Rehabilitative Trial With Transcranial Direct Current Stimulation (tDCS) in Amyotrophic Lateral Sclerosis
Phase: NA
Conditions: Amyotrophic Lateral Sclerosis
Sponsor: Azienda Socio Sanitaria Territoriale degli Spedali Civili di Brescia

 
 
CLINICA NEUROLOGICA - UNIVERSITA’ DEGLI STUDI DI BRESCIA 
UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
Direttore: Prof. Alessandro Padovani 
 
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Title of the study: “Rehabilitative Trial With tDCS in Amyotrophic Lateral Sclerosis 
(tDCS_MND) 
NCT number: NCT03293394 
Date: May 5th, 2017 
 
 
 

 
 
CLINICA NEUROLOGICA - UNIVERSITA’ DEGLI STUDI DI BRESCIA 
UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
Direttore: Prof. Alessandro Padovani 
 
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EXPERIMENTAL CLINICAL PROTOCOL 
 
TITLE OF THE STUDY: "Rehabilitative Trial With tDCS in Amyotrophic Lateral 
Sclerosis" 
 
CODE: tDCS_MND 
 
VERSION AND DATE: Version 1, 05.05.17 
 
PROMOTER: ASST Spedali Civili di Brescia 
 
DIVISION IN WHICH RESEARCH WILL BE PERFORMED: Clinica 
Neurologica, Università degli Studi di Brescia – U.O. Neurologia 2, ASST Spedali 
Civili di Brescia.  
 
PRINICIPAL INVESTIGATOR:  
Prof. Barbara Borroni 
Associate professor 
Clinica Neurologica 
Dipartimento di Scienze Cliniche e Sperimentali 
Università degli Studi di Brescia 
U.O. Neurologia 2 
ASST Spedali Civili di Brescia 
 
OTHER INVESTIGATORS INVOLVED: 
Prof. Alessandro Padovani, Director of the Neurology Division, Università degli Studi 
di Brescia, ASST Spedali Civili di Brescia 
Dr. Antonella Alberici, U.O. Neurologia 2, ASST Spedali Civili di Brescia 
Dr. Alberto Benussi, Medico Specializzando, Università degli Studi di Brescia 
Dr. Valentina Dell’Era, Medico Specializzando, Università degli Studi di Brescia 
Dr. Valentina Cantoni, Psicologa, U.O. Neurologia 2, ASST Spedali Civili di Brescia 
 
INTRODUCTION: 
Transcranial stimulation with direct currents or transcranial direct current stimulation 
(tDCS) represents a neurophysiologic method of non-invasive modulation of the 
excitability of the central nervous system, which is having an increasing diffusion and 

 
 
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UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
Direttore: Prof. Alessandro Padovani 
 
