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Mar Cortes

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Mar Cortesa,b, Alejandra Climenta, Laura Dubreuil Vallc, Giulio Ruffinic  Douglas Labara, Dylan Edwardsa a. Non invasive Brain Stimulation and Human Motor Control Laboratory, Burke Medical Research Institute, Weill Medical College of Cornell University, 785 Mamaroneck Avenue, 10605, White Plains, NY, USA.b. Universitat de Barcelona, Barcelona, Spain. c. Neuroelectrics Corporation, Cambridge (MA), USA. Corresponding author:Mar CortesNon invasive Brain Stimulation and Human Motor Control Laboratory, Burke Medical Research Institute, Weill Medical College of Cornell University, 785 Mamaroneck Avenue, 10605, White Plains, NY, USA.mac2083@med.cornell.eduPhone: +1 914 368 3181 Abstract: (250)[MC1]  Background: Existing strategies to enhance motor function following Spinal Cord Injury (SCI) are suboptimal leaving patients with considerable disability. Available evidence suggests that transcranial direct current stimulation (tDCS) is a promising method to improve motor dysfunction. How tDCS affects resting brain activity monitored by EEG is little explored. Objective: Investigate the effects of anodal tDCS on brain signaling (EEG) and neurophysiology (TMS) when targeting forearm muscles below the level of the lesion in chronic SCI subjects. Methods: We conducted a randomized, single blind, sham-controlled, cross-over study in seven chronic SCI subjects with cervical lesions. We investigated the effects of 20-minute anodal tDCS applied over the left primary motor cortex (M1) on electroencephalography (EEG) power spectrum density, coherence and frequency band power. Subjects were randomized to receive either 1mA or sham stimulation.  The EEG data acquisition pre and post stimulation comprised 5-minute takes of 24 bit, 500 S/s 8-channel EEG using StarStim Ag/AgCl EEG electrodes (at F3, F4, Cz, C4, P3 and P4; and Pi Ag/AgCl electrodes at C3, anode, AF8, return). Results: In comparison to sham stimulation, 20-minutes of active 1mA tDCS induced a pattern of faster activity around the anodal stimulating electrode, and slowing activity near the return electrode in the frequency (full band) and mean power domain (gamma band). In addition, tDCS increased coherence in the fastest bands (gamma, beta 2) and decreased coherence in slower frequency bands (theta, SMR), with no relation with brain topography or the stimulation electrode polarity. Conclusions: These findings show that tDCS is capable of inducing modulation of ongoing oscillatory brain rhythms captured by EEG, in spinal cord injury patients. The combined use of EEG and tDCS sets the stage for optimizing tDCS protocols targeting motor cortex and may have application in treatment of motor dysfunction and chronic pain.  Key words: spinal cord injury, EEG, tDCS, motor cortex, TMS Abbreviations:    Introduction[JC2]  Transcranial direct current stimulation (TDCS) delivered over primary motor cortex (M1) can increase or decrease corticomotor excitability as determined by the amplitude of the motor evoked potential (MEP) from stimulating M1 with supra-threshold transcranial magnetic stimulation (TMS). The effect on MEP amplitude depends on the duration and intensity of stimulation and the polarity and spatial arrangement of the TDCS electrodes, and these effects can persist for up to an hour after TDCS is ceased. It is assumed that the TDCS after-effect results from some form(s) of neuromodulation at the cortical level such as synaptic potentiation or depression, or effects on ion channels. However, further insight into the cortical mechanisms in the human is limited experimentally. The electroencephalogram (EEG) measures far-field potentials from synchronized neural populations over cortical regions that extend beyond M1, and so offers a method for measuring the effect of TDCS on neural activity across the cortex.  In particular, such measurements do not depend on spinal cord conduction and alpha-motoneuron activation, which is the case for MEP recordings. This could have advantages for determining the effect of TDCS at the cortical level in spinal cord injury (SCI). While most SCI studies focus on the level of injury, there is increasing interest in the compensatory changes that might occur in the cortex, and whether they might facilitate or impede recovery. In the present study we measured the effect of EEG after 20 min of anodal or sham TDCS applied to the dominant M1 in chronic cervical SCI. We show that there are changes in EEG power, frequency and coherence that are spatially-related to the TDCS electrode configuration, but that spread widely across the hemispheres. [MC3]  Materials and methods Participants and study designThe randomized, single blind, sham controlled, cross over study was conducted on seven chronic SCI subjects, all males with an average age of 51.14 ± 10.57 years (mean ±SD, range 34 to 65 years). Participants presented traumatic SCI at the cervical level (C4-C8); some degree of motor function in wrist extension (score 1-5 over 5 on the Medical Research Council (MRC) scale for motor strength in the right extensor carpi radialis (ECR) muscle); chronic injury (>9 months after injury); and tolerance to sitting upright for at least one hour (see Table 1 for baseline characteristics). Patients were excluded if they presented with: progressive