Acupuncture in migraine prophylaxis: a randomized sham-controlled trial

Blackwell Science, LtdOxford, UKCHACephalalgia0333-1024Blackwell Science, 2005?? 2005265520529Original ArticleAcupuncture in migraine prophylaxisJ Alecrim-Andrade et al.
Acupuncture in migraine prophylaxis: a randomizedsham-controlled trial J Alecrim-Andrade1,2, JA Maciel-Júnior2, XC Cladellas3, HR Correa-Filho4 & HC Machado5
1Universitat Autonòma de Barcelona, Medicina Interna, Barcelona, Catalunya, Spain, 2State University of Campinas, Neurology, Campinas, São
Paulo, Brazil,
3Hospital Clinic, Clinical Pharmacology Unit, Barcelona, Catalunya, Spain, 4State University of Campinas, Social and Preventive
Medicine and
5State University of Campinas, School of Medical Sciences, Campinas, São Paulo, Brazil
Alecrim-Andrade J, Maciel-Júnior JA, Cladellas XC, Correa-Filho HR & Machado HC. Acupuncture in migraine prophylaxis: a randomized sham-controlled trial.
Cephalalgia 2006; 26:520–529. London. ISSN 0333-1024The purpose of the present trial was to evaluate semi-standardized acupuncture efficacy in migraine prophylaxis. Twenty-eight subjects with migraine were randomized to the real or sham acupuncture groups. Semi-standardized and standardized minimal acupuncture were used, respectively, in the two groups of patients. They were all treated with 16 acupuncture sessions in 12 weeks. Both groups exhibited similar reductions in: percentage of patients with reduction of migraine ≥40% and ≥50% regarding frequency of migraine attacks, days with migraine, frequency of migraine attacks, average duration of a migraine attack, rate of rescue medication used, average headache severity rate and other parameters compared with the baseline period. Associated symptoms, such as nausea and vomiting, also showed equal estimates in both groups. These findings showed that semi-standardized acupuncture shows no difference from sham acupuncture in preventing migraine attacks. ᮀAcupuncture, efficacy, headache, migraine, prophylaxis, randomized controlled trial Dr Jerusa Andrade, State University of Campinas, Neurology, Campinas, São Paulo, Brazil. Tel. + 55 19 3242 1492, e-mail Received 11 January 2005, Introduction
accordingly to their own experience and feelings. In fact, acupuncture can be applied in different ways.
Acupuncture has been largely used for migraine suf- Traditional Chinese acupuncture basis has been dis- ferers in western countries, although the role it may regarded in most trials. Usually, a standard treat- play in migraine treatment remains unknown (1–3).
ment has been adopted regardless of the previous Some trials have pointed out some effects of acu- TCM diagnosis (9, 10). Furthermore, several acu- puncture in migraine, but no conclusive results have puncture techniques have also been applied, been reached, probably because there have been although they cannot be matched to the original only a few high-quality trials in this area (4, 5).
technique (7, 11). Therefore, comparisons of acu- Migraine does not exist as an entity in the theory puncture trials are very difficult (7). Is there a mid- of traditional Chinese medicine (TCM) (6). The way betwwen traditional concepts and the scientific basis of acupuncture follows the TCM grounding.
approach? The authors thought that the semi- However, there are serious methods of diagnosis, standardized acupuncture treatment could be the treatment and practice as far as acupuncture is con- best way, because TCM would be partially respected cerned. In western countries practice sometimes fol- and the treatment would be easily reproduced in lows the paths of neurophysiology, neuromuscular other trials in the future as well as in clinical practice.
and other modern concepts and principles (7).
The present trial was developed to evaluate According to TCM’s principles, treatment should be the efficacy of semi-standardized acupuncture in individualized (8). Each acupuncturist should treat Blackwell Publishing Ltd Cephalalgia, 2006, 26, 520–529
Acupuncture in migraine prophylaxis the patients were evaluated by a general physician, who is also an acupuncture specialist (J.A-A.). At this time, the clinical history and physical evalua- tion based on the principles of Chinese medicine The present study was developed in the Headache were recorded, including examination of the pulse Clinic of the Department of Neurology, in the and tongue. The evaluation was done and the Clinical Hospital, State University of Campinas probable treatment following TCM principles was (UNICAMP), Brazil. The protocol and supporting planned for all patients in order not to break the documents (information sheet, consent form, proto- col) were approved by the institutional Ethics Com- After the acupuncture doctor’s evaluation, patients were randomly assigned to two acupunc- The recruitment of patients was done from August ture groups, group C and group D, by a research 2002 to March 2003, and the trial was completed assistant. The research assistant prepared opaque, in February 2004 (including all follow-up periods).
