Articles

Abdominal Acupuncture for Non-Responding TMD Patients: a Retrospective Observational Study in General Practice
Department of Oral and Maxillofacial Sciences, “Sapienza” University of Rome, Rome, Italy
Correspondence to:This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Acupunct Meridian Stud 2023; 16(2): 56-64
Published April 30, 2023 https://doi.org/10.51507/j.jams.2023.16.2.56
Copyright © Medical Association of Pharmacopuncture Institute.
Abstract
Objectives: To analyze the efficacy of AA for the treatment of patients with subacute and chronic pain related to TMD and non-responding to previous conventional therapies (occlusal splint, medications, physical therapy).
Methods: Twenty-eight patients, 24 F and four M (mean age 49.36 years), were recruited from January 2019-February 2021. All patients underwent AA treatment: two sessions per week for four weeks, for a total of eight sessions. At the beginning of therapy (T0) and at the end of the cycle (T1) the following data were evaluated: maximum mouth opening (MMO); cranio-facial pain related to TMD (verbal numeric scale, VNS); pain interference with normal activities and quality of life of patients (Brief Pain Inventory, BPI); oral functioning (Oral Behavior Checklist, OBC); impression of treatment effectiveness (Patients' Global Impression of Improvement, PGI-I Scale). Statistical comparison of data before and after the AA treatment was performed by Wilcoxon's signed-rank test (significance level p < 0.05).
Results: The MMO values were significantly improved after one cycle of AA (p = 0.0002). In addition, TMD-related pain had a statistically significant decline following AA treatment (all p < 0.001). Patients’ general activity and quality of life (BPI) were described as improved following a course of AA, with statistically significant values for all aspects considered (all p < 0.05).
Conclusion: Abdominal acupuncture resulted in effective treatment of subacute/chronic-resistant pain related to TMD, capable of improving mandibular function and facial pain, and reduced the interference of pain affecting patients' quality of life.
Keywords
INTRODUCTION
Temporomandibular disorders (TMD) refer to a group of conditions affecting the masticatory muscles, temporomandibular joint (TMJ), and associated surrounding structures such as connective tissues and ligaments [1]. These pathologies are characterized by a complex symptomatology consisting mainly of joint noises, joint and/or muscle pain, and impaired or limited mandibular movements [1-3]. TMD cause great suffering in patients and are a widespread problem in clinical practice, representing the most common orofacial pain (OP) diagnosis [3-5]. Patients with TMD have painful symptoms associated with the purely articular or muscular type, such as headache and neck pain. As with many other types of chronic pain, TMD-related pain, and especially myalgia/myofascial pain, often limits the daily life activities of patients [2,4,6,7]. TMD-related pain is described as one of the most common forms of musculoskeletal pain [8]. Subjects with chronic pain conditions experience other comorbid chronic pain conditions; painful TMD has been frequently associated with widespread pain [9]. In fact, patients with painful TMD seem to be more sensitive to experimental pain stimuli and have a lower pain tolerance than non-TMD subjects [10-12].
Acupuncture therapy (AT) is an effective method of pain management, especially for pain of musculoskeletal origin, including TMD [13,14]. Several clinical studies analyzed the effects of different types of acupuncture treatments on pain related to TMD and proven its effectiveness, especially for pain of muscular origin, such as myalgia and myofascial pain [15-17]. In the OP field, classic somatic AT is the most investigated AT method, followed by laser acupuncture, electroacupuncture and several microsystems AT methods, such as ear, scalp, mouth, and fingers [16-19].
Abdominal acupuncture (AA) is a relatively new microsystem technique, which was developed in the last 30 years by Prof. Bo Zhi-Yun [20]. Bo’s Abdominal Meridian System (AMS) resulted from the combination of traditional Chinese meridian theory and his innovation in considering the acupoint Shenque (CV8), which corresponds to the navel, as the “mother-point” for regulating the distribution of
Given the well-known clinical effectiveness on chronic and resistant pain conditions, we applied AA at our University outpatient acupuncture clinic, as elective AT treatment for our patients with TMD affected by cranio-cervico-facial pain that does not respond to conventional OP treatment. The study presented below was performed retrospectively, to investigate and verify the effects of AA on pain and its interference with common activities and quality of life of patients with TMD, through the analysis of medical records and questionnaires that are routinely used in our specialistic ward.
MATERIALS AND METHODS
This study was a retrospective observational study in general practice. Patients referred to our acupuncture clinic in a University Hospital specialistic Unit of Orofacial Pain, between January 2019 to February 2021, were included. Forty-three patients were assessed and enrolled for AA treatment. Due to the COVID-19 pandemic during the period March-November 2020 (10 patients), or the distance to the hospital or job difficulties (2 patients), a total of 12 patients were excluded from the analysis for not finishing the full AT treatment; three patients were excluded for not completing the provided questionnaires. In total, 28 patients with subacute and chronic TMD-related pain non-responding to conventional treatments were fully treated and data analyzed. All patients were diagnosed with TMD pain (ICD-9 729.1; ICD-9 524.62; ICD-9 339.89; ICD-9 524.63) greater than or equal to 30 on the Numeric Verbal Scale (NVS), according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) classification [1]; the frequency of pain was greater than or equal to 1 time/week. All patients had an history of previous unsuccessful treatments such as advice or counseling regarding oral functioning, administration or topical use of pain medication, physical therapy, and/or an occlusal appliance.
As standard practice in our ward, all patients signed an informed consent form before undergoing acupuncture therapy, concerning both the treatment and the possible anonymous use of their clinical data for scientific and publication purposes. Participants also filled in validated questionnaires regarding their oral and general health conditions, before and after the AA treatment. The study was retrospectively approved by the Institutional Ethics Committee (N.112/22/0000868) and followed STROBE guidelines [23].
1. Abdominal acupuncture treatment
The patients were treated two times per week for four weeks with abdominal acupuncture (AA) by an experienced physician and acupuncture licensed specialist (ES). The acupuncture points used were according to the Bo’s Method of Abdominal Acupuncture [20-22]: CV 12 (Zhongwan), CV10 (Xiawan), CV6 (Qihai), CV4 (Guanyuan), ST24 (Huaroumen) bilaterally, EX - Ab1 (Shangfengshi) bilaterally, KI17 (Shangqu) and KI19 (Yindu) mono or bilaterally, according to pain location (Fig. 1).