neurodegenerative disorder; concomitant traumatic brain injury or stroke; clinically significant cognitive impairment; medically unstable; change in medication during the study; or presented contraindications to brain stimulation (history of seizures/epilepsy, presence of metallic implants in the brain, pacemaker, pregnancy). The subjects were randomized in two groups depending on whether they were receiving stimulation or not:  a) Sham or control group, b) 1mA or active group. We investigated the effects of 20-minute anodal t-DCS applied over the primary motor cortex (M1) on: a) quantitative electroencephalography (EEG) power spectrum density, coherence and frequency band power. At Baseline we collected: clinical and functional evaluations were performed prior the brain stimulation intervention, and included the upper extremity motor score (UEMS), American Spinal Injury Association impairment scale (AIS), spinal cord independence measure (SCIM III) and visual analog scale (VAS) pain questionnaires. [DE4]  The study was approved by the Burke Medical Rehabilitation Institutional Review Board and conformed to the standards set out by the 1964 Declaration of Helsinki.                  AddTable 1 Transcranial direct current stimulation (t-DCS) interventionParticipants remained seated in their own wheelchair or were provided with a comfortable chair. The StarStimNE noninvasive wireless t-DCS/EEG neurostimulator (NE Neuroelectrics®, Barcelona, Spain) was used to both record EEG data and deliver the direct current sequentially. The StarStimNE neurostimulator included a wireless neoprene cap, based on the International 10-20 system, which was placed on the participants’ heads by aligning the central CZ electrode position with the vertex (intersection of nasion-inion and inter-aural line mid-point).Small Ag/AgCl gelled electrodes, with a surface contact area of 3.14 cm2 specific to the StarStimNE device (Pi electrodes, Neuroelectrics®), were placed over the left M1 at C3 (anode) and contralateral supraorbital area, (AF8; cathode) (Figure 1). The electrodes were connected to a control box device, which was wirelessly connected to a computer and communicated with the NIC software (version 1.2, Neuroelectrics®).  Add Figure 1 During anodal stimulation, direct current was delivered from a current-control circuit in the battery-driven stimulator within the control box device. The current was set at 1mA intensity and applied for 20 minutes. [DE5] For the sham stimulation, electrodes were placed in the same position and participants received a short ramp (30sec total up / down[DE6] ) at the beginning and end of the stimulation period.  Electroencephalography (EEG)The StarStimNE multichannel wireless device (NE Neuroelectrics®, Barcelona, Spain), which allows for simultaneous electroencephalography (EEG) and tDCS[DE7] , was used to record EEG data. The EEG data consisted of 5-minute takes (pre and post stimulation) with 24 bit resolution, 500 S/s 8-channel EEG collected with StarStimNE Ag/AgCl EEG electrodes (geltrodes, NE022; positions at F3, F4, Cz, C4, P3 and P4; Pi stimulating electrodes at C3 and AF8), based on the International 10-20 system, with the cap aligned to the central CZ electrode position (vertex).[DE8]  The electrodes were connected to a control box device, which was wirelessly connected to a computer and communicated with the NIC software (version 1.2, Neuroelectrics®). Transcranial magnetic stimulation (TMS) Electromyography (EMG): A bipolar surface EMG electrode (1 cm diameter, 2 cm inter-pole distance; Biometrics Ltd, UK) was placed over the right ECR muscle, with the forearm relaxed in a pronated position and supported by a cushion. The EMG activity was amplified and filtered on site (x1000 gain, band-pass filter 20-460 Hz; SX230-1000), digitized at 2 kHz (CED 1401, Cambridge Electronic Design, Cambridge, UK) and stored for offline analysis using Spike 2.6 software. Measurements were performed at rest. During the experiment, free running EMG was continuously monitored with visual feedback of EMG to ensure complete muscle relaxation.The right ECR muscle was selected for clinical relevance; where restoration of motor function in this muscle can help increase independence in quadriplegic subjects with activities of daily living.  Transcranial Magnetic Stimulation (TMS): A figure-of-eight coil (Model DB-80, Tonika Elektronik A/S, Farum, Denmark), connected to a MagPro X100 series (MagVenture A/S, Farum, Denmark) magnetic stimulator, was placed congruent with the head and the handle rotated 45° lateral from mid-sagittal so as to induce currents in the brain approximately perpendicular to the central sulcus. Resting motor threshold (RMT) was established at C3, and was defined as the minimum TMS intensity required to elicit a reliable MEP in the contralateral ECR amplitude of >50 µV in at least 50% of consecutive trials.[DE9]  EEG data processing/statistical analysisResting quantitative EEG and corticospinal excitability (transcranial magnetic stimulation; MEP) were recorded before (PRE) and at the end (POST) of each intervention. Quantitative EEG measures included: mean power, mean frequency and mean coherence. The three measures were normalized as the percentage change between PRE and POST stimulation, for each frequency band and electrode. The frequency bands were defined as: Theta= [4 8] Hz, Alpha-1=[8 10] Hz, Alpha-2=[10 12] Hz, SMR=[12 16] Hz, Beta-1=[16 25] Hz, Beta-2=[25 35] Hz, Gamma=[35 40] Hz.The mean power is defined as the average power in uV2 in a given frequency band. The mean frequency is defined as the average frequency in a given band, weighting each frequency value by their corresponding power at that frequency. The mean coherence of a given electrode is defined as the average coherence of this electrode with all the other ones (i.e., the similarity of that signal with all the other electrodes).