This study is a monocentric, non-pharmacological, longitudinal, randomized, blind, controlled
study.
Subjects The Investigators will study 40 right-handed MS patients with Expanded Disability
Status Scale (EDSS) score ≤6 and an indication to perform a physiotherapy treatment by the
treating physician. Patients will be recruited from the Department of Neurology, San Raffaele
Hospital. Forty sex- and age-matched right-handed healthy individuals (HC) will also be
enrolled. The HC will be recruited from the patients' relatives or acquaintances of the study
personnel. The enrolment of HC is crucial to define whether post-treatment changes observed
in MS patients are adaptive or maladaptive and to estimate the magnitude of these
alterations.
Subjects who will satisfy the inclusion criteria will then be randomized through a sequence
generated by the computer to determine their assignment to the conventional motor
rehabilitation therapy group (control group) or to the aerobic training (experimental group).
The computerized randomization software will generate personal codes to allocate every
patient to a treatment arm. These codes will be placed in opaque envelopes and delivered to
the patient by an operator external to the study.
Thus, participants will be split into 4 groups of 20 subjects per group:
1. Experimental group of HC: aerobic training by treadmill at moderate intensity;
2. Control group of HC: training of passive mobility, stretching and balance;
3. Experimental group of MS patients: aerobic training by treadmill at moderate intensity;
4. Control group of MS patients: training of passive mobility, stretching and balance.
Inclusion criteria (All)
- - Age: 18-65 years;
- Native italian language speaking;
- Right-handed;
- No particular motor skills;
- No additional neurologic, psychiatric, orthopaedic or rheumatologic diseases;
- Normal or corrected-to-normal vision;
- No MRI contraindications;
- Ability to understand the purpose of the study and provide signed informed consent.
For MS patients, the following additional inclusion criteria will be applied:
- - Patients with a MS diagnosis;
- EDSS ≤6.0;
- Stable MS treatment from ≥1 month prior to study enrolment;
- Relapse- and steroid-free from ≥3 months before screening visit;
- An indication to perform a physiotherapy treatment by the treating physician.
Exclusion criteria.
- - Persons who perform regularly a structured training;
- Patients who performed a physiotherapy treatment for at least 3 months;
- Concomitant therapy with antidepressant, baclofen, psychoactive, and steroids drugs
as well as symptomatic treatment for fatigue;
- History of alcohol or substance abuse;
- Pregnancy or breastfeeding.
Clinical and functional assessment All subjects will undergo a screening questionnaire
and a cardiologic evaluation, including electrocardiogram, followed by a graded exercise
test, in order to exclude possible contraindications to the inclusion in the study.
The graded exercise test will permit to measure the following parameters:
- - peak oxygen consumption (VO2);
- maximum wattage;
- heart rate (isolabour and maximum);
- rate of perceived exertion (Borg scale of rating of perceived exertion (RPE)).
Subjects who will satisfy the inclusion criteria will be randomized to the four groups
previously described.
All the subject will be assessed with clinical, cardiologic, neuropsychological and MRI
evaluations at:
- - baseline (before the start of the rehabilitation treatment) - T0,
- Week 4 - T1,
- Week 8 (end of the study) - T2.
A clinical follow-up has been planned 3 months
after the end of the treatment.
At each time-point, MS patients will be evaluated with the following assessments:
- - Graded exercise test;
- Complete neurological evaluation by a neurologist with the definition of the EDSS
score and the MS Functional Composite (MSFC);
- Assessment of autonomy in daily life activities, through the "Functional
Independence Measurement" (FIM);
- Spasticity, evaluating the modified Ashworth scale;
- Fatigue, through the Modified Fatigue Impact Scale (MFIS);
- Depression, using the Beck Depression Inventory II (BDI-II);
- Perceived quality of life, through the Multiple Sclerosis Quality of Life Scale
(MSQOL-54).
HC will be evaluated with the MSFC, MFIS and BDI-II. All subjects will also be evaluated
on additional motor aspects by the "6 minutes walking test" and the "Time up and go"
test. Finally, participants will undergo a neuropsychological assessment, using the
Brief Repeatable Battery of Neuropsychological Tests, the Digit Span (forward and
backward) and the Brief Test of Intelligence.
At the same time-points, all subjects will perform a MR scan, using a 3.0 Tesla scanner,
available at the San Raffaele Hospital. The following brain MRI sequences will be
acquired:
1. Dual-echo (DE) turbo spin echo (SE);
2. 3D T1-weighted fast field echo;
3. Pulsed-SE gradient echo planar imaging (EPI) with SENSE (acceleration factor=2) and
diffusion gradients applied in 35 non-collinear directions;
4. functional MRI (fMRI) in Resting State (RS) condition;
5. fMRI with an active cognitive task (Stroop task): using an event-related design, it
will be proposed different type of stimuli, i.e. congruent, incongruent and neutral
stimuli. Participants will be asked to respond to the color of the ink through four
buttons (red, green, yellow, blue) of a fMRI compatible response-box, which will
record reaction times and accuracy.
Assessors of clinical, neuropsychological and MRI evaluations will be blind with respect
to participants allocation.