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an ever-increasing range of potential therapeutic applications such as, for example, 
post-stroke rehabilitation, depression and pain, already established (Lefaucheur et al., 
2017). This method, being non-invasive, low cost and simple to perform, has the 
potential to be widely used both as a research tool and as a tool to support rehabilitation 
treatment. A long-term effect of tDCS treatment on synaptic plasticity of the motor 
cortex, which depends on modulation of NMDA receptors and GABAergic systems, 
has been demonstrated (Nitsche et al., 2005). 
The application of these techniques within the project of "Identification of a possible 
rehabilitative protocol through the use of direct current brain stimulation (tDCS) in 
patients affected by cerebellar ataxia of neurodegenerative origin." Approved by this 
Ethics Committee allowed to identify a clinical improvement also in patients affected 
by cerebellar ataxia subjected to cerebral transcranial electrical stimulation, also 
supported by neurophysiology data (Benussi et al., 2015a; Benussi et al., 2017). 
This application could also be applied in amyotrophic lateral sclerosis (ALS), a 
progressive chronic neurodegenerative pathology in which there is an irreversible 
degeneration of the first and second motoneurons at the level of the motor cortex and 
of the spinal cord, respectively. ALS is characterized by muscle stiffness, muscle 
contractions and gradual progressive weakness secondary to muscular atrophy. This 
results in difficulty in speech, swallowing and, finally, breathing (Salameh et al., 2015). 
At present, there is no known cure for ALS. One drug, riluzole, can extend life 
expectancy by about two or three months (Miller et al., 2012). 
For this purpose, cortico-spinal transcranial electrical stimulation techniques have been 
developed, in which a simultaneous stimulation of the cerebral cortex (in this case in 
the area of the scalp corresponding to the motor cortex of both sides) and of the spinal 
cord (in this case in the area of the back corresponding to the dorsal-lumbar spinal cord) 
is applied (Fiocchi et al., 2016; Parazzini et al., 2014). 
In order to evaluate an effect of tDCS on the progression of the pathology, some 
transcranial magnetic stimulation techniques (TMS) will be used in addition to the 
clinical and life quality scales. In fact, TMS has been widely used in the study of some 
neurodegenerative diseases, in particular in Parkinson's disease and in dementia due to 
Alzheimer's disease. Over the years, numerous TMS protocols have been developed to 
obtain information on neurophysiological parameters with the aim of investigating 
both the pathogenesis and the evolution of some neurodegenerative diseases (Benussi 
et al., 2015b). 
Short-interval intracortical inhibition (SICI) is currently used to investigate 
intracortical inhibitory circuits. From neuropharmacology studies, it has been 
hypothesized that SICI is dependent on the activity of GABAA receptors (Kujirai et al., 

 
 
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UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
Direttore: Prof. Alessandro Padovani 
 
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1993). In this way, alterations of the intracortical inhibitory circuits have been 
identified in many neurological diseases, in particular in dystonia and in parkinsonism. 
The study of intracortical facilitation (ICF) is used to investigate the combined effect 
of secondary facilitation on inhibitory and excitatory mechanisms mediated by GABAA 
and NMDA receptors respectively (Ziemann et al. 1998). 
In particular, in patients suffering from ALS, a deficiency of SICI and ICF has been 
observed, due to an imbalance in the mechanisms of inhibition and cortical facilitation 
mediated by GABAergic and glutamatergic interneurons (Menon et al., 2015). Based 
on these premises, treatment with tDCS is proposed in patients with ALS. 
 
TARGETS: 
The aim of the study is to evaluate the long-term effects of tDCS on clinical, biological 
and neurophysiological parameters in patients with ALS. 
In particular, the clinical and quality of life scales currently used in clinical trials on 
ALS will be used (ALSFRS-R, ALSAQ-40, CBI, EQ-5D-5L, muscle strength assessed 
with the MRC scale). Moreover, SICI and ICF protocols will be evaluated through 
TMS. 
 
 
PROCEDURES: 
Subjects enrolled at the Neurological Clinic will be enrolled with the diagnosis of 
probable ALS, laboratory-supported, or defined according to the current clinical 
criteria (World Federation of Neurology Revised El Escorial criteria - Brooks et al., 
2000). 
Subjects will sign an informed consent and will be subjected to a neurological clinical 
evaluation using the clinical and quality of life scales currently used in clinical trials 
on ALS (ALSFRS-R, ALSAQ-40, CBI, EQ-5D -5L, muscle strength evaluated with 
the MRC scale in different muscle groups). 
Clinical assessment: Neuromuscular impairment will be quantified by the five-point 
MRC scale megascore (shoulder abductors, elbow flexors and extensors, wrist flexors, 
thumb opponent, hip flexors, knee flexors and extensors, and ankle dorsiflexors and 
extensors on both sides for a total of 100). Good reliability and reproducibility for 
manual muscle testing in patients with ALS have previously been shown. 
Clinical status will be evaluated with the ALSFRS-R, quality of life with the 
amyotrophic lateral sclerosis assessment questionnaire-40-item scale (ALSAQ-40), 
EQ-5D-5L and EQ-VAS, while caregiver burden will be evaluated with the caregiver 
burden inventory (CBI). 