numbered and sealed envelopes containing one of Patients were recruited via a media campaign. They the two above letters. They were divided into six were scheduled by the research assistants and eval- blocks. A block had six letters, three letter Cs and uated by an experienced neurologist.
three letter Ds. The random digits (14) were used to Twenty-eight patients suffering from migraine, define the sequence of the letters in each block.
with or without aura, in accordance with the diag- Therefore, the order of the letters was different nostic criteria of the International Headache Society for each block. That sequence was unknown by (IHS) (12), present for at least 1 year, male and the medical acupuncturist. The identification of female, aged 18–50 years, were enrolled. Other in- each letter was unknown by the first and second clusion criteria were: patients with only migraine, research assistants, the statistician, the neurologist patients who had not used drugs with migraine pro- (evaluator) and the patients. Only the medical acu- phylactic effects or acupuncture in the previous puncturist knew the meaning of each letter, but she 3 months, patients who could come to the clinical had not revealed any code before completing all hospital 17 times only in the following 12 weeks the data analysis. The second research assistant was (acupuncture treatment period) and patients who responsible for sending the randomization list to had accepted inclusion in the study after oral and the statistician at the end of the trial. Group C written explanations about the clinical trial. The received the real acupuncture and group D the exclusion criteria were patients with any other sham acupuncture. The acupuncturist doctor did chronic pain syndrome, who were unable to under- not give any information to the patients, neurolo- stand and maintain headache diaries, who were mis- gist or research assistants about the acupuncture using drugs or alcohol and who occasionally used a Real and sham acupuncture are defined in World Prior to enrolment there was a 4-week prelimi- Health Organization (WHO) Guidelines for Clini- nary phase (baseline period) when patients kept a cal Research on Acupuncture (15) as being ‘acu- diary recording the frequency and intensity of head- puncture given as a real clinical treatment’ and ache, menstruation, drug intake and associated ‘inappropriate acupuncture for the condition being symptoms in each migraine attack. By that time, the treated taking into account the acupuncture selected patients had received a written text with information about all aspects of the trial, including The patients were submitted to the acupuncture possible adverse events and their right to withdraw treatment for 12 consecutive weeks. That included from the trial at any time without giving reasons.
16 sessions, twice a week during the first 4 weeks During that period at least three, but not more than and weekly during the following 8 weeks. Patients six migraine attacks, had to occur. A new attack was had to keep a headache diary from the baseline considered as such when the patient had been head- period (diary 1) up to follow-up period (diaries ache free for at least 48 h (13). Patients with non- 5–10), which was for 24 weeks after the last acu- migraine types of headaches were excluded as well puncture session (approximately 6 months). The as patients who had used drugs for the treatment of follow-up period was designed to test the longer- migraine attacks for more than 10 days in 4 weeks.
lasting effects of acupuncture on migraine. During After the baseline period, during the second neurol- the acupuncture treatment, the patients completed ogist visit, the selected patients signed the written three diaries (diaries 2–4). Neurologist visits were informed consent. Before acupuncture treatment, scheduled at weeks 0, 5, 18, 25, 33 and 42.
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Treatment of migraine attacks, if they happened, as De Qi. The ‘Qi sensation’ was explained to the patients of this group in the first session as an patient. The neurologist withheld any suggestions awareness of numbness, strange aching or tingling as to changes in the ongoing rescue medication radiating from the point of needling.
and any comments on the acupuncture treatment 1 The skin was disinfected with 70% alcohol.
2 Sterile disposable and steel needles (0.25 × 40 mm) 3 Moxa or electrical stimulation was not used.
1 Twelve needles (six on each side) were inserted in 4 The patients were kept lying down for 30 min with 2 Minimal acupuncture was used and consisted of 5 Body acupuncture was done bilaterally. very shallow needle insertion in the acupuncture 4 The points in the sham acupuncture group were Efficacy was measured by comparing the first diary, selected after an extensive consultation of the clas- which was made in the baseline period, with the sical acupuncture literature to confirm that they diaries of the treatment period (diaries 2–4) and of had minimal or no influence on headaches (16–21).
the follow-up period (diaries 5–10). Each diary cov- 5 Standardized and fixed acupuncture treatments 1 The treatment was semi-standardized (see 1 The percentage of patients with a reduction of Table 2). The chosen points were related to the ≥40% in migraine attack frequency from the pain topography of the most frequent migraine second to the fourth diaries compared with the attacks in each patient and based on some princi- ples of traditional Chinese medicine.
