use echo-planar imaging (EPI) sequence,which is equipped with TR/TE/FA=2000 ms/40 ms/90°,33 slices with 5 mm thickness,interval of 1.0 mm,4 × 4 × 4 mm voxel size,FOV of 240 × 240 mm2,a matrix size of 64 × 64 mm.

2.12. Outcomes

McGill Pain Questionnaire (MPQ) is used to assess pain severity and its characteristics of MPS,and Modified Japanese Orthopaedic Association scale (M-JOA),visual analog scale (VAS) are designed to measure intensity scores of LDH.24.25Fear-Avoidance Beliefs Questionnaire (FABQ) is used to assess the pain affects to the physical activity and working.26The outcome measured above will be assessed before treatment,7,14,and 21 d.Therapeutic effect was assessed by comparing baseline and final conditions reported by the patient.Adverse effects during treatment will be recorded on the trial.Once it happens,stop the research immediately and take relevant measures to ensure the safety of the participants.

2.13.Adverse events

We define adverse events as unfavorable or unintended signs,symptoms or disease occurring after treatment.Such as blister,scald,swelling,dry cough and so on.In every visit,adverse events will be reported by participants and examined by the practitioner.

2.14.Data acquisition and preprocessing

The process will be conducted by DPARSFA2.2 (Data Processing Assistant for rfMRI) and REST V1.8.Before the data analysis,we should adjust the data form from DICOM images to NIFTI in order to facilitate data processing and matching corresponding software functions.We will respectively move the structural image and functional images into the T1Raw file and FunRaw file.Data preprocessing includes removing first time point,slice timing,realignment,image registration,T1 segmentation,normalization to the MNI space,smoothing,linear detrending and bandpass temporal filtering (0.01-0.08 Hz).

2.15.Data analyses

Demographic and clinical data (e.g.,MPQ,M-JOA,VAS and FABQ) will be analyzed by SPSS 24.0 (IBM Corp.,Armonk,NY,USA).The level of significance was set asP ＜0.05.We select seed of S1,S2,IC,ACC and PAG as the regions of interest (ROI) to analyze the relationship between brain functional connectivity of pain-related networks and clinical data.Mean fractional amplitude of low-frequency fluctuation (mfALFF)analysis and ReHo analysis will be performed by Statistical Parametric Mapping SPM8 (https://www.fil.ion.ucl.ac.uk/spm/software/spm8/) and REST 1.827(http://www.restfmri.net/ forum/ REST_V1.8).Pairedttest will be used to assess the difference in mfALFF and ReHo between the patients with LDH and MPS.Significant differences are set at the threshold of voxelwiseP ＜0.05 (corrected),with a cluster size ≥ 85 voxels.In addition,pearson correlation coefficients will be used to estimate the connection between the seed region and all of the voxels within the whole brain separately based on different patients and conditions.The standardized ReHo Z-maps apply to subsequent group-level analysis.Significant differences are set at the threshold of voxelwiseP ＜0.01 (corrected),cluster size ＞ 2295 mm3.Intragroup standardized values of mfALFF and ReHo maps will be constructed by computing a pairedt-test between baseline and after HSM mfALFF and ReHo values (AlphaSim correction atP ＜0.05,edge connected cluster connectivity criterion,rmm=5,continuous voxel≥ 85).

3.DISCUSSION

Pain is an unpleasant sensation when people receive the actual or potential tissue injury which is detected by nerve endings of nociceptive neurons.28The negative cognition and emotion accompanied by pain bring a great burden to the life and work of patients.Statistically,chronic pain affects about a fifth of adults worldwide,about 30%-45% of people with chronic pain suffer from severe depression.29.30Clinical trials showed that the appearance of heat-sensitive phenomenon is highly related to the disease state.Under the pathological condition,the state of acupoint become sensitive,as the disease improving,the rate of heat-sensitive phenomenon could reduce by 15%.31The relationship between sensitized acupoints and external intervention summarizes as “small stimuli with big responses”.23

There are some studies about HSM for pain diseases.Effect of multiple brain regions with significant change is basically consistent with the somatic pain temperature conduction path after HSM,and the somatosensory system activation may be the objective basis of meridian sensation transmission phenomenon.32The network of multiple brain regions achieves the brain functional regulation on the intervention of HSM.It regulates the emotional part of the network central control through ACC-IC medial pain system.17

We conduct the study to explore the analgesic brain effect of the moxibustion at heat-sensitized Yaoyangguan (GV3) in patients with LDH and MPS by rfMRI.The technique of rfMRI based on changes in brain function during resting states,or spontaneous neural activity.It is uncomplicated to operate with high compliance for without repeated task stimulation.Considering the characteristics of noninvasive,high spatial and temporal resolution,it is widely used in many fields of neuroscience and psychology to clarify the brain functional connections.

We select various method to analyze the data and images.The mfALFF analysis is applicable to rfMRI,it could directly reflect the brain function changes and amplitude of BOLD oscillations on the resting state without the time interference.33ReHo analysis can effectively reflect the homogeneity between different voxel time series in a functional area.The pain sensation regulated from peripheral (spinal cord),subcortical and cortical three levels.34When people receiving the pain signals,they are transmitted by ascending projection fibers to the various centers and received multiple integration.On the process,the ACC and IC play great role.The IC receives signals from the thalamus and involves the regulation of sensorimotor,pain and emotion from multiple angles.35It is known as the center of the endosensory system with closely connection of emotion expression.ACC mainly accepts the signals from the medial thalamus nucleus and involves in the processing and modulation of pain information.36The metabotropic glutamate receptor 5(mGluR5) in the PAG maintain an appropriate sensory perception in normal conditions.While in the neuropathic pain condition,the disrupted of persistent mGluR5 activity would result in chronic pain.37

There is great significant in chronic visceral pain and sensitivity of functional connectivity to PAG localization.38So we choose HSM as a sensory stimulus and select S1-S2-IC-ACC-PAG seed as ROI to analyze functional connections.It can assess the strength of significant connections between functional brain regions and reflect the function of brain as network structure.

We hope our study could disclose key brain targets and central response characteristics of the analgesic brain effect and the brain functional connection of HSM.It would be helpful for the clinical application of HSM in pain relieving.