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Figure 1.Bo’s abdominal acupuncture scheme: the lines in the left hemiabdomen show the “abdominal turtle pattern” according to Bo [20]. The selected acupoint are circled in red: CV-12 (4 cun above the navel); CV-10 (2 cun above the navel); CV-6 (1.5 cun below the navel); CV-4 (3 cun below the navel CV-8); KI-19 (0.5 cun lateral CV-12); KI-17 (0.5 cun lateral CV-10); ST-24 (2 cun lateral CV-9, located 1 cun above the navel); Ex-ab1 (0.5 cun lateral and 0.5 cun above ST-24).
Needles were inserted at the penetration site after asepsis of the skin with 70% alcohol. The needles were disposable and sterilized, individually packed, size 0.22 × 40 mm (TEWA, ASIA-MED GMBH & CO. KG). According to Bo’s acupuncture method, the depth of needle penetration was “standard depth” for all selected points, except for KI19 (“superficial depth”) [20]. The needles were not manipulated, and they remained in place for 30 minutes and were then removed.
2. Outcome measures and data analysis
Temporomandibular joint (TMJ) pain, masticatory muscle pain, headache, and neck pain were the symptoms assessed, and each one was measured through the 0-100 VNS. The maximum mouth opening (MMO) was measured in all patients, recording the interincisal distance in mm using a ruler (mouth opening without assistance).
All patients were requested to complete the following questionnaires. The Oral Behavior Checklist (OBC) estimated oral functioning. The questionnaire consisted of 21 items rated on a five-point Likert scale (0-4) asking about the frequency of parafunctional behaviors within the last month [1]. The Short Form of the Brief Inventory Pain (BPI) assessed the severity of pain and its impact on daily function, and was comprised of nine items rated on a 0-10 Visual Numeric Scale (VNS) asking the patient to rate the severity of pain and of pain-related disability in the last 24 hours [24]. The Patients’ Global Impression of Improvement (PGI-I) Scale is a global index indicating the response of a condition to a therapy; it evaluated patients’ impression of treatment effectiveness [1].
Craniofacial pain intensity (NVS), pain and pain-related disability (BPI) and MMO were measured at the following times:
T0: Baseline, before AA treatment
T1: End of AA treatment, four weeks after T0 (after the last acupuncture session)
At T1, also the patient’s impression of treatment effectiveness (PGI-I) was determined.
The statistical processing software SPSS (version 23) was used for data analysis. Normality of data distributions was examined using the Shapiro-Wilk test. The Wilcoxon signed-rank test was used to compare the VNS, MMO, OBC and BPI results before treatment (T0) with those obtained after AA (T1). For all tests, the level of significance was set at
RESULTS
A total of 28 patients, 24 females (85.7%) and four males (14.3%), with an average age of 49.36 years, were fully treated and analyzed. Nineteen (19) patients had bilateral TMD, and nine had unilateral TMD. From the onset of pathology, the range in the duration of symptoms was 4-144 months. Symptoms lasted for ≤ 6 months (subacute TMD) in five patients, and for > 6 months (chronic TMD) in the remaining 23 patients. Ten (10) patients were affected by a systemic painful condition (Table 1). No side effects or complications were observed with respect to the AA procedure itself and all patients tolerated the treatment well.
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Table 1. Patients’ demographic and clinical baseline characteristics
No. Gender Age Diagnosis Side MMOb (mm) Pain duration (month) Systemic disease Past treatment Arthralgia Myalgia DDWRa 1 F 49 × B 39 5 PT, ST 2 F 65 × R 39 4 ST 3 F 52 × × × B 36 144 FM, UCTD D, PT, ST 4 F 46 × × L 42 18 D, PT, ST 5 F 54 × × B 41 84 FM D, PT, ST 6 F 49 × B 40 60 FM, PsA D, PT, ST 7 F 57 × B 42 120 FM, UCTD D, PT, ST 8 F 44 × B 44 24 D, PT, ST 9 F 63 × R 39 48 OP D, ST 10 F 73 × × × L 40 18 RA D, PT, ST 11 F 61 × B 41 6 OP D, ST 12 F 45 × R 42 18 PT, ST 13 F 27 × B 34 42 D, PT, ST 14 M 28 × B 43 12 PT, ST 15 F 48 × B 40 4 D, ST 16 M 53 × B 43 36 D, PT, ST 17 F 56 × × L 38 96 FM D, PT, ST 18 F 37 × B 37 24 PT, ST 19 F 62 × × B 49 84 RA D, PT, ST 20 M 55 × B 43 30 D, PT, ST 21 F 48 × × × R 35 60 D, PT, ST 22 F 61 × B 38 120 D, PT, ST 23 M 32 × B 39 7 PT, ST 24 F 18 × B 38 24 ST 25 F 45 × × L 36 18 D, PT, ST 26 F 60 × B 42 144 FM D, ST 27 F 52 × L 40 60 D, PT, ST 28 F 42 × × B 42 4 ST aDDWR = disc displacement with reduction; bMMO = maximum mouth opening.
D = drugs; FM = fibromyalgia; OP = osteoporosis; PsA = psoriatic arthritis; PT = physical therapy; RA = rheumatoid arthritis; differentiated Connective Tissue Disease.
