Brain Stimulation 7 (2014) 332e343
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Letters to the Editor
Transcranial Direct Current Stimulation (tDCS) and Lymphocytes Introduction Despite the growing interest in non-invasive brain stimulation techniques for modulating cortical excitability in healthy subjects and patients, few data are available about their immunological safety. A novel data is that repetitive transcranial magnetic stimulation (rTMS) modulates lymphocytes . These ﬁndings indirectly conﬁrm earlier observations that peripheral lymphocyte number increases after TMS . Another promising non-invasive brain stimulation technique is transcranial direct current stimulation (tDCS). An electric ﬁeld can induce directional cell migration (i.e. electrotaxis) in epithelial cells, endothelial cells, ﬁbroblasts, and neutrophils. Human lymphocytes respond in vitro to applied direct current (DC) by migrating toward the cathode . Additionally, video microscopic tracings show that murine T lymphocytes in vivo actively migrate toward the cathode in an applied DC electric ﬁeld  and cathodal and anodal multisession tDCS both induce pro-inﬂammatory changes in rat brain . Knowing more about possible tDCS-induced changes in peripheral lymphocytes is essential to guarantee the immunological safety of the technique. Our aim in this study was to investigate possible tDCS-induced changes in circulating human lymphocytes. Methods and materials Eight healthy right-handed subjects (six women and two men aged 23e39 years; mean þ SD: 29.6 5.64) participated in the study. All participants gave their informed consent and the procedures had the approval of the hospital review board. The experimental procedure was conducted in accordance with the declaration of Helsinki. Subjects underwent anodal and sham extra-cephalic reference tDCS and extracranial DCS in a cross over randomized design. tDCS was delivered using a constant DC stimulator (HDCStim Newronika) connected to a pair of saline-soaked surface sponge electrodes. For anodal tDCS the active electrode (anode) was placed over the left temporal lobe (T3) and the reference (cathode) over the right arm. The same position was used for sham tDCS but the stimulator was turned off after 30 s. Extracranial DCS was delivered with the anode placed medially to the patellar tendon and the cathode over the ipsilateral ankle. Current was delivered at 2 mA intensity for 20 min (electrode area: 35 cm2). Peripheral blood samples for immunological testing were collected in ethylenediamine tetracetate tubes at baseline (T0) and one (T1) and 4 h (T2) after stimulation offset. The procedure was repeated at the same time in all the experimental sessions. 1935-861X/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved.
The samples were processed to establish the total lymphocyte count and the lymphocyte subsets were determined with the FACScan ﬂow cytometer (Becton Dickinson, San Jose, CA, USA). Monoclonal antibodies identiﬁed the following lymphocyte subsets: T-lymphocytes (CD3þ, CD4þ3þ, CD8þ3þ and CD3þDRþ), B-lymphocytes (CD20þ), IL-2 receptor (CD25þ) and natural killer cells (CD56þ16þ3). Lymphocyte subset counts were expressed for each subset as absolute values. Lymphocyte count changes were expressed for each subject as a percentage change (%) from baseline values for each stimulation condition (anodal tDCS, sham tDCS and extracranial DCS) at each time point (T1 and T2). A two-way repeatedmeasures analysis of variance (ANOVA) for each lymphocyte subset was run with factors “stimulation condition” (three levels: anodal tDCS, sham tDCS and extracranial DCS) and “time point” (two levels: T1 and T2). Tukey honest signiﬁcant test was used for post hoc analysis; differences were considered signiﬁcant at P < 0.05. Values in the text are expressed as means standard deviation (SD). Results Anodal tDCS did not induce signiﬁcant changes on total lymphocyte count and lymphocyte subsets compared to sham tDCS and extracranial DCS (factor “stimulation”: P: >0.05). Conversely, total lymphocytes, CD3þ, CD4þ3þ, CD8þ3þ, and CD20þ changed signiﬁcantly over time with no differences between stimulation conditions (factor “time point”: total lymphocytes, P: 0.0004; CD3þ, P: 0.0004; CD4þ, P: 0.002; CD8þ3þ, P: 0.003; and CD20þ, P: 0.002) (Table 1). Discussion Anodal tDCS over the dominant temporal lobe does not affect circulating lymphocyte counts compared to sham stimulation. Total lymphocytes, CD3þ, CD4þ3þ, CD8þ3þ, CD3þDRþ and CD20þ, change over time without signiﬁcant differences between stimulation conditions. These ﬁndings suggest that tDCS is a promising immunologically safe technique. Our paper is one of the few to investigate changes induced by non-invasive brain stimulation on the immune system. TMS applied to animal and human cerebral cortex modulates lymphocyte activation and blood lymphocyte numbers within hours [2,5]. The differences in the effects induced by the two neuromodulation techniques on the human immune system may depend on their different mechanisms of action in the CNS . Given that the central nervous system is thought to modulate immune functions also through the autonomic nervous system (ANS)  lymphocyte changes in subjects undergoing non-invasive brain stimulation might partly depend on the ANS. Whether and if so how tDCS could inﬂuence autonomic functions nevertheless remain controversial. Anodal tDCS over the temporal cortex affects cardiovascular variables such as peak power output,
Letters to the Editor / Brain Stimulation 7 (2014) 332e343
Table 1 Transcranial direct current stimulation (tDCS) delivered over the dominant temporal lobe using an extra-cephalic reference electrode setup leaves circulating lymphocyte counts in healthy subjects unchanged. Values in the table are expressed as mean standard deviation (n ¼ 8) of lymphocyte count changes from baseline values expressed as percentage [%]. Lymphocyte counts
Anodal tDCS T1
Natural killer cells Human leukocyte antigens B-cells
Total number CD3+ CD25+ CD4+3+ CD8+3+ Ratio CD56+16+3CD3+DR+ CD20+
3,49 0,96 40,46 3,03 5,35 3,81 15,79 2,69 13,96
Sham tDCS T2
19,35 21,3 73,45 23,27 27,10 14,92 23,80 38,15 25,30
10,33 16,16 14,86 19,98 4,80 9,28 17,19 3,41 32,90
T1 17,78 15,95 62,49 17,84 28,12 12,50 26,57 26,37 21,14
parasympathetic vagal withdrawal, and heart rate interval and variability in athletes thus suggesting that non-invasive brain stimulation modulates ANS activity [8,9] though these ﬁndings received no conﬁrmation in non athlete and healthy subjects . Why circulating lymphocyte numbers changed under all the tDCS conditions we tested remains hard to explain. Changes may depend on lymphocyte circadian variations. Another possible explanation might involve the known relationship between peripheral blood lymphocytes and endogenous plasma cortisol. A study by Raimundo et al. (2012) supports these ﬁndings showing that anodal and sham tDCS both induce changes in plasma cortisol levels . Our study has several limitations. First, although we tested how acute DC stimulation affects lymphocyte numbers, chronic stimulation or multiple session protocols could induce different effects. Second, we provided no information on a tDCS setup using a cephalic reference electrode. Third, cortical modulation of circulating lymphocytes should be assessed also in different behavioral states that can inﬂuence cell counts. To assess tDCS-induced changes better, future research should also test cytotoxic activity and mitogen responses. In conclusion, anodal tDCS does not induce any signiﬁcant effect on peripheral lymphocyte numbers compared to sham stimulation in healthy subjects thus arguing in favor of its safety.
