Lasers in Surgery and Medicine 45:648–653 (2013)
Transcranial Application of Near-Infrared Low-Level Laser Can Modulate Cortical Excitability Ljubica M. Konstantinovic´,1,2 Milan B. Jelic´,3 Aleksandra Jeremic´,2 Vuk B. Stevanovic´,3 Sladjan D. Milanovic´,3 and Sasˇa R. Filipovic´3 1 Department of Rehabilitation, Faculty of Medicine, University of Belgrade, 11000, Belgrade, Serbia 2 Klinika za Rehabilitaciju “Dr Miroslav Zotovic´”, 11000, Belgrade, Serbia 3 Department of Neurophysiology, Institute for Medical Research, University of Belgrade, 11000, Belgrade, Serbia
Background and Objective: Near-infrared low-level laser (NIR-LLL) irradiation penetrates scalp and skull and can reach superficial layers of the cerebral cortex. It was shown to improve the outcome of acute stroke in both animal and human studies. In this study we evaluated whether transcranial laser stimulation (TLS) with NIRLLL can modulate the excitability of the motor cortex (M1) as measured by transcranial magnetic stimulation (TMS). Methods: TLS was applied for 5 minutes over the representation of the right first dorsal interosseal muscle (FDI) in left primary motor cortex (M1), in 14 healthy subjects. Motor evoked potentials (MEPs) from the FDI, elicited by single-pulse TMS, were measured at baseline and up to 30 minutes after the TLS. Results: The average MEP size was significantly reduced during the first 20 minutes following the TLS. The pattern was present in 10 (71.5%) of the participants. The MEP size reduction correlated negatively with the motor threshold at rest. Conclusions: TLS with NIR-LLL induced transitory reduction of the excitability of the stimulated cortex. These findings give further insights into the mechanisms of TLS effects in the human cerebral cortex, paving the way for potential applications of TLS in treatment of stroke and in other clinical settings. Lasers Surg. Med. 45:648–653, 2013. ß 2013 Wiley Periodicals, Inc.
found that LLL can reduce brain damage from acute traumatic brain injury in mice [6] and rabbits [7]. Moreover, two recently completed large randomized controlled clinical trials showed a potential of TLS with NIR LLL to improve the outcome in human cases of acute stroke [8,9]. It is thought that LLL acts by inducing a photochemical reaction in the cell, a process referred to as biostimulation or photobiomodulation. Absorption of red or infrared (IR) photons by cytochrome c oxidase in the mitochondrial respiratory chain [10] causes an increase in cellular respiration with increasing of ATP synthesis [11–14]. Further cellular effects induced by LLL also include changes in intracellular calcium [14], modulation of activity of NaþKþATPase [15], photodissociation of nitric oxide [16], and increasing concentration of cyclic adenosine monophosate [17]. All these changes can affect a number of important cellular processes which are supposed to be related to the therapeutic effects of LLL application in various medical conditions [1]. Some of the processes affected by the photobiomodulation are involved into maintaining of neuronal membrane excitability. To explore this issue further, in this study we aimed to evaluate whether and how a short course of transcranially applied NIR LLL modulates motor cortex excitability as measured by transcranial magnetic stimulation (TMS).
Key words: neuronal excitability; membrane potential; transcranial magnetic stimulation; photobiomodulation; human
INTRODUCTION Low-level laser irradiation (LLL) with near infrared (NIR) light has been shown to produce beneficial cellular and physiological effects in animal and human controlled trials [1]. Given its good safety profile and long-standing successful use for wound healing and pain relief, there is an increasing interest for expanding the indications for the medical applications of LLL, including central nervous system pathological conditions [2]. It has been shown that LLL can significantly reduce damages from experimentally induced stroke in rats [3] and rabbits [4,5]. It was also ß 2013 Wiley Periodicals, Inc.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported. Contract grant sponsor: Ministry for Education, Science and Technological Development of Republic of Serbia; Contract grant number: 175012. Correspondence to: Professor S. R. Filipovic´, MD, PhD, Department of Neurophysiology, Institute for Medical Research, University of Belgrade, PO Box 39, 11129 Beograd 102, Serbia. E-mail: [email protected] Accepted 21 September 2013 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/lsm.22190
TRANSCRANIAL LASER STIMULATION
METHODS Participants Eighteen healthy right-handed women were recruited for the study. Results from four of them had to be excluded from analysis due to technical reasons (explained later), and thus, results presented here were based on the data collected from 14 women (mean age 35.0 11.2 years, range 19–50 years). None of them had any history of neurological conditions or other serious medical issues, they were not taking any medications, and none of them was in the menstrual phase. All participants gave informed written consent to participate in the study. The study was approved by the Ethics Committee of the Clinic for Rehabilitation of the Faculty of Medicine, University of Belgrade. Transcranial Magnetic Stimulation (TMS) Motor cortex excitability was assessed by measuring motor evoked potentials (MEPs) elicited in first dorsal interosseus muscle (FDI) of the dominant right hand by a single pulse transcranial magnetic stimulation (TMS) applied over left primary motor cortex (M1). The MEPs were identified in the electromyography (EMG) data collected via Ag–AgCl surface electrodes placed over the muscle in a belly-tendon montage. The EMG activity was amplified (1,000), filtered (10 Hz to 1 kHz), and then sampled at 2 kHz (“CED 1401 plus,” Cambridge Electronic Design, Cambridge, UK). Data were stored on a computer for off-line analysis. For all TMS procedures a Magstim Rapid stimulator (Magstim, Dyfed, Wales, UK) and a 70mm figure-of-eight coil were used. The optimal scalp location (“hot-spot”) for FDI stimulation was determined using single TMS pulses by moving the coil over the scalp in a 1 cm steps around the spot 1 cm anterior to the C4 site from the 10 to 20 EEG Electrode Placement Method. The handle of the coil was oriented posterior to the midline at a 458 angle, in order for the electromagnetic currents to flow perpendicular to the central sulcus. For each participant, at the beginning of the experimental session, a resting motor threshold (RMT) was determined as the lowest stimulus intensity sufficient to produce motor evoked potentials (MEPs) of 50 mV peak-to-peak amplitude, in 5 of 10 subsequent trials in a muscle at rest [18]. Cortical excitability was probed by measuring peak-to-peak amplitudes of MEPs elicited by TMS pulses with intensity 120% of RMT. Participants were comfortably seated in a chair and instructed to remain relaxed throughout the experiment. During the recording, muscle relaxation was monitored by giving subjects visual feedback of their EMG, and trials in which background EMG activity was present were excluded from analysis.
