Main aim of this study will be the evaluation of the neurophysiological techniques of Transcranial Magnetic Stimulation (TMS) via electroencephalography (EEG) co-registration (TMS-EEG) with the study of TEPs (TEP: transcranial evoked potentials) as surrogates of white matter and grey matter functional integrity in patients with Multiple Sclerosis (MS). Data will be compared with those obtained from a group of healthy control subjects. Secondary aim will be the longitudinal evaluation of these neurophysiological parameters in MS patients during routine clinical and radiological evaluations, performed according to clinical practice, for 12 months.

To this aim a longitudinal multicenter study will be carried out, interventional (for neurophysiological techniques) and observational (for clinical and radiological evaluations), which involves the enrollment of 64 patients diagnosed with MS. Patients will keep their usual therapeutic regimen and their usual clinical-radiological checks according to clinical practice. The control group will consist of 64 healthy subjects, enrolled with prior written informed consent, age and sex-matched with MS patients and selected among the caregivers of the patients. Healthy subjects will only undergo neurophysiological assessment at baseline.

The neurophysiological evaluation will include the study of the propagation of potentials induced by stimulation. This method allows the study of cortical responses in terms of time domain and frequency, obtaining a measurement of interhemispheric connectivity and of microstructural and functional integrity of white matter. In the same way, these methods allow the assessment of grey matter integrity through the study of intracortical excitability.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) whose pathogenesis involved both demyelinating events and neurodegeneration. It is on of the most frequent cause of disability in young adults. It is characterized by different clinical phenotypes: currently, the relapsing-remitting form (RR), the most common, and the progressive forms of MS (primary progressive -PP, secondary progressive -SP) are recognized. The relapsing-remitting form is characterized by the presence of acute/subacute onset of clinical events, the appearance of new lesions on magnetic resonance imaging (MRI), or the uptake of gadolinium by a new or pre-existing lesion. The progressive forms, on the other hand, present with a slow accumulation of disability from the onset (PP-MS), or following a relapsing-remitting trend (SP-MS). The clinical scale mainly used for evaluating patients with MS is the Expanded Disability Status Scale (EDSS). Currently, there are several treatments available for the control of the disease and the identification of the correct therapeutic choice can lead to an important slowdown, up to stabilization, of the clinical course of the disease. It is therefore essential, given the existence of different therapeutic strategies, to recognize early those patients who respond in a sub-optimal manner to therapy. To date, the evaluation of the effectiveness of the treatment is based on clinical and radiological data. Several studies have tried to identify new markers of disability, but none of these have entered the routine clinical use. In this context, the possible role of neurophysiology in early identify markers of inflammatory/ degenerative disease activity is outlined.

Among the neurophysiological methods potentially able to identify the inflammatory or neurodegenerative phase of the disease, the most promising results were obtained with transcranial magnetic stimulation (TMS) and electroencephalography (EEG). These methods, already widely used in the clinical setting, are characterized by being reproducible, non-invasive and low-cost. Thanks to the development of EEG systems compatible with magnetic stimulation, it is possible to study the cortical potentials evoked by TMS (TEPs). The TEPs constitute a sensitive and reproducible experimental index of intracortical excitability and allow the identification of specific alterations of different neurological conditions. The study of TEPs offers a better performance than that obtainable using the single techniques, TMS and EEG, separately.

Neurophysiological estimates of white matter integrity Several evidence have confirmed that the EEG indices of functional connectivity are influenced by the degree of myelination of the white matter. Among these indices of EEG connectivity, cortico-cortical coherence is a linear correlation index between the oscillatory signal of two cortical areas and has been shown to be a sensitive index of the myelination state of the brain in physiological conditions and in various neurological pathologies.

Distinct EEG oscillations in the different frequency bands revealed many robust relationships to behavioral, cognitive, and clinical states in several studies. Spatial-temporal oscillatory dynamic patterns recorded by EEG are important brain state-dependent measures of neocortical dynamics, including functional connectivity. Myelin is critical for sustaining oscillatory neural activity and entrainment between "generators" (e.g., cell assemblies or networks at multiple scales) in brain regions separated by substantial conduction delays. Functional connectivity as assessed by scalp EEG at large scales is believed to be strongly influenced by white matter tracts, especially the cortico-cortical projections. The standard Fourier transform-based multi-channel signal measure coherence is a squared correlation coefficient expressed as a function of frequency; it can provide robust measures of cognitive state and white matter (WM) maturation or disease. WM integrity determines propagation delays (i.e. the timing) of synaptic inputs in a determined brain network thus allowing phase synchrony of local oscillations. Relatively small changes in conduction delays can have significant effects on oscillatory coupling and phase synchrony between distant brain regions. Disruption in brain synchronization contributes to dysfunction in many neurological and psychiatric disorders. Compared to scalp EEG, high-resolution EEG (HR-EEG) methods employ computer algorithms (e.g., Laplacian or dura image) to provide estimates of brain or dural surface potentials at roughly the 2-3 cm scale. HR-EEG functional connectivity measures, like narrow band (e.g., 1 Hz) alpha and theta coherence, has been linked to cortico-cortical signal propagation via (mostly) myelinated axons. The propagation time between hemispheres is about 30 ms through myelinated callosal fibers and 150-300ms through unmyelinated fibers. An investigation of scalp-registered inter-hemispheric coherence between the left and right sensorimotor hand areas using HR-EEG revealed a superposition of both bilaterally coherent and incoherent rhythmic activities within the alpha band. Synaptic integration can be expected to be strongly influenced by the degree of myelination of intercallosal axons. Combined EEG and HR-EEG can provide complementary functional connectivity estimates that are maximally sensitive to large/global (~5-10 cm) and intermediate/local {-2-3 cm) spatial scale source regions, respectively. White matter integrity is important for cortico-cortical connections and is critical to these functional connectivity estimates, especially scalp oscillatory coupling and distant sources phase coherence.