The application of AA resulted in a general improvement in MMO, oral functioning, patients’ pain perception and pain interference with normal activities and quality of life (Table 2, Fig. 2, 3). The difference in MMO and the improvement in oral functioning (OBC) and pain perception, according to both NVS and BPI, at T0 and T1, resulted in statistical significance for all the considered variables (all
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Table 2. Effect of abdominal acupuncture in the study population on: mouth opening (MMO), oral functioning (OBC), TMD-related pain (VNS), general pain and pain interference (BPI)
Variable Before treatment–T0 After treatment–T1 Z p -valueMMO 40.07 ± 3.13 41.82 ± 2.52 –3.724 0.0002 OBC 45.43 ±13.85 37.68 ±13.99 –3.959 < 0.0001 Pain (VNS) TMJ pain 34.64 ± 39.11 7.50 ± 15.54 –3.408 0.0006 Muscle pain 37.5 ± 4.88 14.28 ± 23.32 –3.296 0.0009 Headache 62.14 ± 33.81 22.50 ± 27.16 –4.197 < 0.0001 Neck pain 64.46 ± 35.68 28.21 ± 26.39 –4.197 < 0.0001 Brief pain inventory (BPI) Pain (VAS) 1 6.43 ± 2.20 3.46 ± 2.22 –4.372 < 0.0001 2 3.21 ± 2.29 1.11 ± 1.42 –3.602 0.0003 3 5.21 ± 1.83 2.32 ± 1.87 –4.493 < 0.0001 4 4.78 ± 2.68 1.36 ± 1.61 –4.2378 < 0.0001 Pain interference (VAS) General activity 4.03 ± 2.38 1.89 ± 2.02 –4.107 < 0.0001 Mood 5.68 ± 2.88 2.68 ± 2.11 –4.197 < 0.0001 Walking ability 1.89 ± 2.79 0.89 ± 1.69 –2.665 0.0084 Normal work 3.64 ± 2.69 1.61 ± 1.90 –3.919 0.0001 Relations with other people 3.11 ± 2.30 1.93 ± 2.29 –3.296 0.0009 Sleep 4.71 ± 2.76 3.00 ± 2.71 –3.621 0.0003 Enjoyment of life 3.28 ± 2.55 1.82 ± 1.90 –3.621 0.0003 1 = WORST pain in the last 24 h; 2 = MINIMUM pain in the last 24 h; 3 = AVERAGE pain in the last 24 h; 4 = pain value in this moment.
Z,
p -value: Wilcoxon signed-rank test results.
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Figure 2.Patients TMD-related pain distribution at T0 and T1, according to different locations: TMJ, Masticatory muscle, Head and Neck. Higher NVS scores indicate more severe pain. The horizontal bar inside the boxes indicates the median, the x inside the boxes indicates the mean, and the lower and upper ends of the boxes are the first and third quartiles. The whiskers indicate the minimum and the maximum ranges, and data more extreme than the whiskers are plotted individually as outliers (circles).
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Figure 3.Distribution at T0 and T1, according to BPI, of (A) TMD-related pain and (B) pain interference in common activities and quality of life. (A) 1 = WORST pain in the last 24 h; 2 = MINIMUM pain in the last 24 h; 3 = AVERAGE pain in the last 24 h; 4 = pain value IN THIS MOMENT. (B) A = General activity; B = Mood; C = Walking ability; D = Normal work; E = Relations with other people; F = Sleep; G = Enjoyment of life. Higher BPI scores indicate more severe pain (A) and more severe pain interference in quality of life’s aspects (B). The horizontal bar inside the boxes indicates the median, the x inside the boxes indicates the mean, and the lower and upper ends of the boxes are the first and third quartiles. The whiskers indicate the minimum and the maximum ranges, and single data or data more extreme than the whiskers (outliers) are plotted individually as circles.
Table 1 displays the participants’ baseline demographic and clinical characteristics. Table 2 illustrates the exact results from data analysis. Fig. 2, 3 indicate the distribution of patients’ pain and interference of pain perceptions, at T0 and T1, according to NVS (Fig. 2) and BPI (Fig. 3) scales. Fig. 4 presents the results of patients’ self-evaluations of treatment effectiveness using the PGI-I scale.
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Figure 4.Patients’ impression of the treatment effectiveness at T1, according to the PGI-I Scale.
DISCUSSION
According to Traditional Chinese Medicine (TCM), the abdomen is categorized as a type of acupuncture microsystem, i.e., a well-defined anatomical area in which all the structural and functional components of the human organism are represented, a miniature projection of the human body and its functions. The microsystems are somatotopic areas, in which each specific point represents a well-defined point of correspondence in the human body [25]. However, compared with other microsystems, such as the auricle, the scalp, the foot, or the hand, the abdomen is considered unique in its location. For TCM theory, the abdominal area (located behind the acupoints Qihai (CV6) and Guanyuan (CV4)) is the source of the human body’s qi (vital energy) and jing (essence) resides [20-22,26]. In addition to the traditional meridian system, Dr. Bo Zhi-Yun discovered two other regulatory systems at different layers of the abdomen wall [20,26]. These systems develop from the fetal circulatory system, which delivers blood and nutrients to the fetus via the umbilical cord, and which serves as a regulatory system of the human body during uterine life. Immediately after birth, fetal circulation undergoes a rapid transition to accommodate extra-uterine life [27]. However, the umbilicus has many embryological remnants, and these elements of the prenatal system and its ability to distribute qi and blood still exist in adults, and are centred at the navel, where the acupuncture point Shenque (CV8) is located [28]. Therefore, the abdominal region is an extremely valuable energetic area in acupuncture practice, center and origin of all the body’s dynamic forces and functional activities, through which it is possible to treat all parts of the body. Thanks to these unique characteristics, Bo’s method of abdominal acupuncture has been widely applied by acupuncture practitioners since its systematization in 1991, and has rapidly spread throughout the world. The few studies investigating the clinical effects of AA in the current scientific literature support the efficacy of this method and have led to results of general improvement of pain symptoms, including musculoskeletal disorders [29-33]. This evidence is in line with the results obtained in the present study, which reports for the first time, the application of AA for treating TMD-related pain and symptoms.
In addition, in modern scientific science, the abdomen represents a crucial part of the human body. This anatomical area has been intensely investigated in the scientific literature, and it is referred to as a “second brain”– the “abdominal brain” [34,35]. The relationship between the enteric nervous system (ENS), gut microbiota and neurological diseases, including chronic pain, is receiving increasing attention from researchers. The enteric nervous system shares many sensory and motor neurons, information processing circuits, neurotransmitters, receptors, and transcription factors with the central nervous system (CNS) [36]. Different studies on gastrointestinal behavior revealed a highly complex interaction among the microbiota, the bowels, and the brain through the so-called “gut-brain axis”, a bidirectional neurohumoral connection system regulated by spinal and vagal visceral pathways [35,37]. It was noted that ENS and its extended communication system could influence irritable bowel syndrome, psychiatric disorders, and neurological conditions among others [36,37]. In addition, ENS plays an important role in inflammatory and nociceptive processes, influencing neurogenic inflammation in visceral pain [38,39]. Furthermore, gut microbiota and its modulation has been described as anti-nociceptive in visceral, inflammatory, and neuropathic pain models in animal studies [36,38,39]. Its potential critical role was suggested in many other types of chronic pain, including inflammatory pain, neuropathic pain, headache and widespread musculoskeletal pain [40-43].