6,23 6,62 25,78 3,25 8,32 7,69 14,96 13,01 8,47
Extracranial DCS T2
1721, 17,36 53,75 16,19 20,52 14,92 27,32 24,42 17,75
10,47 11,15 39,49 13,76 5,08 4,96 13,44 8,99 30,82
24,59 22,65 49,70 16,97 23,88 27,70 51,54 22,72 23,21
3,85 2,39 11,28 3,27 5,29 10,22 24,23 6,53 15,69
T2 15,31 14,18 52,14 16,95 20,14 24,62 36,65 50,10 16,57
22,73 23,80 60,88 32,40 18,02 13,91 2,28 10,95 52,63
29,12 29,31 90,98 29,78 34,23 21,56 51,30 39,33 28,50
S. Garlaschi Unità Operativa di Neuroﬁsiopatologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy S. Barbieri Unità Operativa di Neuroﬁsiopatologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy A. Priori* Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy Centro Clinico per la Neurostimolazione, le Neurotecnologie ed i Disordini del Movimento, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy * Corresponding
author. Dipartimento di Fisiopatologia MedicoChirurgica e dei Trapianti, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, Milan 20122, Italy. Tel.: þ39 (0)2 50320438; fax: þ39 (0) 2 55033855. E-mail address: [email protected]
Received 1 October 2013 Available online 22 December 2013 http://dx.doi.org/10.1016/j.brs.2013.11.007
References G. Ardolino Unità Operativa di Neuroﬁsiopatologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy E. Scelzo Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy F. Cogiamanian Unità Operativa di Neuroﬁsiopatologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy P. Bonara Dipartimento di Medicina Interna, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy A. Nozza Dipartimento di Oncologia Medica ed Ematologia, Istituto Clinico Humanitas, Milan, Italy M. Rosa Centro Clinico per la Neurostimolazione, le Neurotecnologie ed i Disordini del Movimento, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
 Wang HY, Crupi D, Liu J, Stucky A, Cruciata G, Di Rocco A, et al. Repetitive transcranial magnetic stimulation enhances BDNF-TrkB signaling in both brain and lymphocyte. J Neurosci 2011;31:11044e54.  Amassian VE, Henry K, Durkint H, Chicet S, Cracco JB, Somasundaram M, et al. Differing immune functions, including natural killer cell activity, after left versus right magnetic stimulation of human T-P-O cortex. J Physiol; 1994: 143e4.  Lin F, Baldessari F, Gyenge CC, Sato T, Chambers RD, Santiago JG, et al. Lymphocyte electrotaxis in vitro and in vivo. J Immunol 2008;181:2465e71.  Rueger MA, Keuters MH, Walberer M, Braun R, Klein R, Sparing R, et al. Multisession transcranial direct current stimulation (tDCS) elicits inﬂammatory and regenerative processes in the rat brain. PLoS One 2012;7(8):e43776.  Moshel YA, Durkin HG, Amassian VE. Lateralized neocortical control of T lymphocyte export from the thymus I. Increased export after left cortical stimulation in behaviorally active rats, mediated by sympathetic pathways in the upper spinal cord. J Neuroimmunol 2005;158:3e13.  Priori A, Hallett M, Rothwell JC. Repetitive transcranial magnetic stimulation or transcranial direct current stimulation? Brain Stimul 2009;2:241e5.  Nance DM, Sanders VM. Autonomic innervation and regulation of the immune system (1987-2007). Brain Behav Immun 2007;21(6):736e45.  Okano AH, Fontes EB, Montenegro RA, Farinatti PD, Cyrino ES, Li LM, et al. Brain stimulation modulates the autonomic nervous system, rating of perceived exertion and performance during maximal exercise. Br J Sports Med 2013;27.  Montenegro RA, Farinatti Pde T, Fontes EB, Soares PP, Cunha FA, Gurgel JL, et al. Transcranial direct current stimulation inﬂuences the cardiac autonomic nervous control. Neurosci Lett 2011;497(1):32e6.
Letters to the Editor / Brain Stimulation 7 (2014) 332e343
 Raimundo RJ, Uribe CE, Brasil-Neto JP. Lack of clinically detectable acute changes on autonomic or thermoregulatory functions in healthy subjects after transcranial direct current stimulation (tDCS). Brain Stimul 2012;5(3): 196e200.