2 The percentage of patients with a reduction of 2 From 6 to 10 points were used in each session (12– ≥50% in migraine attack frequency from the second to the fourth diaries compared with the 3 Each patient had a fixed treatment in their 16 3 Total migraine days from the second to the fourth 4 The needles were manipulated by rotation meth- diaries compared with the first diary (baseline ods to produce a characteristic sensation known Table 1 Protocol treatment in the sham acupuncture group (points)
At the level of the depression below the spinous process C7. 12.7 mm lateral to the Posterior to the lobule of the ear, in the depression between the mandible and Directly above the auricular apex, on the hairline 15 cm proximal to the proeminence of the medial malleolus, dorsal to the medial 76 mm inferior to St36 (described above), one middle finger breadth lateral to the In the cubital fold in the depression at the radial side of the biceps brachii muscle Lian YL, Chen CY, Hammes M, Kolster BC. The Seirin pictorial atlas of acupuncture. An illustrated manual of acupuncture points. Cologne: Könemann Verlagsgesellschaft mbH Press 2000.
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Acupuncture in migraine prophylaxis Table 2 Protocol treatment in the real acupuncture group (points)
In the depression dorso-cranial to the mastoid process In the depression between the start of the sternocleidomastoid At the midpoint between the depression below the spinous 12.7 mm within the ideal anterior hairline, 33 mm lateral to the midline in the depression at the lateral edge of thetrapezium muscle At the midpoint between the prominence of the lateral With the patient’s fist loosely clenched, at the ulnar end of the proximal crease of the fifth metacarpophalangeal joint, onthe dividing line between red and white flesh In the depression at the medial end of the eyebrow in the One middle fingerbreadth lateral to the anterior crest of the tibia, at the level of distal edge of the tuberosity of the tibia On the midline, 25 mm within the midpoint of the ideal On the dorsum of the hand, to the side of the midpoint of the second metacarpal bone, in the pollicis muscle adductor 5 cm proximal to the dorsal wrist crease between the ulna and In the depression ventral and distal to the head of the fibula With the patient’s fist loosely clenched, at the ulnar end of the proximal crease of the fifth metacarpophalangeal joint, on thedividing line between red and white flesh At the middle of the vertex, at the midpoint between the two On the dorsum of the foot, in the depression distal to the proximal corner between the first and second metatarsalbones 50 mm proximal to the distal wrist crease, between the palmaris longus and flexor carpi radialis tendons On the dorsum of the foot, in the depression distal to the proximal corner between the first and second metatarsalbones Lian YL, Chen CY, Hammes M, Kolster BC. The Seirin pictorial atlas of acupuncture. An illustrated manual of acupuncture points. Cologne: Könemann Verlagsgesellschaft mbH Press 2000.
= migraine not interfering with daily activities; 2, moderate headache = migraine interfering with daily activities; and 3, severe headache = migraine 2 Average duration of a migraine attack.
making normal daily activities impossible) follow- 4 Total duration of migraine pain in hours per diary.
The patients were instructed to report all adverse The severity of headache was evaluated on a 4- events to the medical acupuncturist in each session point scale (0, no headache; 1, mild headache as well as in a paper that they had received before Blackwell Publishing Ltd Cephalalgia, 2006, 26, 520–529
the first session. Adverse events were listed Table 3 Patient demographic characteristics
descriptively and comparison between groups was done, ascribing some statistical significant difference Patients were fully informed that two kinds of acu- puncture would be done. One would be real and the other would be false, placebo. No additional infor- At the end of the study, patients were invited to fill in a questionnaire evaluating the acupuncture treatment and to give their impression about which treatment group (real or sham acupuncture) they were included in the statistical analysis. The flow of participants through each stage of the trial is presented in Fig. 1. There was no significant differ- ence between study completers and drop-outs in The sample size was determined considering the diary scores, group of treatment, age or headache results shown in an unpublished pilot trial that had been done before this present trial. The endpoint The groups’ demographic data are presented in considered to calculate this sample was the percent- Table 3. There was only one statistically significant age of patients with reduction of ≥40% in their difference between groups, the age mean. The migraine attacks in the second month of treatment patients in the sham acupuncture group were older with sham or real acupuncture. The equivalence than in the real acupuncture group (P = 0.024).