649
(Fig. 1). At each of the points, laser probe was held stationary for 60 seconds keeping skin contact at 908 angle and with the following settings: wavelength 905 nm, pulse frequency 3 kHz, power density 50 mW/cm2, light-beam spot area surface 1 cm2, and single dose 3 J/cm2 (total dose per participant was 15 J/cm2). The optical output of the light-emitting probe was tested before and after the end of the each trial. Design of the Study In order to asses M1 cortical excitability ten MEPs were collected at baseline (before TLS delivery), immediately after the completion of the TLS, and every 5 minutes thereafter (for up to 30 minutes), that is, in total seven times following the TLS. The TMS pluses were delivered with average inter-stimulus interval of 5 seconds that varied randomly 1 seconds. The stimulus delivery was controlled by the “Signal 5” software (Cambridge Electronics Design) which was also used for data collection and analyses. Data Analysis Peak-to-peak MEP amplitudes were measured from the individual traces and the values were subsequently averaged for each of the eight time points. Average of the first 10 MEP measurements, taken before the TLS, was defined as baseline (T0). However, to simplify further statistical analyses and to control for short-term stochastic fluctuations in excitability, we collapsed seven post-TLS time points’ results into three overlapping blocks, each including three successive time-points, covering first, second and third 10-minutes time segments following the TLS (i.e., average measurements taken at 0, 5, and 10 minutes after the TLS formed the block T10; average measurements taken at 10, 15, and 20 minutes after the TLS formed the block T20; and finally, average measurements taken at 20, 25, and 30 minutes after the TLS
Transcranial Laser Stimulation (TLS) The pulsed-mode LLL was applied using standard commercial apparatus Endolaser 476 (Enraf Nonius, Rotterdam, The Netherlands), over the M1 brain area at five adjoined points covering circular area, 3 cm in diameter, centered at the hot-spot for the FDI muscle
Fig. 1. The area covered by the NIR LLL. It consisted of five adjoined circles, 1 cm in diameter each, covering circular area, 3 cm in diameter, centered at the hot-spot for the FDI muscle. Numbers represent the order of stimulation.
650
´ ET AL. KONSTANTINOVIC
formed the block T30). For some of the analyses, relative change from the baseline (dT) was also calculated for each of the post-TLS blocks (i.e., dT10, dT20, and dT30) following the formula: dTx ¼ (Tx T0)/T0 (Tx stands for one of the post-TLS blocks—T10, T20, or T30). Significance of the variability of the excitability measurements over time was evaluated using a repeated measures analysis of variance with analysis of covariance (ANCOVA), where TIME was repeated measures factor (levels: T0, T10, T20, T30) and RMT was a covariate. Subsequent pair-wise analyses of differences between post-TLS blocks and T0 were carried out using post-hoc Fisher least-square difference test. Correlations were evaluated using Pearson’s correlation coefficients. Significance criterion was P < 0.05.
A
B
RESULTS Of 18 participants that were recruited in the study, results from four had to be discarded. For one participant it was due to unusually high baseline MEPs (>3 mV), while for other three it was due to their inability to relax appropriately (i.e., background EMG activity could be seen in too many of the traces). Participants did not report any adverse effects either during or following the TLS. The group average results of the 14 participants included in analyzes showed clear drop in MEP size following the TLS, which was the most pronounced for the T20 (Fig. 2a). The variability of the MEP sizes over time was found to be significant (factor TIME effect: F(3,36) ¼ 4.58, P ¼ 0.008). The post-hoc pair-wise comparisons of post-TLS blocs with T0 showed as significant T0– T10 (P ¼ 0.023) and T0–T20 (P ¼ 0.003) differences, while T0–T30 difference was not (P ¼ 0.18). In addition, there was a significant TIME RMT interaction (F(3,36) ¼ 3.80, P ¼ 0.018). At the individual participants’ level, post-TLS excitability changes did not conform to the group average pattern in four of them (28.6%). In those participants the MEP sizes in at least two post-TLS blocks were near the baseline or above it. In contrast, post-TLS excitability changes followed the pattern found for the group, that is, the MEP sizes in at least two post-TLS blocks were smaller than the baseline, in 10 participants (71.4%); all three blocks below baseline had three participants (21.4%), T10 and T20 were below baseline in four participants (28.6%), and T20 and T30 below baseline were in three participants (21.4%). At the level of individual post-TLS blocks (Fig. 2b), the T20 was most frequently below the baseline (in 10 participants, 71.4%). The T10 and T30 were below baseline with relatively similar frequencies (in 7 and 8 participants, 50.0% and 57.1%, respectively). Given the significant TIME RMT interaction possible correlations were tested between RMT and relative changes from the baseline at each of the post-TLS blocks (Fig. 3). The results showed as significant dT10 versus RMT correlation (R ¼ 0.653, P ¼ 0.011) only, while there was a trend for dT20 versus RMT correlation (R ¼ 0.508, P ¼ 0.064); no correlation was found between dT30 and RMT (R ¼ 0.262, P ¼ 0.202).
Fig. 2. A: Average MEP amplitudes (in mV) at each of the time points; vertical lines are standard errors (SE). B: Numbers of participants with MEP amplitudes at or above the baseline (white bars) contrasted with number of participants with below baseline MEP amplitudes (grey bars) at each time point; numbers at Y-axes represent number of participants, negative numbers are symbolical to highlight that those participants have had MEP sizes below the baseline.
DISCUSSION In this preliminary proof-of-concept study we found that short course of transcranial LLL applied over M1 caused reduction of M1 excitability, as measured by TMS elicited MEP amplitudes, which lasted up to 30 minutes following the end of the laser stimulation. Besides providing another proof for biomodulatory effect of TLS, the results suggest presence of a direct transcranial LLL neuromodulatory effect on cortical excitability that may be further exploited either in physiological research or in treatment of the neurological conditions. Because of the complex nature of the way how an MEP is elicited by TMS, changes in MEP amplitude may reflect change in membrane excitability of either pyramidal neurons or cortical interneurons or change in the synaptic efficacy between the neurons [19]. Nevertheless, whatever may have happened in this study, an MEP is always a result of an action potential generated in a cortical pyramidal cell. Therefore, the obtained results may be interpreted as if LLL increased resistance to the TMS induced depolarization of the cortical pyramidal cells.