 
 
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Through the use of TMS techniques, the effects of tDCS on neurophysiological 
parameters will be evaluated, in particular SICI and ICF. 
Transcranial Magnetic Stimulation assessment: TMS will be performed with a figure-
of-eight coil (each loop diameter 70 mm) connected to a Magstim Bistim2 system 
(Magstim Company, Oxford, UK). Motor evoked potentials (MEPs) will be recorded 
from the right first dorsal interosseous muscle (FDI) through surface Ag/AgCl 
electrodes placed in a belly-tendon montage and acquired using a Biopac MP-150 
electromyograph (BIOPAC Systems Inc., Santa Barbara, CA, USA). 
The TMS coil will be held tangentially over the scalp region corresponding to the 
primary hand motor area contralateral to the target muscle, with the coil handle pointed 
45° posteriorly and laterally to the sagittal plane. The motor hot spot will be defined as 
the location where TMS consistently produced the largest MEP size at 120% of the 
resting motor threshold (rMT) in the target muscle and was marked with a felt tip pen 
on the scalp to ensure constant placement of the coil throughout the experiment. 
Resting motor threshold (rMT) will be defined as the minimal stimulus intensity 
needed to produce MEPs with an amplitude of at least 50 μV in 5 out of 10 consecutive 
trails during complete muscle relaxation, which will be controlled by visually checking 
the absence of EMG activity at high-gain amplification. 
SICI and ICF will be studied at rest via a paired-pulse paradigm, delivered in a 
conditioning-test design, as previously reported. Briefly, the conditioning stimulus 
(CS) will be set at an intensity of 70% of the rMT, while the test stimulus (TS) will be 
adjusted to evoke a MEP approximately 1 mV peak-to-peak in the relaxed FDI. 
Different interstimulus intervals (ISIs) between the CS and TS will be employed to 
investigate preferentially both SICI (1, 2, 3 ms) and ICF (7, 10, 15 ms). 
Ten stimuli will be delivered for each ISI and fourteen control MEPs in response to the 
TS alone will be recorded, for each paradigm, in all participants in a pseudorandomized 
sequence. The amplitude of the conditioning MEPs will be expressed as a ratio of the 
mean unconditioned response. The inter trial interval will be set at 5 sec (±10%). All 
protocols will be performed in a randomized order in all participants. Throughout the 
experiment, complete muscle relaxation will be monitored by audio-visual feedback 
where appropriate. 
All clinical and, neurophysiological evaluations will be performed at baseline (T0), 
after 10 tDCS applications i.e. at two weeks (T1), at two months (T2) and at six months 
(T3). 
After the first evaluation (T0), the patients will be divided into two groups randomly. 
The first group will undergo a tDCS stimulation protocol, while the second group will 
undergo a placebo (sham) treatment. tDCS will be delivered by a battery-driven 

 
 
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Study Potoicol. tDCS_MND _Version 1, 05.05.17 
 
constant current stimulator (HDCstim, Newronika, Milan, Italy) through a pair of 
saline-soaked (0.9% NaCl) surface sponge electrodes. The anodes will be placed on 
the scalp over the motor cortex area and the cathode over the spinal cervical 
enlargement (over C6) (see Fig. 1B). The electrodes will be secured using elastic 
gauzes and an electroconductive gel will be applied to electrodes to reduce contact 
impedance (<5 kΩ for all sessions). During real stimulation a constant current of 2 mA 
per each anodal electrode (4.0×6.5 cm2, current density 0.077 mA/cm2) and 4 mA per 
cathodal spinal electrode (5.0×7.5 cm2, current density 0.107 mA/cm2) will be applied 
for 20 minutes. For the sham condition, the electrode placement will be the same, but 
the electric current will be ramped-down 5 seconds after the beginning of the 
stimulation to make this condition indistinguishable from the experimental stimulation. 
To detect differences in the perception of the stimulation, patients will be asked 
whether they thought they were receiving real or sham stimulation at the end of the 
two-weeks’ treatment. 
 