Transcranial magnetic stimulation (TMS) is a non-invasive brain-stimulation technique. By producing high-intensity magnetic pulse, TMS induces brief electric currents that can excite or inhibit a small area of the cerebral cortex. This activating property is classically used on the primary motor cortex (M1) to produce action potentials along the cortico-spinal bundle and evoke a motor potential (MEP, motor evoked potential) in the contralateral musculature. In MS patients, EEG and TMS are widely used as diagnostic tools to prove demyelination by means of abnormal conduction time along white matter tracts, even in subjects with normal MRI scans. Furthermore, when the magnetic stimulus is delivered during voluntary muscle contraction of the stimulation target muscle, the TMS of M1 can generate a brief interruption of voluntary electromyography (EMG) activity both contralateral (CSP: contralateral silent period) and ipsilateral (the " ipsilateral silent period" -IpSP). lpSP is a measure of interhemispheric motor inhibition that has been found to be altered in patient with MS and callosal lesions.

Compared to the separate recording of the EEG signals and those generated by the TMS, the simultaneous execution of the two methods - TMS-EEG - allows the recording directly from the scalp of the potentials (TEP) evoked by the TMS. The recording of TEPs from areas distant from the stimulation site allows to obtain information about the connectivity of the stimulated cortex. The "interhemispheric signal propagation" (ISP) is a measure of interhemispheric connectivity based on the propagation of TMS-EEG responses from the stimulated hemisphere to the contralateral. The ISP correlates with the microstructural integrity of the callosal microfibers and with the hand dexterity during motor development.

Neurophysiological estimates of grey matter Integrity:

Regarding gray matter (GM) pathology, evidences obtained from TMS and EEG studies have contributed to unveil the role of cortical dysfunction in MS. Alterations in EEG oscillations considered important in sensorimotor integration and motor control have been associated with clinical disorders and radiological changes in patients with MS. Furthermore, paired-pulse TMS protocols have shown alterations in M1 excitability in MS patients and that these alterations correlate with clinical disability. Among the measures of TMS-EEG, the locally recorded TEPs reflect the excitability and activation state of the stimulated cortex and therefore the degree of integrity of the cortical gray matter.

In conclusion, EEG and TMS-EEG measures are powerful tools to assess WM and GM functional integrity in patients with MS. As such, EEG and TMS-EEG also provide a potential objective framework to systematically assess the efficacy of Disease modifying therapies (DMTs) in MS.

Study aims and hypotheses Main aim of this study is the evaluation of TMS-EEG neurophysiological measures as surrogates of functional integrity of both gray and white matter in MS patients. The data obtained will be compared with those of a group of healthy subjects comparable for sex and age. Healthy subjects will be identified as those with normal neurological examination and negative medical history for morbidity. By combining the neurophysiological variables with the clinical ones, an attempt will be made to identify neurophysiological markers expressing the degree of disability of patients with MS (primary objective). The secondary objective is to identify the neurophysiological variables that may play a predictive role in the clinical or radiological relapses of the pathology and of the long-term clinical-radiological status. To this aim, the neurophysiological TMS-EEG measurements will be repeated longitudinally in the patients participating in the study and correlated with the clinical and radiological according to clinical practice. The neurophysiological markers identified may offer support in identifying patients in clinical deterioration or with a sub-optimal response to therapy.

Enrollment: To guarantee an adequate number of study participants and obtain data that are more traceable to real-life conditions, the investigators will collaborate in recruiting with other MS Centers (list with the relevant Managers attached). All neurophysiological evaluations will be performed at our Department and our Center will be the coordinator.

Tolerability of the neurophysiological evaluation The selected neurophysiological methods are non-invasive procedures, widely used and based on tools currently in use in clinical practice. These methods are painless and therefore easily implemented in a large population of subjects as they are safe and free of side effects.

Inclusion Criteria:

• MS diagnosis according to the latest McDonald criteria

Exclusion Criteria:

• other neurological or immunological diseases
• clinical relapses in the 30 days prior to the clinical and neurophysiological evaluation;
• presence of conditions that contraindicate the execution of Transcranial Magnetic Stimulation (TMS) methods (history of epilepsy, pacemaker, recent head injury).

all collected IPD will be shared at the end of the study by request from any qualified investigator.

starting 3 months after data pubblication

research group focused in neurophysiological investigations in patients with multiple sclerosis