range was 11.8–57.9% for patients with a reduction of the attack rate of 40% and with α = 0.05 and Efficacy and long-term follow-up (24 weeks) = 0.20. The number of patients required was 26 divided into two groups. As a drop-out rate of 15% No statistically significant difference between was expected, 30 patients had to be enrolled into the groups was observed in any pain parameter evalu- ated in this trial. Nevertheless, comparisons within Statistical comparisons were done using univari- each group found that all migraine pain parameters ate analysis with the χ2 test and Fisher’s exact test.
had improved with statistically significant differ- Analysis of variance (Anova) for repeated measures ences in both groups. There was one exception: was used to compare both groups. Comparisons headache severity when the patients were experienc- within groups for the migraine parameters in each one of the periods were done using the Tukey’s post- hoc test. Differences within each group were estimated using the profile test by contrasts. The significant level used for the statistical analysis was The Tukey test showed that the real acupuncture 5%, therefore P < 0.05 indicates significance.
group had significantly reduced nausea during the follow-up period when compared with the sham acupuncture group (F(9,198) = 2.09; P = 0.0372). The variance analysis showed that the variability in the graph lines is not attributable to chance.
There was no statistically significant difference The selection of patients for the trial took place from between groups regarding the number and fre- August 2002 to March 2003. The follow-up period was from July 2003 to February 2004. Thirty-one migraine sufferers were enrolled in the study. They were randomly assigned to the real (16 patients) and sham acupuncture (15 patients) groups. However, No statistically significant difference appeared only 28 patients completed the treatment and they between groups or within each group.
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Acupuncture in migraine prophylaxis Not randomized (n = 388) Declined participation (n = 40) Excluded (n = 348) Other diagnosis (n = 165) Associated headache (n = 61) Insufficient severity (n = 68) Upper severity (n = 6) Prophylactic medication (n = 12) Excluded in the 1st month oftreatment. He did not come regularly to the sessions (n = 1) The patient lost diaries (n = 1).
Figure 1 The flow of participants
ecchymosis, small haematomas, nodule and local paraesthesia during session. General effects were No serious adverse effect (AE) was notified. Most sleepiness and relaxing sensation; however, a statis- AEs observed were related to the local insertion of tically significant difference was observed in sleepi- the needles, such as: local pain after session, ness in the real acupuncture group (P = 0.008), as Blackwell Publishing Ltd Cephalalgia, 2006, 26, 520–529
well as in local nodules (P = 0.008) and local pain Table 3). The age of the patients selected to the real acupuncture group varied from 22 to 50 (mean 32.50) and in the sham acupuncture group from 23 to 49 (mean 39.14). Despite sparse longitudinal prospec- tive epidemiological data and information about the All patients announced their intention to be treated prognosis and natural history of migraine (22, 23), with acupuncture in future. When they were asked authors considered that this difference did not in- how they would classify the treatment, patients who fluence the results. Also, in the present trial the age received real acupuncture classified it as good span for patient selection was not large (18–50 years (28.6%), very good (42.8%) and excellent (28.6%).
old). Therefore, no statistical correction for age was Patients who were in the sham acupuncture group rated it as regular (14.3%), good (35.7%) and very good (50%). In the sham acupuncture group nobody classified the treatment as excellent. Likewise, in the real acupuncture group, every patient qualified the There were no statistically significant differences treatment as good although there was no statistically between the real and the sham acupuncture groups significant difference between groups.