TRANSCRANIAL LASER STIMULATION
A
B
651
C
Fig. 3. Correlation between motor threshold at rest (X-axis), presented as percentage of the maximal stimulator output (MSO), and post-TLS relative (related to baseline; baseline is 0%) MEP amplitude change (Y-axis), at each of the three post-TLS time segments. Gray dashed line represents baseline level; dotted black line is regression line.
It has long been hypothesized that one of the major underlying mechanisms involved in the beneficial clinical effects empirically observed after LLL treatment [3–6] is increased mitochondrial function, with consequent increase in ATP production, in neural cells irradiated. It is supposed that increased intracellular availability of ATP would diminish likelihood of cell death due to ischemia and/or injury. Increase in the level of ATP was indeed reported in cultured cells’ studies [13,20]. Moreover, a recent TLS study showed a clear direct dose-dependent relationship between the level of cortical fluence (energy density) delivered by NIR LLL irradiation and cortical ATP content following experimentally induced embolic strokes in rabbits [21]. Unfortunately, there has been no study published so far on effects of transcranial LLL on cortical excitability in humans. The existing data on LLL effects on neuronal excitability were gathered mostly from animal experiments and peripheral nerves. Nevertheless, constant fluctuation of excitability is a fundamental characteristic of neurons in both central and peripheral nervous system. The homeostatic regulation of neuronal excitability is mainly effected through the mechanisms involved in maintenance of the membrane potential. These mechanisms critically depend on supply of ATP necessary for the activity of the pumps that maintain trans-membrane ion gradients [22,23], and of these the most important is the Naþ/KþATPase, or sodium pump [24].
In view of the later, it was shown that NIR LLL irradiation affects several ATP dependent ion membrane pumps. When applied transcutaneously over rat saphenous nerve for 6–15 seconds, LLL caused increased activity of NaþKþATPase [25]. Similar was found also in a cultured cell study [15]. Our previous studies in adult rabbits have shown that transcranial LLL irradiation at the level of brainstem caused significant increase of the activity of the sodium pump in the cortex and brainstem [26]. Increased activity of both of these pumps leads to greater membrane stability and resistance to depolarization. Given that excitotoxic depolarization of the neurons in the ischemic penumbra is one of the main causes of the cell death and expansion of the stroke lesion size [27], this membrane stabilization effect may be an important contributory factor behind recently reported beneficial clinical effects of the TLS with NIR LLL in acute stroke [8,9]. Alternatively, NIR LLL TLS may target only a selected inter-neuronal sub-population within M1, which through their projections modulate excitability of the corticospinal neurons. In this case, TLS would resemble other TMSbased plasticity-inducing protocols currently used in healthy humans to elicit long-term changes in M1 excitability [28]. The TLS induced reduction of excitability in this study, particularly in the early post-TLS period, was negatively correlated with baseline TMS MEP threshold at rest, the RMT. It has been show that RMT is directly proportional to
652
´ ET AL. KONSTANTINOVIC
the distance from the coil to the cortical surface [29,30]. It may be assumed that the observed link between smaller TLS effects on MEP size reduction with higher RMT is a reflection of the similar phenomenon—the larger distance from the laser diode to the cortical surface in participants with higher RMT may have caused smaller post-TLS effects. The apparent shortcoming of this study may be a lack of placebo control. However, we were of opinion that at this early exploratory stage a placebo control would not be necessary due to the fact that the presence of placebo effect on motor cortex excitability has never been shown. There have been numerous studies so far, using various modalities of sham transcranial stimulation, from early studies with repetitive TMS [31,32] to the latest attempts with transcranial static magnetic field stimulation [33], that have never shown any consistent change in motor cortex excitability following application of a sham transcranial stimulating device. It is hard to believe that 5-minute-long application of a small pen-like device that gives no local sensation would have a different effect. Obviously, there might have been other confounding factors such as postintervention sleepiness or decreased level of attention, which could cause fluctuations of excitability. However, the spontaneous post-intervention reduction of excitability has not been found in previous studies with other types of sham noninvasive brain stimulation, as mentioned earlier [31–33]. Nevertheless, to further the use of LLL TLS in experimental and clinical settings, it will be necessary to confirm the results by testing larger population of subjects, and to compare the effects of 905 nm LLL TLS with effects of LLL TLS with other wavelengths from NIR range and from other light regions that would not be expected to penetrate the cranium (the latter could in fact serve as a sham stimulation too). Similarly, it would be also worthwhile to test the effects of exposure of different areas of the brain not expected to elicit a response in the area being measured for change in activity. In conclusion, the results of this study support possibility of developing a transcranial laser stimulation (TLS) protocols for modulation of brain cortical activity that may be used in research and treatment. The results also provide a physiological background that may contribute towards better understanding of the mechanisms behind reported beneficial effects of TLS in acute stroke patients. ACKNOWLEDGMENTS This study was supported by project grant (#175012) from the Ministry for Education, Science and Technological Development of Republic of Serbia. Authors have nothing else to disclose. REFERENCES 1. Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, Hamblin MR. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 2012;40:516–533. 2. Lapchak PA. Transcranial near-infrared laser therapy applied to promote clinical recovery in acute and chronic neurodegenerative diseases. Expert Rev Med Devices 2012; 9:71–83.