INCLUSION CRITERIA for patients: 
- Male or female patients diagnosed with probable ALS, laboratory-supported, or 
defined according to current clinical criteria (World Federation of Neurology 
Revised El Escorial criteria - Brooks et al., 2000). 
- Age over 18 years; 
- Disease duration ≤ 48 months; 
- Score ≥ 2 at the item "swallowing" of the ALS Functional Rating Scale-Revised 
(ALSFRS-R); 
- Score ≥ 1 at the item "walking" of the ALSFRS-R; 
- Score ≥ 1 at the item "respiratory insufficiency" of the ALSFRS-R 
- Signature of informed consent. 
 
EXCLUSION CRITERIA for patients: 
- Motor neuron diseases other than ALS; 
- Diagnosis of dementia according to current clinical criteria; 
- Severe head trauma in the past; 
- Persons carrying static electrical stimulators (e.g. cardiac stimulators, nerve 
stimulators, auditory implants) that would not work or would be damaged by the 
magnetic field; 
- Persons carrying special metallic foreign bodies (e.g. splinters, some prostheses, 
screws and nails) that could move if placed inside the magnetic field; 
- People with a history of epilepsy ore seizures; 

 
 
CLINICA NEUROLOGICA - UNIVERSITA’ DEGLI STUDI DI BRESCIA 
UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
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Study Potoicol. tDCS_MND _Version 1, 05.05.17 
 
- History of ischemic stroke or hemorrhage; 
- Severe comorbidity 
- Given that the effects of TMS on the developing fetus are not known, pregnant 
women will be excluded from the study. 
 
STUDY DESIGN: 
This is a non-pharmacological, randomized double-blind controlled clinical trial. This 
study does not present sub studies. Patients will be randomized into two groups: each 
group will receive anodal bilateral motor cortex tDCS and cathodal spinal tDCS (real 
tDCS) or sham stimulation for 5 days/week for 2 weeks, in a 2:1 ratio respectively. 
 
END POINTS: 
The long-term effect of tDCS on clinical parameters (ALSFRS-R, ALSAQ-40, CBI, 
EQ-5D-5L, muscle strength assessed with the MRC scale in different muscle districts), 
and neurophysiological measures (SICI and ICF). 
For efficacy analyses, the primary end point will be the defined as change from baseline 
in global MRC scores. Secondary end points will be defined as changes from baseline 
in the ALSFRS-R, ALSAQ-40, EQ-5D-5L, EQ-VAS, CBI and intracortical 
connectivity (SICI and ICF). 
 
STATISTICAL PLAN: 
Parametric analysis (t test) will be used to compare the demographic variables (e.g. 
age, duration of illness, education) of the various groups at the baseline. A p-value 
<0.05 will be considered significant. 
In order to estimate the sample size, in order to obtain a significant difference in the 
primary outcome (Global MRC Score), we performed a "power analysis" based on the 
effects observed in a small group of patients (pilot study). The "effect size" f (V) was 
calculated by direct method. Taking into account from previous observations a "partial 
η2" = 0.31, equal to an "effect size" f (V) = 0.67 and, considering alpha = 0.05 and the 
power (1-β) = 0.80, considering a variable as covariate, the sample size expected for 
two patient groups (real and sham) is 25 total subjects using a repeated-measure 
ANCOVA study. The proposed sample size of 30 patients should be adequate to 
achieve the main objective of the study and would allow monitoring for confounding 
factors or drop-out patients. 
Considering the unneglectable number of drop-out patients, that characterize most 
trials in ALS, we performed intention-to-treat and per-protocol analysis to reduce any 
bias due to the possible unbalanced drop-out rates between treatment groups. For 

 
 
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patients with missing values, data were assigned using mixed effects models for 
repeated measures without any ad hoc imputation. 
To assess the effect of tDCS treatment on clinical scores over time, we will use a two-
way repeated measure ANCOVA with TIME (T0, T1, T2 and T3) as within-subject 
factors and TREATMENT (sham vs real stimulation) as between-subjects factor, and 
the baseline values of each score as covariate, to reduce possible effects of baseline 
impairment on clinical score changes over time. 
To assess the effect of tDCS treatment on TMS parameters we will use a two-way 
mixed ANOVA with TIME (T0, T1, T2 and T3) as within-subject factors and 
TREATMENT (sham vs real stimulation) as between-subjects factor. 
When a significant main effect was reached, post hoc tests with Bonferroni correction 
for multiple comparisons will be conducted to analyze group-differences at respective 
time points. Mauchly’s test will be used to assess for assumption of sphericity, while 
Greenhouse–Geisser epsilon determination will be used to correct in case of sphericity 
violation. Spearman’s rank-order correlations will be used to assess associations 
between the improvement in clinical scores and neurophysiological parameters. 
Statistical analyses will be performed using SPSS version 21 (SPSS, Inc., Chicago, IL, 
USA). 
 