Some animal studies investigated the effects of somatic acupuncture on ENS and gut microbiota during the treatment of different diseases [44-46]. Jang et al. (2020) [45] suggested an association between the effects of acupuncture in enhancing motor function and protecting dopaminergic neurons, and the regulation of the gut microbial dysbiosis, determining the inhibition of neuroinflammation in mice affected by Parkinson’s disease. Hong et al. (2020) [46] revealed a potential role of the gut-brain axis in increasing appetite and improving insomnia during acupuncture therapy. Visceral hyperalgesia was attenuated by electroacupuncture (EA), through down-regulation of central serotonergic activities in the brain-gut axis. Other studies discovered that EA at auricular points could increase mRNA expression of the 5-HT1a receptor in both the colon (peripheral) and raphe nuclei (central) in rats with induced visceral pain [47,48].
This evidence may be the basis for explaining the mechanisms behind the clinical efficacy of Bo’s AA technique, particularly for visceral pathologies, pain, and neurological disorders. This method resulted in the effective management of TMD pain and related interference on life aspects like “sleep” in the present study. Furthermore, the insertion of needles directly on abdominal acupuncture points could suggest an amplified action of the ENS, thanks to the localized acupuncture-mediated anti-inflammatory effects already demonstrated in the literature [49,50]. This could further explain the recognized clinical evidence of a short-term therapeutic response to this acupuncture technique, especially for pain, that is reinforced by the results of the present study. Indeed, even after only one course of AA treatment (eight sessions) our patient population, characterized by subacute/chronic TMD pain non-responding to conventional gnathological therapy, conveyed a statistically significant decrease in pain, and pain-related disability in common activities and quality of life, for all the variables considered except for the aspect “walking ability” (Table 2). The parameters concerning mouth opening and general oral functioning was significantly improved (Table 2). These results are consistent with various clinical studies that support the abdominal acupuncture technique for the treatment of other kinds of musculoskeletal disorders such as cervical spondylosis, prolapse of lumbar intervertebral disc, knee osteoarthritis and shoulder periarthritis [30,31].
Furthermore, the AA clinical methodology has two important characteristics: (1) needles, after their insertion into the acupoints, are not manipulated; and (2) the location of the acupuncture points is particularly accurate, because the abdominal points are located by the practitioner measuring the horizontal and vertical lines of the AMS scheme with a ruler and converting the corresponding traditional measure (cun) into centimeters [26]. So this technique, while avoiding needle stimulation and using a millimetric definition of acupoint location, is an easy-to-be-standardized repeatable method, as required by research methodology.
There are significant limitations to this study. Despite the evidence of clinical efficacy of AA on dysfunctional non-responder patients, this is a retrospective study on a small population of subjects. In addition, the data are not homogeneous regarding the presence or absence of painful systemic diseases. The promising results obtained, however, lay the basis for a randomized controlled clinical trial with a placebo group. Clinical trials are necessary to confirm the effectiveness of AA and to better explore the therapeutic possibilities of a microsystem method that is currently little investigated, but has strong performance in clinical acupuncture practice, especially for chronic pain.
AUTHORS’ CONTRIBUTIONS
Conceptualization: E. Serritella, C. Di Paolo; Data curation and Formal analysis: E. Serritella, G. Galluccio; Investigation: E. Serritella; Project administration: C. Di Paolo; Supervision: E. Serritella, G. Galluccio, C. Di Paolo; Validation: E. Serritella, C. Di Paolo; Writing - original draft: E. Serritella; Writing - review & editing: E. Serritella, G. Galluccio, C. Di Paolo.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Related articles in JAMS

Article
Research Article
J Acupunct Meridian Stud 2023; 16(2): 56-64
Published online April 30, 2023 https://doi.org/10.51507/j.jams.2023.16.2.56
Copyright © Medical Association of Pharmacopuncture Institute.
Abdominal Acupuncture for Non-Responding TMD Patients: a Retrospective Observational Study in General Practice
Emanuela Serritella *, Gabriella Galluccio
, Carlo Di Paolo
Department of Oral and Maxillofacial Sciences, “Sapienza” University of Rome, Rome, Italy
Correspondence to:Emanuela Serritella
Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
E-mail emanuela.serritella@uniroma1.it
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Pain related to Temporomandibular Disorders (TMD) is severe, negatively affecting patients' quality of life, and often resistant to conventional treatments. Abdominal Acupuncture (AA) is known to be particularly effective for pain, especially chronic and musculoskeletal pain, but it is still poorly studied and never investigated in TMD patients.
Objectives: To analyze the efficacy of AA for the treatment of patients with subacute and chronic pain related to TMD and non-responding to previous conventional therapies (occlusal splint, medications, physical therapy).
Methods: Twenty-eight patients, 24 F and four M (mean age 49.36 years), were recruited from January 2019-February 2021. All patients underwent AA treatment: two sessions per week for four weeks, for a total of eight sessions. At the beginning of therapy (T0) and at the end of the cycle (T1) the following data were evaluated: maximum mouth opening (MMO); cranio-facial pain related to TMD (verbal numeric scale, VNS); pain interference with normal activities and quality of life of patients (Brief Pain Inventory, BPI); oral functioning (Oral Behavior Checklist, OBC); impression of treatment effectiveness (Patients' Global Impression of Improvement, PGI-I Scale). Statistical comparison of data before and after the AA treatment was performed by Wilcoxon's signed-rank test (significance level p < 0.05).
Results: The MMO values were significantly improved after one cycle of AA (p = 0.0002). In addition, TMD-related pain had a statistically significant decline following AA treatment (all p < 0.001). Patients’ general activity and quality of life (BPI) were described as improved following a course of AA, with statistically significant values for all aspects considered (all p < 0.05).
Conclusion: Abdominal acupuncture resulted in effective treatment of subacute/chronic-resistant pain related to TMD, capable of improving mandibular function and facial pain, and reduced the interference of pain affecting patients' quality of life.