in any pain parameters evaluated in this trial during When they were asked about the kind of treatment the treatment or follow-up periods, such as: percent- that they received, patients in the real acupuncture age of patients with reduction ≥40% and ≥50% in group answered: ‘real treatment’ (28.7%), ‘placebo migraine attack frequency, total migraine days, acupuncture’ (7%) and ‘I don’t know’ (64.3%). In the frequency of migraine attacks, mean duration of a sham acupuncture group, they answered: ‘real treat- migraine attack, mean headache severity and total ment’ (28.6%) and ‘I don’t know’ (71.4%). There duration of migraine pain in hours per diary (see was no significant statistical difference between the Figs 2, 3 and 4). No differences were observed replies from the two groups, indicating that the between groups in the rate of rescue medication blinding (keeping the patients unaware of their used or frequency of nausea and vomiting. These results are in accordance with three sham-controlled trials testing the efficacy of acupuncture in treating Discussion
Only one statistically significant difference appeared between groups in demographics characteristics (see observed during the treatment up to 20 weeks after the last acupuncture session (most of the follow-up Figure 2 Percentage of responders (reduction ≥40% in
migraine attack frequency) in each diary (4 weeks) compared with the baseline period (diary 1) in the real (᭿) and sham Figure 3 Total duration of pain in each diary (4 weeks) in the
(ᮀ) acupuncture groups. Diaries 2, 3 and 4 correspond to the real (᭿) and sham (᭹) acupuncture groups. Diaries 2, 3 and treatment period (12 weeks). Diaries 5–10 correspond to the 4 correspond to the treatment period (12 weeks). Diaries 5–10 correspond to the follow-up period (24 weeks) Blackwell Publishing Ltd Cephalalgia, 2006, 26, 520–529
Acupuncture in migraine prophylaxis groups in the present trial was below that of two previous high-quality trials (9, 25). It could be deter- mined by several factors: chance, bias, the sort of semi-standardized acupuncture chosen by medical acupuncturists, the small size of the sample, or large variability of the results observed in both groups. It is important to recall that the patients were aware that the placebo acupuncture treatment was being applied. It is known that in pain studies the response to an active drug is lower in placebo-controlled trials than in open trials or in trials when patients were informed that they would receive only active Persistence of the improvement lasted up to 24 weeks after the period of treatment (approxi- mately 6 months) in both groups, in accordance with the long-lasting acupuncture effects related previ- Figure 4 Total of migraine days in each diary (4 weeks) in the
ously in three well-designed clinical trials (8, 25, 37).
real (᭿) and sham (᭹) acupuncture groups. Diary 1 = baseline Guidelines and an article containing methodological period (4 weeks), diaries 2, 3 and 4 correspond to the suggestions for trials in acupuncture also suggested treatment period (12 weeks) and diaries 5–10 correspond to slightly in the real acupuncture group from the first period) in almost all pain parameters evaluated, month of treatment up to the fifth month of the fol- with the exception of pain intensity during the low-up period, contrasting with the increase of the migraine attack. Although several trials testing acu- same variable in the sham acupuncture group. There puncture or drugs showed improvement of the was a statistically significant difference between frequency of migraine attacks, the same was not ver- them from the third to the fifth month of the follow- ified for headache severity (10, 11, 27–31).
up period and the variance analysis did not attribute this result to chance. Nevertheless, the rate of rescue medication used and vomiting in both groups did 1 The placebo effects are higher in pain sufferers not change in any phase of the trial.
than in patients suffering from other complaints 2 Invasive technical proceedings have higher anal- gesic effects than oral drugs (2, 33, 34).
Effects such as: local nodule (P = 0.008) and local 3 The majority of acupuncture trials that tested pain after session (P = 0.002) appeared with a statis- the efficacy of acupuncture in pain conditions had tically significant difference in the real acupuncture identified that sham acupuncture could result in a group, which is understandable because the depth positive response, reducing the possibility of iden- of needle insertion was very superficial in the sham tifying statistically significant differences between group. In the real group, damage of superficial ves- real and sham acupuncture (2, 4, 5, 7, 15, 35).
sels and nerves is more likely. Sleepiness was signif- Therefore, the improvement achieved for both icantly more frequent in the real acupuncture group groups (real and sham) could be a result of the com- (P = 0.008) and this is also attributable to the depth bination of the three factors referred to above more than the specific effects of the needling. The non- specific effects depend on the patients’ beliefs, such as: the magic effects of the eastern techniques in the western patient’s mind, the patients’ beliefs that they Selection of patients was difficult because of the very are being treated, the acupuncture consultation, the strict inclusion and exclusion criteria adopted in this practioners’ expectations of acupuncture effects and trial, which followed all suggestions presented in the general results of needle insertion (15, 25, 36).
two guides: Guidelines for controlled trials of drugs in migraine (13), edited by the IHS, and Guidelines improvement rate reached for both acupuncture for Clinical Research on Acupuncture (15), edited by Blackwell Publishing Ltd Cephalalgia, 2006, 26, 520–529
the WHO. Most of the volunteers for the trial were tematic review of randomized controlled trials. Cephalal- eliminated because they had other kinds of head- ache, mainly chronic daily headache.