Safety Possible fatigue, dyspnea, pain in the lower limbs. Subjects with possible
contraindications to the execution of an aerobic training (belonging to risk classes
according to World Health Organization (WHO) classification and to the American College
of Sports Medicine) and the execution of the MR (e.g., claustrophobia, pacemakers,
pregnancy, etc.) will not be enrolled in the study. The occurrence of side effects will
be recorded at each clinical visit or treatment session.
Treatment For each subject, the treatment will lasts 8 weeks. Each treatment will
consists of 35 minutes of training, administered 3 times per week. Both experimental and
control treatment will be performed by two experienced physiotherapists (different from
those involved in clinical and functional evaluations). Subjects of the experimental
groups (both patients and HC) will carry out an aerobic training of moderate intensity
(fixed time and variable intensity) on a treadmill. The training will be set
individually via direct method: during the first session, the subject will be trained at
an intensity that gets the heart rate (HR) corresponding to 46-63% of VO2 peak measured
during the exercise test; in subsequent sessions the intensity will increase to maintain
the same HR, which will be always monitored. The intensity workout identified will be
maintained for 30 minutes each session, preceded and followed by a few minutes of
warm-up and cool-down. Control groups of both patients and HC will follow a conventional
non-aerobic physiotherapy training, structured in: 15 minutes of passive mobilization of
upper and lower limbs and spine, 5 minutes of stretching of the upper and the lower
limbs and 10 minutes of balance training.
Duration The treatment period for each patient is 8 weeks. Follow-up visits will occur
at 3 months after the end of treatment.
MRI analysis All anonymized MRI data will be saved on a Linux workstation and coded with
letters (A,B,C,D) according to the study group (to preserve blindness). All image
post-processing will be performed by an experienced observer unaware of subjects
identity and type of treatment.
At baseline, T2 lesion volumes (LV) will be measured. New T2-visible lesions at follow
up will be counted.
On 3D T1 images, the normalized brain volume, as well as the normalized WM and GM
volumes will be quantified using the cross-sectional version of the software Structural
Imaging Evaluation of Normalized Atrophy (SIENAx). Longitudinal changes of brain volumes
will be evaluated with the longitudinal version of the software Structural Imaging
Evaluation of Normalized Atrophy (SIENA).
Definition of the patterns of GM volume changes Voxel-based Morphometry (VBM) with
Diffeomorphic Image Registration Algorithm (DARTEL) method will be applied to determine
the differences of GM volumes between different subgroups of patients and controls at
baseline.
Tensor-based Morphometry (TBM) will be applied to map the longitudinal regional
variations of GM volume at T1 and T2.
Tract-based Spatial Statistics (TBSS) will be used to define the patterns of the
microstructural WM abnormalities at baseline and their variations during the follow up.
Analysis of fMRI data Active and RS fMRI data will be pre-processed using SPM12.
Activations during the Stroop task will be estimated using SPM12. An independent
Component Analysis (ICA) will be used to decompose RS fMRI data into spatially
independent maps and time courses, using the Group ICA Of fMRI Toolbox (GIFT) software.
Statistical analysis Demographic, clinical, functional and neuropsychological variables,
as well as MRI measures at baseline will be compared using Chi-Square, t-test or ANCOVA
models as appropriate. The condition of a normal distribution will be verified using the
Kolmogorov-Smirnov and Shapiro-Wilk, as well as with the visual assessment of the
estimated non-parametric Kernel density and Q-plot.
To assess changes over time of clinical measures, functional and Z-score average of RS
fluctuations, longitudinal linear models will be applied using a statistical design that
takes into account the repeated measures in the context of a bivariate model. The
correlations in each patient will be quantified with a matrix of correlations
unstructured.
The dependent variable will be the vector of the assessment from all participants at
each time point (before and after treatment).
Considering the two groups of patients together, the effects of different treatment will
be evaluated considering the cross-interaction "treatment x time" in the linear model. A
p value <0.05 will be considered statistically significant.
Statistical analyses of the VBM, the TBM and the fMRI active task will be performed
using the SPM12 software (whole brain analysis, p <0.05, family-wise error [FWE],
corrected for multiple comparisons).
Voxelwise differences of mean diffusivity and fractional anisotropy values between
treatment and control groups at baseline, and their within-group changes at follow up
will be tested, using a permutation method ("Randomize" program within FSL) and
two-sample and paired t tests, as appropriate (p<0.05 FWE).
Linear regression analysis (using SPM12) will be used to assess the correlations between
fMRI activations and clinical and neuropsychological data.
Sample size calculation Given the exploratory nature of the project, the sample size of
the study has been calculated also taking into account its feasibility. The power's
study showed that, for two continuous variables, with n=40 subjects and a type I error
alpha= 0.05, we will able to detect a significant Pearson correlation at least equal to
0.48 with a power of 0.90 and a standardized difference between balanced groups equal to
0.8 with a power of 0.90. Furthermore, the sample size planned in this project is
usually considered adequate for the performance of a fMRI analysis.
Ethical and regulatory considerations This clinical study will be conducted in
accordance with the principles laid down by the 18th World Medical Assembly (Helsinki,
1964) and all applicable amendments laid down by the World Medical Assemblies, and the
International Council for Harmonisation of Technical Requirements for Pharmaceuticals
for Human Use (ICH) guidelines for Good Clinical Practice.
This clinical study will be conducted in compliance with all international laws and
regulations, and national laws and regulations of the country(ies) in which the clinical
trial is performed, as well as any applicable guidelines.