3. Oron A, Oron U, Chen J, Eilam A, Zhang C, Sadeh M, Lampl Y, Streeter J, DeTaboada L, Chopp M. Low-level laser therapy applied transcranially to rats after induction of stroke significantly reduces long-term neurological deficits. Stroke 2006;37:2620–2624. 4. Lapchak PA, Wei J, Zivin JA. Transcranial infrared laser therapy improves clinical rating scores after embolic strokes in rabbits. Stroke 2004;35:1985–1988. 5. Lapchak PA, Salgado KF, Chao CH, Zivin JA. Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: An extended therapeutic window study using continuous and pulse frequency delivery modes. Neuroscience 2007;148:907–914. 6. Oron A, Oron U, Streeter J, De TL, Alexandrovich A, Trembovler V, Shohami E. low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits. J Neurotrauma 2007;24:651–656. 7. Konstantinovic´ L, Cernak I, Prokic´ V. Influence of low level laser irradiation on biochemical processes in brainstem and cortex of intact rabbits. Vojnosanit pregl 1997;54:533–540. 8. Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B, Borenstein P, Andersson B, Perez J, Caparo C, Ilic S, Oron U. Infrared laser therapy for ischemic stroke: A new treatment strategy: Results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke 2007;38:1843–1849. 9. Zivin JA, Albers GW, Bornstein N, Chippendale T, Dahlof B, Devlin T, Fisher M, Hacke W, Holt W, Ilic S, Kasner S, Lew R, Nash M, Perez J, Rymer M, Schellinger P, Schneider D, Schwab S, Veltkamp R, Walker M, Streeter J. Effectiveness and safety of transcranial laser therapy for acute ischemic stroke. Stroke 2009;40:1359–1364. 10. Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, Kane M, Whelan HT. Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: Role of cytochrome c oxidase. J Biol Chem 2005;280:4761–4771. 11. Karu T, Pyatibrat L, Kalendo G. Irradiation with He–Ne laser increases ATP level in cells cultivated in vitro. J Photochem Photobiol B 1995;27:219–223. 12. Mochizuki-Oda N, Kataoka Y, Cui Y, Yamada H, Heya M, Awazu K. Effects of near-infra-red laser irradiation on adenosine triphosphate and adenosine diphosphate contents of rat brain tissue. Neurosci Lett 2002;323:207–210. 13. Oron U, Ilic S, De TL, Streeter J. Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture. Photomed Laser Surg 2007;25:180–182. 14. Yu W, Naim JO, McGowan M, Ippolito K, Lanzafame RJ. Photomodulation of oxidative metabolism and electron chain enzymes in rat liver mitochondria. Photochem Photobiol 1997;66:866–871. 15. Yachnev IL, Plakhova VB, Podzorova SA, Shelykh TN, Rogachevsky IV, Krylov BV. Mechanism of pain relief by low-power infrared irradiation: ATP is an IR-target molecule in nociceptive neurons. Med Chem 2012;8:14–21. 16. Lohr NL, Keszler A, Pratt P, Bienengraber M, Warltier DC, Hogg N. Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: Potential role in cardioprotection. J Mol Cell Cardiol 2009;47:256–263. 17. Zungu IL, Hawkins ED, Abrahamse H. Mitochondrial responses of normal and injured human skin fibroblasts following low level laser irradiation—An in vitro study. Photochem Photobiol 2009;85:987–996. 18. Rossini PM, Barker AT, Berardelli A, Caramia MD, Caruso G, Cracco RQ, Dimitrijevic MR, Hallett M, Katayama Y, Lucking CH. Non-invasive electrical and magnetic stimulation of the brain spinal cord and roots: Basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalogr Clin Neurophysiol 1994;91:79–92. 19. Cheeran B, Koch G, Stagg CJ, Baig F, Teo J. Transcranial magnetic stimulation: From neurophysiology to pharmacology, molecular biology and genomics. Neuroscientist 2010; 16:210–221. 20. Sharma SK, Kharkwal GB, Sajo M, Huang YY, De TL, McCarthy T, Hamblin MR. Dose response effects of 810 nm
TRANSCRANIAL LASER STIMULATION
21.
22. 23. 24.
25.
26. 27.
laser light on mouse primary cortical neurons. Lasers Surg Med 2011;43:851–859. Lapchak PA, De TL. Transcranial near infrared laser treatment (NILT) increases cortical adenosine-50 -triphosphate (ATP) content following embolic strokes in rabbits. Brain Res 2010;1306:100–105. Ames A III. CNS energy metabolism as related to function. Brain Res Brain Res Rev 2000;34:42–68. Bean BP. The action potential in mammalian central neurons. Nat Rev Neurosci 2007;8:451–465. Eijkelkamp N, Linley JE, Baker MD, Minett MS, Cregg R, Werdehausen R, Rugiero F, Wood JN. Neurological perspectives on voltage-gated sodium channels. Brain 2012;135: 2585–2612. Kudoh C, Inomata K, Okajima K, Motegi M, Ohshi T. Effects of 830 nm gallium aluminium arsenide diode laser radiation on rat saphernous nerve sodium–potassium-adensine oiphosphaiase activity: A possible attenuation mechanism examinated. Laser Ther 1989;1:63–67. ´ L, Cernak I, Proki C ´ V. Influence of low level Konstantinovi C laser irradiation on biochemical processes in brainstem and cortex of intact rabbits. Vojnosanit Pregl 1997;54:533–540. Brouns R, De Deyn PP. The complexity of neurobiological processes in acute ischemic stroke. Clin Neurol Neurosurg 2009;111:483–495.
653
28. Di Lazzaro V, Profice P, Pilato F, Dileone M, Oliviero A, Ziemann U. The effects of motor cortex rTMS on corticospinal descending activity. Clin Neurophysiol 2010;121: 464–473. 29. McConnell KA, Nahas Z, Shastri A, Lorberbaum JP, Kozel FA, Bohning DE, George MS. The transcranial magnetic stimulation motor threshold depends on the distance from coil to underlying cortex: A replication in healthy adults comparing two methods of assessing the distance to cortex. Biol Psychiatry 2001;49:454–459. 30. Stokes MG, Chambers CD, Gould IC, English T, McNaught E, McDonald O, Mattingley JB. Distance-adjusted motor threshold for transcranial magnetic stimulation. Clin Neurophysiol 2007;118:1617–1625. 31. Chen R, Classen J, Gerloff C, Celnik P, Wassermann EM, Hallett M, Cohen LG. Depression of motor cortex excitability by low-frequency transcranial magnetic stimulation. Neurology 1997;48:1398–1403. 32. Pascual-Leone A, Valls-Sole J, Wassermann EM, Hallett M. Responses to rapid-rate transcranial magnetic stimulation of the human motor cortex. Brain 1994;117:847–858. 33. Oliviero A, Mordillo-Mateos L, Arias P, Panyavin I, Foffani G, Aguilar J. Transcranial static magnetic field stimulation of the human motor cortex. J Physiol 2011;589: 4949–4958.