Keywords: Temporomandibular disorders, Orofacial pain, Acupuncture, Abdominal acupuncture
INTRODUCTION
Temporomandibular disorders (TMD) refer to a group of conditions affecting the masticatory muscles, temporomandibular joint (TMJ), and associated surrounding structures such as connective tissues and ligaments [1]. These pathologies are characterized by a complex symptomatology consisting mainly of joint noises, joint and/or muscle pain, and impaired or limited mandibular movements [1-3]. TMD cause great suffering in patients and are a widespread problem in clinical practice, representing the most common orofacial pain (OP) diagnosis [3-5]. Patients with TMD have painful symptoms associated with the purely articular or muscular type, such as headache and neck pain. As with many other types of chronic pain, TMD-related pain, and especially myalgia/myofascial pain, often limits the daily life activities of patients [2,4,6,7]. TMD-related pain is described as one of the most common forms of musculoskeletal pain [8]. Subjects with chronic pain conditions experience other comorbid chronic pain conditions; painful TMD has been frequently associated with widespread pain [9]. In fact, patients with painful TMD seem to be more sensitive to experimental pain stimuli and have a lower pain tolerance than non-TMD subjects [10-12].
Acupuncture therapy (AT) is an effective method of pain management, especially for pain of musculoskeletal origin, including TMD [13,14]. Several clinical studies analyzed the effects of different types of acupuncture treatments on pain related to TMD and proven its effectiveness, especially for pain of muscular origin, such as myalgia and myofascial pain [15-17]. In the OP field, classic somatic AT is the most investigated AT method, followed by laser acupuncture, electroacupuncture and several microsystems AT methods, such as ear, scalp, mouth, and fingers [16-19].
Abdominal acupuncture (AA) is a relatively new microsystem technique, which was developed in the last 30 years by Prof. Bo Zhi-Yun [20]. Bo’s Abdominal Meridian System (AMS) resulted from the combination of traditional Chinese meridian theory and his innovation in considering the acupoint Shenque (CV8), which corresponds to the navel, as the “mother-point” for regulating the distribution of
Given the well-known clinical effectiveness on chronic and resistant pain conditions, we applied AA at our University outpatient acupuncture clinic, as elective AT treatment for our patients with TMD affected by cranio-cervico-facial pain that does not respond to conventional OP treatment. The study presented below was performed retrospectively, to investigate and verify the effects of AA on pain and its interference with common activities and quality of life of patients with TMD, through the analysis of medical records and questionnaires that are routinely used in our specialistic ward.
MATERIALS AND METHODS
This study was a retrospective observational study in general practice. Patients referred to our acupuncture clinic in a University Hospital specialistic Unit of Orofacial Pain, between January 2019 to February 2021, were included. Forty-three patients were assessed and enrolled for AA treatment. Due to the COVID-19 pandemic during the period March-November 2020 (10 patients), or the distance to the hospital or job difficulties (2 patients), a total of 12 patients were excluded from the analysis for not finishing the full AT treatment; three patients were excluded for not completing the provided questionnaires. In total, 28 patients with subacute and chronic TMD-related pain non-responding to conventional treatments were fully treated and data analyzed. All patients were diagnosed with TMD pain (ICD-9 729.1; ICD-9 524.62; ICD-9 339.89; ICD-9 524.63) greater than or equal to 30 on the Numeric Verbal Scale (NVS), according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) classification [1]; the frequency of pain was greater than or equal to 1 time/week. All patients had an history of previous unsuccessful treatments such as advice or counseling regarding oral functioning, administration or topical use of pain medication, physical therapy, and/or an occlusal appliance.
As standard practice in our ward, all patients signed an informed consent form before undergoing acupuncture therapy, concerning both the treatment and the possible anonymous use of their clinical data for scientific and publication purposes. Participants also filled in validated questionnaires regarding their oral and general health conditions, before and after the AA treatment. The study was retrospectively approved by the Institutional Ethics Committee (N.112/22/0000868) and followed STROBE guidelines [23].
1. Abdominal acupuncture treatment
The patients were treated two times per week for four weeks with abdominal acupuncture (AA) by an experienced physician and acupuncture licensed specialist (ES). The acupuncture points used were according to the Bo’s Method of Abdominal Acupuncture [20-22]: CV 12 (Zhongwan), CV10 (Xiawan), CV6 (Qihai), CV4 (Guanyuan), ST24 (Huaroumen) bilaterally, EX - Ab1 (Shangfengshi) bilaterally, KI17 (Shangqu) and KI19 (Yindu) mono or bilaterally, according to pain location (Fig. 1).
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Figure 1. Bo’s abdominal acupuncture scheme: the lines in the left hemiabdomen show the “abdominal turtle pattern” according to Bo [20]. The selected acupoint are circled in red: CV-12 (4 cun above the navel); CV-10 (2 cun above the navel); CV-6 (1.5 cun below the navel); CV-4 (3 cun below the navel CV-8); KI-19 (0.5 cun lateral CV-12); KI-17 (0.5 cun lateral CV-10); ST-24 (2 cun lateral CV-9, located 1 cun above the navel); Ex-ab1 (0.5 cun lateral and 0.5 cun above ST-24).
Needles were inserted at the penetration site after asepsis of the skin with 70% alcohol. The needles were disposable and sterilized, individually packed, size 0.22 × 40 mm (TEWA, ASIA-MED GMBH & CO. KG). According to Bo’s acupuncture method, the depth of needle penetration was “standard depth” for all selected points, except for KI19 (“superficial depth”) [20]. The needles were not manipulated, and they remained in place for 30 minutes and were then removed.
2. Outcome measures and data analysis
Temporomandibular joint (TMJ) pain, masticatory muscle pain, headache, and neck pain were the symptoms assessed, and each one was measured through the 0-100 VNS. The maximum mouth opening (MMO) was measured in all patients, recording the interincisal distance in mm using a ruler (mouth opening without assistance).
All patients were requested to complete the following questionnaires. The Oral Behavior Checklist (OBC) estimated oral functioning. The questionnaire consisted of 21 items rated on a five-point Likert scale (0-4) asking about the frequency of parafunctional behaviors within the last month [1]. The Short Form of the Brief Inventory Pain (BPI) assessed the severity of pain and its impact on daily function, and was comprised of nine items rated on a 0-10 Visual Numeric Scale (VNS) asking the patient to rate the severity of pain and of pain-related disability in the last 24 hours [24]. The Patients’ Global Impression of Improvement (PGI-I) Scale is a global index indicating the response of a condition to a therapy; it evaluated patients’ impression of treatment effectiveness [1].