Pain Treatment Using tDCS in a Single Patient: Tele-medicine Approach in Non-invasive Brain Simulation Chronic pain often shows insufﬁcient response to pharmacological treatments. Non-invasive brain stimulation (NIBS) of the motor cortex has been proposed as an alternative therapeutic approach . Repetitive transcranial magnetic stimulation (rTMS) is a NIBS technique that could be used as a preoperative tool to predict the outcome of invasive motor cortex stimulation and could also serve as a therapeutic procedure in itself to treat pain disorders. This therapeutic procedure requires repeated rTMS sessions to be performed as well as a maintenance protocol . RTMS devices used for pain treatment are not portable, so for the maintenance protocol the patients need to go to a specialized Hospital with available rTMS devices (and expertise). This is a potential limitation for the widespread use of rTMS in pain treatment. Other studies have also demonstrated the efﬁcacy of transcranial direct current stimulation (tDCS) in relieving chronic pain syndromes . TDCS is a NIBS technique that painlessly delivers electrical current of relatively low intensity through the skull to selected areas of the brain, inducing changes in the excitability of brain neurons and neuronal circuits . The safety proﬁle of tDCS is very high, with very low risk of seizures. Several double-blind studies on tDCS treatment for chronic pain have shown an analgesic effect of anodal tDCS applied over the primary motor cortex, typically with 20 min of stimulation during 5 consecutive days [4e7]. Again, some patients beneﬁt from a maintenance protocol. TDCS devices are portable, which opens the possibility for patients e at least for the maintenance protocol e not to need to go to a specialized Hospital to receive the treatment. Here, we present a case of a patient treated in our Hospital using tDCS with good pain relief after 5 tDCS daily sessions, and with maintenance of the positive analgesic effects for more than one year with a single tDCS session every 7 days. The patient lives very far away from our Hospital and we decided to use modern information technology to start a Tele-NIBS treatment approach (Fig. 1). Case presentation Our patient, a 56 year-old male, was diagnosed of macrophagic myofasciitis. Macrophagic myofasciitis is a rare condition with few hundreds of deﬁnite cases identiﬁed in France, and isolated cases recorded in other countries. The main clinical complaints of our patient were pain (diffuse myalgias) and chronic fatigue. Pain was intense (mean VAS of 8, VAS scale scored pain from 0 to 10, where 10 meant worst possible pain) and the patient was treated with many different protocols of analgesic drugs (AINE, paracetamol, antiepileptic, antidepressants, gabapentin, pregabalin, benzodiazepine, etc.). All these treatments failed. For this reason, the patient was screened as a possible candidate for NIBS. We received the patient 2 years ago and we decided to treat the patient with a tDCS protocol. The NIBS consisted of 20 min (with 8 s of fade in/out) of anodal
Figure 1. Tele-NIBS treatment approach.
stimulation (with an intensity of 1.5 mA) over both motor cortices. This stimulation protocol was repeated on ﬁve consecutive days and the VAS was obtained for the following 15 days (baseline VAS was also obtained for the 15 days before the tDCS started). The stimulation was delivered using a battery-driven stimulator (Newronika, Milan, Italy), with saline-soaked surface sponge electrodes. Two electrodes were bilaterally placed approximately over the hand area of the motor cortex (7 5 cm). The other electrode was placed over the forehead. Mean VAS in the 15 days before the ﬁve tDCS daily sessions was 8 2 and mean VAS in the 15 days following the tDCS sessions was 4 2. We decided to start a maintenance protocol every 7 days (one session with the same stimulation parameters described above). Along time, mean VAS diminished and the patient reached a VAS of 2 1. Once the patient reached a good pain control, he stopped the treatment and VAS started to go up again in few months (VAS ¼ 5). The reason why the patient stopped the treatment was that he lived very far from the Hospital (about 300 km) and it was difﬁcult for him to reach our Hospital. As reported above, the patient also had chronic fatigue and it was impossible for him to cover the distance between home and our Hospital in the same day to receive the maintenance tDCS protocol (even if his wife/caregiver was driving). For this reason, he had to sleep at a hotel every time he had to receive a tDCS session. Few months ago, we repeated the 5 days tDCS sessions with the same protocol and the patient’s pain diminished again (VAS ¼ 2 1). After more than one year of successful treatment, we decided e together with the patient e to use modern information technology to start a Tele-NIBS treatment approach. We explained all the risks related to tDCS at home and we obtained informed consent from patient and caregiver. The Newronika tDCS device consists of two independent parts: a programmer and the stimulator proper. The caregiver came to the Hospital for one week to learn how to ﬁx the electrodes and how to manage the stimulator (not the programmer). She practiced in our lab all the procedure. Once we decided she was ready, we gave a stimulator (not the programmer) to the patient and we had the ﬁrst “at home” trial. The caregiver was allowed to prepare the skin and to ﬁx the electrodes to the patient head. After this preparation, a skype connection was started. We asked the patient how he felt, if he experienced any problem in the last week, if he had fever, headache, or any other medical complaint. We asked for the webcam to show the patient’s face and head (to