Transcranial Application of Near-Infrared Low-Level Laser Can Modulate Cortical Excitability Ljubica M. Konstantinovic´,1,2 Milan B. Jelic´,3 Aleksandra Jeremic´,2 Vuk B. Stevanovic´,3 Sladjan D. Milanovic´,3 and Sasˇa R. Filipovic´3 1 Department of Rehabilitation, Faculty of Medicine, University of Belgrade, 11000, Belgrade, Serbia 2 Klinika za Rehabilitaciju “Dr Miroslav Zotovic´”, 11000, Belgrade, Serbia 3 Department of Neurophysiology, Institute for Medical Research, University of Belgrade, 11000, Belgrade, Serbia
Background and Objective: Near-infrared low-level laser (NIR-LLL) irradiation penetrates scalp and skull and can reach superficial layers of the cerebral cortex. It was shown to improve the outcome of acute stroke in both animal and human studies. In this study we evaluated whether transcranial laser stimulation (TLS) with NIRLLL can modulate the excitability of the motor cortex (M1) as measured by transcranial magnetic stimulation (TMS). Methods: TLS was applied for 5 minutes over the representation of the right first dorsal interosseal muscle (FDI) in left primary motor cortex (M1), in 14 healthy subjects. Motor evoked potentials (MEPs) from the FDI, elicited by single-pulse TMS, were measured at baseline and up to 30 minutes after the TLS. Results: The average MEP size was significantly reduced during the first 20 minutes following the TLS. The pattern was present in 10 (71.5%) of the participants. The MEP size reduction correlated negatively with the motor threshold at rest. Conclusions: TLS with NIR-LLL induced transitory reduction of the excitability of the stimulated cortex. These findings give further insights into the mechanisms of TLS effects in the human cerebral cortex, paving the way for potential applications of TLS in treatment of stroke and in other clinical settings. Lasers Surg. Med. 45:648–653, 2013. ß 2013 Wiley Periodicals, Inc.
found that LLL can reduce brain damage from acute traumatic brain injury in mice [6] and rabbits [7]. Moreover, two recently completed large randomized controlled clinical trials showed a potential of TLS with NIR LLL to improve the outcome in human cases of acute stroke [8,9]. It is thought that LLL acts by inducing a photochemical reaction in the cell, a process referred to as biostimulation or photobiomodulation. Absorption of red or infrared (IR) photons by cytochrome c oxidase in the mitochondrial respiratory chain [10] causes an increase in cellular respiration with increasing of ATP synthesis [11–14]. Further cellular effects induced by LLL also include changes in intracellular calcium [14], modulation of activity of NaþKþATPase [15], photodissociation of nitric oxide [16], and increasing concentration of cyclic adenosine monophosate [17]. All these changes can affect a number of important cellular processes which are supposed to be related to the therapeutic effects of LLL application in various medical conditions [1]. Some of the processes affected by the photobiomodulation are involved into maintaining of neuronal membrane excitability. To explore this issue further, in this study we aimed to evaluate whether and how a short course of transcranially applied NIR LLL modulates motor cortex excitability as measured by transcranial magnetic stimulation (TMS).
Key words: neuronal excitability; membrane potential; transcranial magnetic stimulation; photobiomodulation; human
INTRODUCTION Low-level laser irradiation (LLL) with near infrared (NIR) light has been shown to produce beneficial cellular and physiological effects in animal and human controlled trials [1]. Given its good safety profile and long-standing successful use for wound healing and pain relief, there is an increasing interest for expanding the indications for the medical applications of LLL, including central nervous system pathological conditions [2]. It has been shown that LLL can significantly reduce damages from experimentally induced stroke in rats [3] and rabbits [4,5]. It was also ß 2013 Wiley Periodicals, Inc.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported. Contract grant sponsor: Ministry for Education, Science and Technological Development of Republic of Serbia; Contract grant number: 175012. Correspondence to: Professor S. R. Filipovic´, MD, PhD, Department of Neurophysiology, Institute for Medical Research, University of Belgrade, PO Box 39, 11129 Beograd 102, Serbia. E-mail: [email protected] Accepted 21 September 2013 Published online 17 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/lsm.22190
TRANSCRANIAL LASER STIMULATION
METHODS Participants Eighteen healthy right-handed women were recruited for the study. Results from four of them had to be excluded from analysis due to technical reasons (explained later), and thus, results presented here were based on the data collected from 14 women (mean age 35.0 11.2 years, range 19–50 years). None of them had any history of neurological conditions or other serious medical issues, they were not taking any medications, and none of them was in the menstrual phase. All participants gave informed written consent to participate in the study. The study was approved by the Ethics Committee of the Clinic for Rehabilitation of the Faculty of Medicine, University of Belgrade. Transcranial Magnetic Stimulation (TMS) Motor cortex excitability was assessed by measuring motor evoked potentials (MEPs) elicited in first dorsal interosseus muscle (FDI) of the dominant right hand by a single pulse transcranial magnetic stimulation (TMS) applied over left primary motor cortex (M1). The MEPs were identified in the electromyography (EMG) data collected via Ag–AgCl surface electrodes placed over the muscle in a belly-tendon montage. The EMG activity was amplified (1,000), filtered (10 Hz to 1 kHz), and then sampled at 2 kHz (“CED 1401 plus,” Cambridge Electronic Design, Cambridge, UK). Data were stored on a computer for off-line analysis. For all TMS procedures a Magstim Rapid stimulator (Magstim, Dyfed, Wales, UK) and a 70mm figure-of-eight coil were used. The optimal scalp location (“hot-spot”) for FDI stimulation was determined using single TMS pulses by moving the coil over the scalp in a 1 cm steps around the spot 1 cm anterior to the C4 site from the 10 to 20 EEG Electrode Placement Method. The handle of the coil was oriented posterior to the midline at a 458 angle, in order for the electromagnetic currents to flow perpendicular to the central sulcus. For each participant, at the beginning of the experimental session, a resting motor threshold (RMT) was determined as the lowest stimulus intensity sufficient to produce motor evoked potentials (MEPs) of 50 mV peak-to-peak amplitude, in 5 of 10 subsequent trials in a muscle at rest [18]. Cortical excitability was probed by measuring peak-to-peak amplitudes of MEPs elicited by TMS pulses with intensity 120% of RMT. Participants were comfortably seated in a chair and instructed to remain relaxed throughout the experiment. During the recording, muscle relaxation was monitored by giving subjects visual feedback of their EMG, and trials in which background EMG activity was present were excluded from analysis.