Craniofacial pain intensity (NVS), pain and pain-related disability (BPI) and MMO were measured at the following times:
T0: Baseline, before AA treatment
T1: End of AA treatment, four weeks after T0 (after the last acupuncture session)
At T1, also the patient’s impression of treatment effectiveness (PGI-I) was determined.
The statistical processing software SPSS (version 23) was used for data analysis. Normality of data distributions was examined using the Shapiro-Wilk test. The Wilcoxon signed-rank test was used to compare the VNS, MMO, OBC and BPI results before treatment (T0) with those obtained after AA (T1). For all tests, the level of significance was set at
RESULTS
A total of 28 patients, 24 females (85.7%) and four males (14.3%), with an average age of 49.36 years, were fully treated and analyzed. Nineteen (19) patients had bilateral TMD, and nine had unilateral TMD. From the onset of pathology, the range in the duration of symptoms was 4-144 months. Symptoms lasted for ≤ 6 months (subacute TMD) in five patients, and for > 6 months (chronic TMD) in the remaining 23 patients. Ten (10) patients were affected by a systemic painful condition (Table 1). No side effects or complications were observed with respect to the AA procedure itself and all patients tolerated the treatment well.
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aDDWR = disc displacement with reduction; bMMO = maximum mouth opening..
&md=tbl&idx=1' data-target="#file-modal"">Table 1D = drugs; FM = fibromyalgia; OP = osteoporosis; PsA = psoriatic arthritis; PT = physical therapy; RA = rheumatoid arthritis; differentiated Connective Tissue Disease..
Patients’ demographic and clinical baseline characteristics.
No. Gender Age Diagnosis Side MMOb (mm) Pain duration (month) Systemic disease Past treatment Arthralgia Myalgia DDWRa 1 F 49 × B 39 5 PT, ST 2 F 65 × R 39 4 ST 3 F 52 × × × B 36 144 FM, UCTD D, PT, ST 4 F 46 × × L 42 18 D, PT, ST 5 F 54 × × B 41 84 FM D, PT, ST 6 F 49 × B 40 60 FM, PsA D, PT, ST 7 F 57 × B 42 120 FM, UCTD D, PT, ST 8 F 44 × B 44 24 D, PT, ST 9 F 63 × R 39 48 OP D, ST 10 F 73 × × × L 40 18 RA D, PT, ST 11 F 61 × B 41 6 OP D, ST 12 F 45 × R 42 18 PT, ST 13 F 27 × B 34 42 D, PT, ST 14 M 28 × B 43 12 PT, ST 15 F 48 × B 40 4 D, ST 16 M 53 × B 43 36 D, PT, ST 17 F 56 × × L 38 96 FM D, PT, ST 18 F 37 × B 37 24 PT, ST 19 F 62 × × B 49 84 RA D, PT, ST 20 M 55 × B 43 30 D, PT, ST 21 F 48 × × × R 35 60 D, PT, ST 22 F 61 × B 38 120 D, PT, ST 23 M 32 × B 39 7 PT, ST 24 F 18 × B 38 24 ST 25 F 45 × × L 36 18 D, PT, ST 26 F 60 × B 42 144 FM D, ST 27 F 52 × L 40 60 D, PT, ST 28 F 42 × × B 42 4 ST aDDWR = disc displacement with reduction; bMMO = maximum mouth opening..
D = drugs; FM = fibromyalgia; OP = osteoporosis; PsA = psoriatic arthritis; PT = physical therapy; RA = rheumatoid arthritis; differentiated Connective Tissue Disease..
The application of AA resulted in a general improvement in MMO, oral functioning, patients’ pain perception and pain interference with normal activities and quality of life (Table 2, Fig. 2, 3). The difference in MMO and the improvement in oral functioning (OBC) and pain perception, according to both NVS and BPI, at T0 and T1, resulted in statistical significance for all the considered variables (all
-
&md=tbl&idx=2' data-target="#file-modal"">Table 2Z,
p -value: Wilcoxon signed-rank test results..Effect of abdominal acupuncture in the study population on: mouth opening (MMO), oral functioning (OBC), TMD-related pain (VNS), general pain and pain interference (BPI).
Variable Before treatment–T0 After treatment–T1 Z p -valueMMO 40.07 ± 3.13 41.82 ± 2.52 –3.724 0.0002 OBC 45.43 ±13.85 37.68 ±13.99 –3.959 < 0.0001 Pain (VNS) TMJ pain 34.64 ± 39.11 7.50 ± 15.54 –3.408 0.0006 Muscle pain 37.5 ± 4.88 14.28 ± 23.32 –3.296 0.0009 Headache 62.14 ± 33.81 22.50 ± 27.16 –4.197 < 0.0001 Neck pain 64.46 ± 35.68 28.21 ± 26.39 –4.197 < 0.0001 Brief pain inventory (BPI) Pain (VAS) 1 6.43 ± 2.20 3.46 ± 2.22 –4.372 < 0.0001 2 3.21 ± 2.29 1.11 ± 1.42 –3.602 0.0003 3 5.21 ± 1.83 2.32 ± 1.87 –4.493 < 0.0001 4 4.78 ± 2.68 1.36 ± 1.61 –4.2378 < 0.0001 Pain interference (VAS) General activity 4.03 ± 2.38 1.89 ± 2.02 –4.107 < 0.0001 Mood 5.68 ± 2.88 2.68 ± 2.11 –4.197 < 0.0001 Walking ability 1.89 ± 2.79 0.89 ± 1.69 –2.665 0.0084 Normal work 3.64 ± 2.69 1.61 ± 1.90 –3.919 0.0001 Relations with other people 3.11 ± 2.30 1.93 ± 2.29 –3.296 0.0009 Sleep 4.71 ± 2.76 3.00 ± 2.71 –3.621 0.0003 Enjoyment of life 3.28 ± 2.55 1.82 ± 1.90 –3.621 0.0003 1 = WORST pain in the last 24 h; 2 = MINIMUM pain in the last 24 h; 3 = AVERAGE pain in the last 24 h; 4 = pain value in this moment..
Z,
p -value: Wilcoxon signed-rank test results..