ETHICAL CONSIDERATIONS (RISK-BENEFIT): 
tDCS is a non-invasive, low cost and simple non-invasive brain stimulation method 
that is having an increasing diffusion and an increasingly numerous spectra of potential 
therapeutic applications such as, for example, post-stroke rehabilitation, depression 
and pain, which have already been established. Its effectiveness in modulating the 
excitability of the motor cortex in humans has been demonstrated (Nitsche et al., 2005). 
We therefore hypothesize that tDCS can have a positive effect on the clinical and 
quality of life scales, and restoration of neurophysiological parameters. 
Literature data show that the application of the method according to the current safety 
guidelines (Iyer et al., 2005; Nitsche et al., 2003; Bikson et al., 2016) has little relevant 
side effects, among which mild tingling sensation during or after stimulation, moderate 
fatigue, mild itching sensation at the electrode sites, mild burning or pain, headache, 
nervousness, nausea, blistering at the electrode application points in rarer cases. 
As for the TMS method, it is a non-invasive brain stimulation that allows the study of 
neurophysiological parameters that can provide information on the pathophysiological 
mechanisms involved in neurodegenerative diseases. 
Literature data show that the application of the method according to the current safety 
guidelines has minor side effects: a certain number of subjects participating in TMS 

 
 
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experiments (up to 20%) suffer from headaches or backaches, due to likely to an 
excessive muscle tension and a stiff position of the head and / or neck during TMS 
application. These effects are temporary and, in most cases, do not require any 
treatment. 
With the parameters that we will use in this study, epileptic seizures have never been 
reported (Rossi et al., 2009). If an epileptic seizure occurs, this would occur during 
TMS, not later. However, this is an extremely rare complication. To prevent this, all 
TMS sessions will be performed in the presence of a neurologist and by personnel 
trained to give first aid and to immediately recognize the first signs of epileptic 
seizures. In addition, all TMS sessions will be carried out within the Neurology Unit, 
equipped with all the emergency material needed to deal with epileptic seizures and to 
prevent any damage to the patient (emergency trolley organized according to the 
treatment of neurological emergencies, antiepileptic drugs). 
The occurrence of an epileptic seizure during a TMS experiment does not imply that 
seizures will reoccur in the future, nor that you should start taking a therapy for the 
prevention of seizures, nor that we should modify an eventual therapy already 
underway due to a seizure. The very few cases of TMS-induced seizures described in 
the literature no longer had episodes later, nor did they report health problems 
attributable to the event. 
Since the TMS produces a magnetic field, people carrying static electric stimulators 
(e.g. cardiac stimulators, nerve stimulators, auditory implants) that would not work or 
would be damaged by the magnetic field cannot participate in the study. Also excluded 
from the examination are the carriers of particular metallic foreign bodies (for splinters, 
some prostheses, screws and nails) that could move if placed inside the magnetic field. 
Because the effects of TMS on the developing fetus are not known, pregnant women 
cannot participate in the study. 
 
ADVERSE EVENT DETECTION: 
The Principal Investigator will promptly inform the Local Ethics Committee of the 
Brescia Hospital of any adverse event incurred during the study. 

 
 
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CLINICA NEUROLOGICA - UNIVERSITA’ DEGLI STUDI DI BRESCIA 
UO Neurologia 2 - AZIENDA SOCIO SANITARIA TERRITORIALE degli SPEDALI CIVILI DI BRESCIA 
 
Direttore: Prof. Alessandro Padovani 
 
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