649
(Fig. 1). At each of the points, laser probe was held stationary for 60 seconds keeping skin contact at 908 angle and with the following settings: wavelength 905 nm, pulse frequency 3 kHz, power density 50 mW/cm2, light-beam spot area surface 1 cm2, and single dose 3 J/cm2 (total dose per participant was 15 J/cm2). The optical output of the light-emitting probe was tested before and after the end of the each trial. Design of the Study In order to asses M1 cortical excitability ten MEPs were collected at baseline (before TLS delivery), immediately after the completion of the TLS, and every 5 minutes thereafter (for up to 30 minutes), that is, in total seven times following the TLS. The TMS pluses were delivered with average inter-stimulus interval of 5 seconds that varied randomly 1 seconds. The stimulus delivery was controlled by the “Signal 5” software (Cambridge Electronics Design) which was also used for data collection and analyses. Data Analysis Peak-to-peak MEP amplitudes were measured from the individual traces and the values were subsequently averaged for each of the eight time points. Average of the first 10 MEP measurements, taken before the TLS, was defined as baseline (T0). However, to simplify further statistical analyses and to control for short-term stochastic fluctuations in excitability, we collapsed seven post-TLS time points’ results into three overlapping blocks, each including three successive time-points, covering first, second and third 10-minutes time segments following the TLS (i.e., average measurements taken at 0, 5, and 10 minutes after the TLS formed the block T10; average measurements taken at 10, 15, and 20 minutes after the TLS formed the block T20; and finally, average measurements taken at 20, 25, and 30 minutes after the TLS
Transcranial Laser Stimulation (TLS) The pulsed-mode LLL was applied using standard commercial apparatus Endolaser 476 (Enraf Nonius, Rotterdam, The Netherlands), over the M1 brain area at five adjoined points covering circular area, 3 cm in diameter, centered at the hot-spot for the FDI muscle
Fig. 1. The area covered by the NIR LLL. It consisted of five adjoined circles, 1 cm in diameter each, covering circular area, 3 cm in diameter, centered at the hot-spot for the FDI muscle. Numbers represent the order of stimulation.
650
´ ET AL. KONSTANTINOVIC
formed the block T30). For some of the analyses, relative change from the baseline (dT) was also calculated for each of the post-TLS blocks (i.e., dT10, dT20, and dT30) following the formula: dTx ¼ (Tx T0)/T0 (Tx stands for one of the post-TLS blocks—T10, T20, or T30). Significance of the variability of the excitability measurements over time was evaluated using a repeated measures analysis of variance with analysis of covariance (ANCOVA), where TIME was repeated measures factor (levels: T0, T10, T20, T30) and RMT was a covariate. Subsequent pair-wise analyses of differences between post-TLS blocks and T0 were carried out using post-hoc Fisher least-square difference test. Correlations were evaluated using Pearson’s correlation coefficients. Significance criterion was P < 0.05.
A
B
RESULTS Of 18 participants that were recruited in the study, results from four had to be discarded. For one participant it was due to unusually high baseline MEPs (>3 mV), while for other three it was due to their inability to relax appropriately (i.e., background EMG activity could be seen in too many of the traces). Participants did not report any adverse effects either during or following the TLS. The group average results of the 14 participants included in analyzes showed clear drop in MEP size following the TLS, which was the most pronounced for the T20 (Fig. 2a). The variability of the MEP sizes over time was found to be significant (factor TIME effect: F(3,36) ¼ 4.58, P ¼ 0.008). The post-hoc pair-wise comparisons of post-TLS blocs with T0 showed as significant T0– T10 (P ¼ 0.023) and T0–T20 (P ¼ 0.003) differences, while T0–T30 difference was not (P ¼ 0.18). In addition, there was a significant TIME RMT interaction (F(3,36) ¼ 3.80, P ¼ 0.018). At the individual participants’ level, post-TLS excitability changes did not conform to the group average pattern in four of them (28.6%). In those participants the MEP sizes in at least two post-TLS blocks were near the baseline or above it. In contrast, post-TLS excitability changes followed the pattern found for the group, that is, the MEP sizes in at least two post-TLS blocks were smaller than the baseline, in 10 participants (71.4%); all three blocks below baseline had three participants (21.4%), T10 and T20 were below baseline in four participants (28.6%), and T20 and T30 below baseline were in three participants (21.4%). At the level of individual post-TLS blocks (Fig. 2b), the T20 was most frequently below the baseline (in 10 participants, 71.4%). The T10 and T30 were below baseline with relatively similar frequencies (in 7 and 8 participants, 50.0% and 57.1%, respectively). Given the significant TIME RMT interaction possible correlations were tested between RMT and relative changes from the baseline at each of the post-TLS blocks (Fig. 3). The results showed as significant dT10 versus RMT correlation (R ¼ 0.653, P ¼ 0.011) only, while there was a trend for dT20 versus RMT correlation (R ¼ 0.508, P ¼ 0.064); no correlation was found between dT30 and RMT (R ¼ 0.262, P ¼ 0.202).
Fig. 2. A: Average MEP amplitudes (in mV) at each of the time points; vertical lines are standard errors (SE). B: Numbers of participants with MEP amplitudes at or above the baseline (white bars) contrasted with number of participants with below baseline MEP amplitudes (grey bars) at each time point; numbers at Y-axes represent number of participants, negative numbers are symbolical to highlight that those participants have had MEP sizes below the baseline.
DISCUSSION In this preliminary proof-of-concept study we found that short course of transcranial LLL applied over M1 caused reduction of M1 excitability, as measured by TMS elicited MEP amplitudes, which lasted up to 30 minutes following the end of the laser stimulation. Besides providing another proof for biomodulatory effect of TLS, the results suggest presence of a direct transcranial LLL neuromodulatory effect on cortical excitability that may be further exploited either in physiological research or in treatment of the neurological conditions. Because of the complex nature of the way how an MEP is elicited by TMS, changes in MEP amplitude may reflect change in membrane excitability of either pyramidal neurons or cortical interneurons or change in the synaptic efficacy between the neurons [19]. Nevertheless, whatever may have happened in this study, an MEP is always a result of an action potential generated in a cortical pyramidal cell. Therefore, the obtained results may be interpreted as if LLL increased resistance to the TMS induced depolarization of the cortical pyramidal cells.