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Figure 2. Patients TMD-related pain distribution at T0 and T1, according to different locations: TMJ, Masticatory muscle, Head and Neck. Higher NVS scores indicate more severe pain. The horizontal bar inside the boxes indicates the median, the x inside the boxes indicates the mean, and the lower and upper ends of the boxes are the first and third quartiles. The whiskers indicate the minimum and the maximum ranges, and data more extreme than the whiskers are plotted individually as outliers (circles).
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Figure 3. Distribution at T0 and T1, according to BPI, of (A) TMD-related pain and (B) pain interference in common activities and quality of life. (A) 1 = WORST pain in the last 24 h; 2 = MINIMUM pain in the last 24 h; 3 = AVERAGE pain in the last 24 h; 4 = pain value IN THIS MOMENT. (B) A = General activity; B = Mood; C = Walking ability; D = Normal work; E = Relations with other people; F = Sleep; G = Enjoyment of life. Higher BPI scores indicate more severe pain (A) and more severe pain interference in quality of life’s aspects (B). The horizontal bar inside the boxes indicates the median, the x inside the boxes indicates the mean, and the lower and upper ends of the boxes are the first and third quartiles. The whiskers indicate the minimum and the maximum ranges, and single data or data more extreme than the whiskers (outliers) are plotted individually as circles.
Table 1 displays the participants’ baseline demographic and clinical characteristics. Table 2 illustrates the exact results from data analysis. Fig. 2, 3 indicate the distribution of patients’ pain and interference of pain perceptions, at T0 and T1, according to NVS (Fig. 2) and BPI (Fig. 3) scales. Fig. 4 presents the results of patients’ self-evaluations of treatment effectiveness using the PGI-I scale.
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Figure 4. Patients’ impression of the treatment effectiveness at T1, according to the PGI-I Scale.
DISCUSSION
According to Traditional Chinese Medicine (TCM), the abdomen is categorized as a type of acupuncture microsystem, i.e., a well-defined anatomical area in which all the structural and functional components of the human organism are represented, a miniature projection of the human body and its functions. The microsystems are somatotopic areas, in which each specific point represents a well-defined point of correspondence in the human body [25]. However, compared with other microsystems, such as the auricle, the scalp, the foot, or the hand, the abdomen is considered unique in its location. For TCM theory, the abdominal area (located behind the acupoints Qihai (CV6) and Guanyuan (CV4)) is the source of the human body’s qi (vital energy) and jing (essence) resides [20-22,26]. In addition to the traditional meridian system, Dr. Bo Zhi-Yun discovered two other regulatory systems at different layers of the abdomen wall [20,26]. These systems develop from the fetal circulatory system, which delivers blood and nutrients to the fetus via the umbilical cord, and which serves as a regulatory system of the human body during uterine life. Immediately after birth, fetal circulation undergoes a rapid transition to accommodate extra-uterine life [27]. However, the umbilicus has many embryological remnants, and these elements of the prenatal system and its ability to distribute qi and blood still exist in adults, and are centred at the navel, where the acupuncture point Shenque (CV8) is located [28]. Therefore, the abdominal region is an extremely valuable energetic area in acupuncture practice, center and origin of all the body’s dynamic forces and functional activities, through which it is possible to treat all parts of the body. Thanks to these unique characteristics, Bo’s method of abdominal acupuncture has been widely applied by acupuncture practitioners since its systematization in 1991, and has rapidly spread throughout the world. The few studies investigating the clinical effects of AA in the current scientific literature support the efficacy of this method and have led to results of general improvement of pain symptoms, including musculoskeletal disorders [29-33]. This evidence is in line with the results obtained in the present study, which reports for the first time, the application of AA for treating TMD-related pain and symptoms.
In addition, in modern scientific science, the abdomen represents a crucial part of the human body. This anatomical area has been intensely investigated in the scientific literature, and it is referred to as a “second brain”– the “abdominal brain” [34,35]. The relationship between the enteric nervous system (ENS), gut microbiota and neurological diseases, including chronic pain, is receiving increasing attention from researchers. The enteric nervous system shares many sensory and motor neurons, information processing circuits, neurotransmitters, receptors, and transcription factors with the central nervous system (CNS) [36]. Different studies on gastrointestinal behavior revealed a highly complex interaction among the microbiota, the bowels, and the brain through the so-called “gut-brain axis”, a bidirectional neurohumoral connection system regulated by spinal and vagal visceral pathways [35,37]. It was noted that ENS and its extended communication system could influence irritable bowel syndrome, psychiatric disorders, and neurological conditions among others [36,37]. In addition, ENS plays an important role in inflammatory and nociceptive processes, influencing neurogenic inflammation in visceral pain [38,39]. Furthermore, gut microbiota and its modulation has been described as anti-nociceptive in visceral, inflammatory, and neuropathic pain models in animal studies [36,38,39]. Its potential critical role was suggested in many other types of chronic pain, including inflammatory pain, neuropathic pain, headache and widespread musculoskeletal pain [40-43].
Some animal studies investigated the effects of somatic acupuncture on ENS and gut microbiota during the treatment of different diseases [44-46]. Jang et al. (2020) [45] suggested an association between the effects of acupuncture in enhancing motor function and protecting dopaminergic neurons, and the regulation of the gut microbial dysbiosis, determining the inhibition of neuroinflammation in mice affected by Parkinson’s disease. Hong et al. (2020) [46] revealed a potential role of the gut-brain axis in increasing appetite and improving insomnia during acupuncture therapy. Visceral hyperalgesia was attenuated by electroacupuncture (EA), through down-regulation of central serotonergic activities in the brain-gut axis. Other studies discovered that EA at auricular points could increase mRNA expression of the 5-HT1a receptor in both the colon (peripheral) and raphe nuclei (central) in rats with induced visceral pain [47,48].