2 Melchart D, Linde K, Berman B, White A, Vickers A, Allais The treatment adopted in the real acupuncture G, Brinkhaus B. Acupuncture for idiopatic headache(Cochrane Review). In: The Cochrane Library, Issue 2.
group (the semi-standardized treatment) was based on the experience of three medical doctors who have 3 Steiner TJ. Acupuncture for recurrent headaches. Editorial worked with acupuncture from 14 to 20 years. The Commentary. Cephalalgia 1999; 19:765.
treatment protocol was based on the topography of 4 Manias P, Tagaris G, Klementine K. Acupuncture in head- the headache related to the theory of traditional ache: a critical review. Clin J Pain 2000; 16:334–9.
Chinese medicine. Therefore, our therapeutic 5 Ezzo J, Berman B, Hadhazy VA, Jadad AR, Lao L, Singh scheme would not be the widespread treatment BB. Is acupuncture effective for the treatment of chronicpain? A systematic review. Pain 2000; 86:217–25.
applied for acupuncturists. In fact, acupuncture 6 García E, Ristol A. Acupuntura y Neurología. Rev Neurol practice is very different between acupuncturists throughout the world. The advantage of the semi- 7 Ernst E, White R. A review of problems in clinical acu- standardized treatment is that it could easily be puncture research. Am J Chinese Med 1997; 25:3–11.
reproduced in future trials, had the results been pos- 8 Tavola T, Constanzo G, Conte G, Invernizzi G. Traditional itive. In fact, acupuncturists know that there is a gap Chinese acupuncture in tension-type headache: a con- between their practice and what has been done in trolled study. Pain 1992; 48:325–9.
9 Allais G, Lorenzo C, Quirico PE, Airola G, Tolardo G, research. It is a common criticism when acupun- Mana O, Benedetto C. Acupuncture in the prophylactic cturists and some researchers comment on the scien- treatment of migraine without aura: a comparison with tific approach to the evaluation of acupuncture flunarizine. Headache 2002; 42:855–61.
10 Melchart D, Thormaehelen J, Hager S, Liao J, Linde K, Weidenhammer W. Acupuncture versus placebo versus Conclusion
sumatriptan for early treatment of migraine attacks: a ran-domized controlled trial. J Intern Med 2003; 253:181–8.
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12 Headache Classification Committee of the International ated showed that this approach is not better than Headache Society. Classification and diagnostic criteria sham acupuncture to prevent attacks in migraine for headache disorders, cranial neuralgias and facial pain.
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yet published, where authors used the individual- 13 International Headache Society Members’ Handbook.
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clinical signs and symptoms presented by each 14 Daniel WD. Biostatistics—a foundation for analysis in the health sciences. New York: John Wiley & Sons, Inc.
patient. However, it is a complex therapeutic scheme 15 WHO. Guidelines for clinical research on acupuncture, Series no. 15. Manila: Regional Publications Western Acknowledgements
16 Maciocia G. The foundations of Chinese medicine. Edin- We would like to thank Drs Lo Sz Hsien and Ling Tung Yang for their suggestions about the treatment through Traditional 17 Nghi NV, Nguyen CR. Medicina Tradicional Chinesa— Chinese Medicine. We would also like to thank Leda Acupuntura-moxibustión & masajes, 1. Barcelona: IBB Fernandes and Heloisa de Lima Gomes for their help in orga- nizing the study and the data. The authors also thank the staff 18 Chenggu Y, Yi J, Biying H. Tratamiento de Las Enfer- from UNICAMP Press Assessory, the Clinic Hospital Public medades Mentales por Acupuntura y Moxibustión.
Relations, Mr Antonio Alberto Ravagnani and the journalist Ani Seixas for their great help with the media. The study 19 Ross J. The organ systems of tradicional Chinese medicine, protocol was written by the principal investigator (JAA). This 2nd edn. Edinburgh: Churchill Livingstone 1985.
work was supported by the State of São Paulo Research Foun- 20 Cheng X, Liangyue D, Yijun G, Shuhui H, Xiaoping J, Yang dation (FAPESP, grant no. 00/09985-0).
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