TRANSCRANIAL LASER STIMULATION
A
B
651
C
Fig. 3. Correlation between motor threshold at rest (X-axis), presented as percentage of the maximal stimulator output (MSO), and post-TLS relative (related to baseline; baseline is 0%) MEP amplitude change (Y-axis), at each of the three post-TLS time segments. Gray dashed line represents baseline level; dotted black line is regression line.
It has long been hypothesized that one of the major underlying mechanisms involved in the beneficial clinical effects empirically observed after LLL treatment [3–6] is increased mitochondrial function, with consequent increase in ATP production, in neural cells irradiated. It is supposed that increased intracellular availability of ATP would diminish likelihood of cell death due to ischemia and/or injury. Increase in the level of ATP was indeed reported in cultured cells’ studies [13,20]. Moreover, a recent TLS study showed a clear direct dose-dependent relationship between the level of cortical fluence (energy density) delivered by NIR LLL irradiation and cortical ATP content following experimentally induced embolic strokes in rabbits [21]. Unfortunately, there has been no study published so far on effects of transcranial LLL on cortical excitability in humans. The existing data on LLL effects on neuronal excitability were gathered mostly from animal experiments and peripheral nerves. Nevertheless, constant fluctuation of excitability is a fundamental characteristic of neurons in both central and peripheral nervous system. The homeostatic regulation of neuronal excitability is mainly effected through the mechanisms involved in maintenance of the membrane potential. These mechanisms critically depend on supply of ATP necessary for the activity of the pumps that maintain trans-membrane ion gradients [22,23], and of these the most important is the Naþ/KþATPase, or sodium pump [24].
In view of the later, it was shown that NIR LLL irradiation affects several ATP dependent ion membrane pumps. When applied transcutaneously over rat saphenous nerve for 6–15 seconds, LLL caused increased activity of NaþKþATPase [25]. Similar was found also in a cultured cell study [15]. Our previous studies in adult rabbits have shown that transcranial LLL irradiation at the level of brainstem caused significant increase of the activity of the sodium pump in the cortex and brainstem [26]. Increased activity of both of these pumps leads to greater membrane stability and resistance to depolarization. Given that excitotoxic depolarization of the neurons in the ischemic penumbra is one of the main causes of the cell death and expansion of the stroke lesion size [27], this membrane stabilization effect may be an important contributory factor behind recently reported beneficial clinical effects of the TLS with NIR LLL in acute stroke [8,9]. Alternatively, NIR LLL TLS may target only a selected inter-neuronal sub-population within M1, which through their projections modulate excitability of the corticospinal neurons. In this case, TLS would resemble other TMSbased plasticity-inducing protocols currently used in healthy humans to elicit long-term changes in M1 excitability [28]. The TLS induced reduction of excitability in this study, particularly in the early post-TLS period, was negatively correlated with baseline TMS MEP threshold at rest, the RMT. It has been show that RMT is directly proportional to
652
´ ET AL. KONSTANTINOVIC
the distance from the coil to the cortical surface [29,30]. It may be assumed that the observed link between smaller TLS effects on MEP size reduction with higher RMT is a reflection of the similar phenomenon—the larger distance from the laser diode to the cortical surface in participants with higher RMT may have caused smaller post-TLS effects. The apparent shortcoming of this study may be a lack of placebo control. However, we were of opinion that at this early exploratory stage a placebo control would not be necessary due to the fact that the presence of placebo effect on motor cortex excitability has never been shown. There have been numerous studies so far, using various modalities of sham transcranial stimulation, from early studies with repetitive TMS [31,32] to the latest attempts with transcranial static magnetic field stimulation [33], that have never shown any consistent change in motor cortex excitability following application of a sham transcranial stimulating device. It is hard to believe that 5-minute-long application of a small pen-like device that gives no local sensation would have a different effect. Obviously, there might have been other confounding factors such as postintervention sleepiness or decreased level of attention, which could cause fluctuations of excitability. However, the spontaneous post-intervention reduction of excitability has not been found in previous studies with other types of sham noninvasive brain stimulation, as mentioned earlier [31–33]. Nevertheless, to further the use of LLL TLS in experimental and clinical settings, it will be necessary to confirm the results by testing larger population of subjects, and to compare the effects of 905 nm LLL TLS with effects of LLL TLS with other wavelengths from NIR range and from other light regions that would not be expected to penetrate the cranium (the latter could in fact serve as a sham stimulation too). Similarly, it would be also worthwhile to test the effects of exposure of different areas of the brain not expected to elicit a response in the area being measured for change in activity. In conclusion, the results of this study support possibility of developing a transcranial laser stimulation (TLS) protocols for modulation of brain cortical activity that may be used in research and treatment. The results also provide a physiological background that may contribute towards better understanding of the mechanisms behind reported beneficial effects of TLS in acute stroke patients. ACKNOWLEDGMENTS This study was supported by project grant (#175012) from the Ministry for Education, Science and Technological Development of Republic of Serbia. Authors have nothing else to disclose. REFERENCES 1. Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, Hamblin MR. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 2012;40:516–533. 2. Lapchak PA. Transcranial near-infrared laser therapy applied to promote clinical recovery in acute and chronic neurodegenerative diseases. Expert Rev Med Devices 2012; 9:71–83.