--> Giulio to revise - DONEThe EEG data were referenced to the average of all 8 electrodes. An automated quality check of the data was then carried out using 8 sec epochs. Epochs have been rejected if they do not meet quality criteria (too high mean power at full band or line noise, or motion artifacts as detected by the built in accelerometer). PSD changes and coherence analysis was carried out for each quality passed epoch. Average PSD’s and coherence have then been computed for each subject.EEG data were discarded from analysis for channels presenting bad signal quality during the entire 5-minute recording. Those subjects that did not have more than 5 valid channels were completely discarded. After the discarding process, in order to have the same subjects in all the conditions, 7 subjects were analyzed for Sham and 1mA.Resting MEP amplitude (peak-to-peak; ECR muscle) was measured following single-pulse TMS (10/12) set at 120% [DE12] of the RMT over the C3[DE13] . Raw and normalized values were used for analysis[DE14] . Results are presented as mean ± standard deviation (SD), and standard error of the mean (SEM)[DE15] .After normalizing the EEG features as the percentage change between PRE and POST stimulation, statistical comparison between Sham and Active (1mA) stimulation has been done. The p value in the T tests has been calculated with a Wilcoxon one-tail T test with the assumption of paired samples. Changes are considered statistically significant when p =< 0.05. PLEASE ADD THE STUDY DATA ANALYSIS – GIULIO / LAURA --> Described above Results Effects of tDCS on EEGNormalized pre-post EEG power, frequency and coherence for those subjects who received 1mA tDCS compared to those who received sham stimulation shows: a) significant increase of the mean power in the 1mA group in the Gamma frequency band under C3 (p=0.0035), the anodal stimulating electrode; b) a significant increment in the active group of mean frequency around the anode (stimulating) electrode (C3, F3) (p=0.0047) with a decreased mean frequency in the Alpha band near the return electrode (P4) (p=0.035); and c) significantly increased mean coherence in the active group in the fastest frequency bands Beta2 under Cz and C4 (p=0.007), Gamma band under Cz electrode (p=0.0023), C3 electrode (p=0.0006) and C4 electrode (p=0.0006), and SMR band under C3 electrode (p=0.05). LAURA – ADD THE TYPE OF TEST USED FOR THE RESULTS AND THE P VALUES OBTAINED --> Described  above Effects of tDCS on Corticospinal excitabilityBaseline values for resting MEP amplitude were similar between interventions (average: 0.37±0.05 mV; mean±SEM). No significant changes were observed for 1 mA a-tDCS or sham. The stimulation intensities used to obtain the RMT were not significantly different between 1 mA (64±17% MSO) and sham (66±16% MSO). All participants presented MEP responses.   Discussion1. tdcs does modulate EEG in SCI population. 2. The effect of the TDCS in EEG is location specific and band specific. 3. The fastest bands are more affected. Augmented power and frequency of fast bands (Gamma and B2) under the anode electrode - there is more fast activity under the anodal electrode.  Why is this?3.1. Anodal stimulation leads to increased spontaneous neuronal firing, increase motor cortex excitability. So we would expect more fast activity and higher amplitudes under the anode electrode. 3.2. Brain activity changes (increase fast band) are related to motor learning – changes in Mu rhythm (preparation for movement, getting ready for movement). This is shown in the coherence increase of SMR band under the stimulating electrode.  MAR – ALL OF THE ABOVE 3.2.1. Is there are connection between C3 and P4 in resting state brain? Why is alpha decreased significantly in P4 (cortex parietal) in the contralateral hemisphere.  CONECTIONS BETWEEN MOTOR CORTEX AND SENSORY MOTOR CORTEX?? – JANA 3.3. Anodal tDCS makes C3 to be more related to the rest of the head, by increasing the connectivity of the SMR under the stimulating electrode. INTERPRETANTION OF THIS POINT TO GIULIO/LAURA, ANYTHING PUBLISHED BEFORE?Here you have a reference using EEG coherence: Rafael Polanıia, Michael A. Nitsche, and Walter Paulus. "Modulating Functional Connectivity Patterns and Topological Functional Organization of the Human Brain with Transcranial Direct Current Stimulation". Human Brain Mapping (2010). 3.4. This is an excitability independent change (no TMS change associated). Can EEG pick up changes in the brain than TMS cannot?                                                       LABAR? Conclusion These findings show that t-DCS is capable of inducing modulation of ongoing oscillatory brain rhythms captured by EEG, in spinal cord injury patients. The combined use of EEG and t-DCS sets the stage for optimizing t-DCS protocols targeting motor cortex and may have application in treatment of motor dysfunction and chronic painFigures