This evidence may be the basis for explaining the mechanisms behind the clinical efficacy of Bo’s AA technique, particularly for visceral pathologies, pain, and neurological disorders. This method resulted in the effective management of TMD pain and related interference on life aspects like “sleep” in the present study. Furthermore, the insertion of needles directly on abdominal acupuncture points could suggest an amplified action of the ENS, thanks to the localized acupuncture-mediated anti-inflammatory effects already demonstrated in the literature [49,50]. This could further explain the recognized clinical evidence of a short-term therapeutic response to this acupuncture technique, especially for pain, that is reinforced by the results of the present study. Indeed, even after only one course of AA treatment (eight sessions) our patient population, characterized by subacute/chronic TMD pain non-responding to conventional gnathological therapy, conveyed a statistically significant decrease in pain, and pain-related disability in common activities and quality of life, for all the variables considered except for the aspect “walking ability” (Table 2). The parameters concerning mouth opening and general oral functioning was significantly improved (Table 2). These results are consistent with various clinical studies that support the abdominal acupuncture technique for the treatment of other kinds of musculoskeletal disorders such as cervical spondylosis, prolapse of lumbar intervertebral disc, knee osteoarthritis and shoulder periarthritis [30,31].
Furthermore, the AA clinical methodology has two important characteristics: (1) needles, after their insertion into the acupoints, are not manipulated; and (2) the location of the acupuncture points is particularly accurate, because the abdominal points are located by the practitioner measuring the horizontal and vertical lines of the AMS scheme with a ruler and converting the corresponding traditional measure (cun) into centimeters [26]. So this technique, while avoiding needle stimulation and using a millimetric definition of acupoint location, is an easy-to-be-standardized repeatable method, as required by research methodology.
There are significant limitations to this study. Despite the evidence of clinical efficacy of AA on dysfunctional non-responder patients, this is a retrospective study on a small population of subjects. In addition, the data are not homogeneous regarding the presence or absence of painful systemic diseases. The promising results obtained, however, lay the basis for a randomized controlled clinical trial with a placebo group. Clinical trials are necessary to confirm the effectiveness of AA and to better explore the therapeutic possibilities of a microsystem method that is currently little investigated, but has strong performance in clinical acupuncture practice, especially for chronic pain.
AUTHORS’ CONTRIBUTIONS
Conceptualization: E. Serritella, C. Di Paolo; Data curation and Formal analysis: E. Serritella, G. Galluccio; Investigation: E. Serritella; Project administration: C. Di Paolo; Supervision: E. Serritella, G. Galluccio, C. Di Paolo; Validation: E. Serritella, C. Di Paolo; Writing - original draft: E. Serritella; Writing - review & editing: E. Serritella, G. Galluccio, C. Di Paolo.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

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Table 1 . Patients’ demographic and clinical baseline characteristics.
No. Gender Age Diagnosis Side MMOb (mm) Pain duration (month) Systemic disease Past treatment Arthralgia Myalgia DDWRa 1 F 49 × B 39 5 PT, ST 2 F 65 × R 39 4 ST 3 F 52 × × × B 36 144 FM, UCTD D, PT, ST 4 F 46 × × L 42 18 D, PT, ST 5 F 54 × × B 41 84 FM D, PT, ST 6 F 49 × B 40 60 FM, PsA D, PT, ST 7 F 57 × B 42 120 FM, UCTD D, PT, ST 8 F 44 × B 44 24 D, PT, ST 9 F 63 × R 39 48 OP D, ST 10 F 73 × × × L 40 18 RA D, PT, ST 11 F 61 × B 41 6 OP D, ST 12 F 45 × R 42 18 PT, ST 13 F 27 × B 34 42 D, PT, ST 14 M 28 × B 43 12 PT, ST 15 F 48 × B 40 4 D, ST 16 M 53 × B 43 36 D, PT, ST 17 F 56 × × L 38 96 FM D, PT, ST 18 F 37 × B 37 24 PT, ST 19 F 62 × × B 49 84 RA D, PT, ST 20 M 55 × B 43 30 D, PT, ST 21 F 48 × × × R 35 60 D, PT, ST 22 F 61 × B 38 120 D, PT, ST 23 M 32 × B 39 7 PT, ST 24 F 18 × B 38 24 ST 25 F 45 × × L 36 18 D, PT, ST 26 F 60 × B 42 144 FM D, ST 27 F 52 × L 40 60 D, PT, ST 28 F 42 × × B 42 4 ST aDDWR = disc displacement with reduction; bMMO = maximum mouth opening..
D = drugs; FM = fibromyalgia; OP = osteoporosis; PsA = psoriatic arthritis; PT = physical therapy; RA = rheumatoid arthritis; differentiated Connective Tissue Disease..
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Table 2 . Effect of abdominal acupuncture in the study population on: mouth opening (MMO), oral functioning (OBC), TMD-related pain (VNS), general pain and pain interference (BPI).
Variable Before treatment–T0 After treatment–T1 Z p -valueMMO 40.07 ± 3.13 41.82 ± 2.52 –3.724 0.0002 OBC 45.43 ±13.85 37.68 ±13.99 –3.959 < 0.0001 Pain (VNS) TMJ pain 34.64 ± 39.11 7.50 ± 15.54 –3.408 0.0006 Muscle pain 37.5 ± 4.88 14.28 ± 23.32 –3.296 0.0009 Headache 62.14 ± 33.81 22.50 ± 27.16 –4.197 < 0.0001 Neck pain 64.46 ± 35.68 28.21 ± 26.39 –4.197 < 0.0001 Brief pain inventory (BPI) Pain (VAS) 1 6.43 ± 2.20 3.46 ± 2.22 –4.372 < 0.0001 2 3.21 ± 2.29 1.11 ± 1.42 –3.602 0.0003 3 5.21 ± 1.83 2.32 ± 1.87 –4.493 < 0.0001 4 4.78 ± 2.68 1.36 ± 1.61 –4.2378 < 0.0001 Pain interference (VAS) General activity 4.03 ± 2.38 1.89 ± 2.02 –4.107 < 0.0001 Mood 5.68 ± 2.88 2.68 ± 2.11 –4.197 < 0.0001 Walking ability 1.89 ± 2.79 0.89 ± 1.69 –2.665 0.0084 Normal work 3.64 ± 2.69 1.61 ± 1.90 –3.919 0.0001 Relations with other people 3.11 ± 2.30 1.93 ± 2.29 –3.296 0.0009 Sleep 4.71 ± 2.76 3.00 ± 2.71 –3.621 0.0003 Enjoyment of life 3.28 ± 2.55 1.82 ± 1.90 –3.621 0.0003 1 = WORST pain in the last 24 h; 2 = MINIMUM pain in the last 24 h; 3 = AVERAGE pain in the last 24 h; 4 = pain value in this moment..
Z,
p -value: Wilcoxon signed-rank test results..
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