3. Oron A, Oron U, Chen J, Eilam A, Zhang C, Sadeh M, Lampl Y, Streeter J, DeTaboada L, Chopp M. Low-level laser therapy applied transcranially to rats after induction of stroke significantly reduces long-term neurological deficits. Stroke 2006;37:2620–2624. 4. Lapchak PA, Wei J, Zivin JA. Transcranial infrared laser therapy improves clinical rating scores after embolic strokes in rabbits. Stroke 2004;35:1985–1988. 5. Lapchak PA, Salgado KF, Chao CH, Zivin JA. Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: An extended therapeutic window study using continuous and pulse frequency delivery modes. Neuroscience 2007;148:907–914. 6. Oron A, Oron U, Streeter J, De TL, Alexandrovich A, Trembovler V, Shohami E. low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits. J Neurotrauma 2007;24:651–656. 7. Konstantinovic´ L, Cernak I, Prokic´ V. Influence of low level laser irradiation on biochemical processes in brainstem and cortex of intact rabbits. Vojnosanit pregl 1997;54:533–540. 8. Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B, Borenstein P, Andersson B, Perez J, Caparo C, Ilic S, Oron U. Infrared laser therapy for ischemic stroke: A new treatment strategy: Results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke 2007;38:1843–1849. 9. Zivin JA, Albers GW, Bornstein N, Chippendale T, Dahlof B, Devlin T, Fisher M, Hacke W, Holt W, Ilic S, Kasner S, Lew R, Nash M, Perez J, Rymer M, Schellinger P, Schneider D, Schwab S, Veltkamp R, Walker M, Streeter J. Effectiveness and safety of transcranial laser therapy for acute ischemic stroke. Stroke 2009;40:1359–1364. 10. Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, Kane M, Whelan HT. Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: Role of cytochrome c oxidase. J Biol Chem 2005;280:4761–4771. 11. Karu T, Pyatibrat L, Kalendo G. Irradiation with He–Ne laser increases ATP level in cells cultivated in vitro. J Photochem Photobiol B 1995;27:219–223. 12. Mochizuki-Oda N, Kataoka Y, Cui Y, Yamada H, Heya M, Awazu K. Effects of near-infra-red laser irradiation on adenosine triphosphate and adenosine diphosphate contents of rat brain tissue. Neurosci Lett 2002;323:207–210. 13. Oron U, Ilic S, De TL, Streeter J. Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture. Photomed Laser Surg 2007;25:180–182. 14. Yu W, Naim JO, McGowan M, Ippolito K, Lanzafame RJ. Photomodulation of oxidative metabolism and electron chain enzymes in rat liver mitochondria. Photochem Photobiol 1997;66:866–871. 15. Yachnev IL, Plakhova VB, Podzorova SA, Shelykh TN, Rogachevsky IV, Krylov BV. Mechanism of pain relief by low-power infrared irradiation: ATP is an IR-target molecule in nociceptive neurons. Med Chem 2012;8:14–21. 16. Lohr NL, Keszler A, Pratt P, Bienengraber M, Warltier DC, Hogg N. Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: Potential role in cardioprotection. J Mol Cell Cardiol 2009;47:256–263. 17. Zungu IL, Hawkins ED, Abrahamse H. Mitochondrial responses of normal and injured human skin fibroblasts following low level laser irradiation—An in vitro study. Photochem Photobiol 2009;85:987–996. 18. Rossini PM, Barker AT, Berardelli A, Caramia MD, Caruso G, Cracco RQ, Dimitrijevic MR, Hallett M, Katayama Y, Lucking CH. Non-invasive electrical and magnetic stimulation of the brain spinal cord and roots: Basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalogr Clin Neurophysiol 1994;91:79–92. 19. Cheeran B, Koch G, Stagg CJ, Baig F, Teo J. Transcranial magnetic stimulation: From neurophysiology to pharmacology, molecular biology and genomics. Neuroscientist 2010; 16:210–221. 20. Sharma SK, Kharkwal GB, Sajo M, Huang YY, De TL, McCarthy T, Hamblin MR. Dose response effects of 810 nm
TRANSCRANIAL LASER STIMULATION
21.
22. 23. 24.
25.
26. 27.
laser light on mouse primary cortical neurons. Lasers Surg Med 2011;43:851–859. Lapchak PA, De TL. Transcranial near infrared laser treatment (NILT) increases cortical adenosine-50 -triphosphate (ATP) content following embolic strokes in rabbits. Brain Res 2010;1306:100–105. Ames A III. CNS energy metabolism as related to function. Brain Res Brain Res Rev 2000;34:42–68. Bean BP. The action potential in mammalian central neurons. Nat Rev Neurosci 2007;8:451–465. Eijkelkamp N, Linley JE, Baker MD, Minett MS, Cregg R, Werdehausen R, Rugiero F, Wood JN. Neurological perspectives on voltage-gated sodium channels. Brain 2012;135: 2585–2612. Kudoh C, Inomata K, Okajima K, Motegi M, Ohshi T. Effects of 830 nm gallium aluminium arsenide diode laser radiation on rat saphernous nerve sodium–potassium-adensine oiphosphaiase activity: A possible attenuation mechanism examinated. Laser Ther 1989;1:63–67. ´ L, Cernak I, Proki C ´ V. Influence of low level Konstantinovi C laser irradiation on biochemical processes in brainstem and cortex of intact rabbits. Vojnosanit Pregl 1997;54:533–540. Brouns R, De Deyn PP. The complexity of neurobiological processes in acute ischemic stroke. Clin Neurol Neurosurg 2009;111:483–495.
653
28. Di Lazzaro V, Profice P, Pilato F, Dileone M, Oliviero A, Ziemann U. The effects of motor cortex rTMS on corticospinal descending activity. Clin Neurophysiol 2010;121: 464–473. 29. McConnell KA, Nahas Z, Shastri A, Lorberbaum JP, Kozel FA, Bohning DE, George MS. The transcranial magnetic stimulation motor threshold depends on the distance from coil to underlying cortex: A replication in healthy adults comparing two methods of assessing the distance to cortex. Biol Psychiatry 2001;49:454–459. 30. Stokes MG, Chambers CD, Gould IC, English T, McNaught E, McDonald O, Mattingley JB. Distance-adjusted motor threshold for transcranial magnetic stimulation. Clin Neurophysiol 2007;118:1617–1625. 31. Chen R, Classen J, Gerloff C, Celnik P, Wassermann EM, Hallett M, Cohen LG. Depression of motor cortex excitability by low-frequency transcranial magnetic stimulation. Neurology 1997;48:1398–1403. 32. Pascual-Leone A, Valls-Sole J, Wassermann EM, Hallett M. Responses to rapid-rate transcranial magnetic stimulation of the human motor cortex. Brain 1994;117:847–858. 33. Oliviero A, Mordillo-Mateos L, Arias P, Panyavin I, Foffani G, Aguilar J. Transcranial static magnetic field stimulation of the human motor cortex. J Physiol 2011;589: 4949–4958.
