CB1 receptor affects cortical plasticity and response to physiotherapy in multiple sclerosis

Standard protocol approvals, registrations, and patient consents.

The study was approved by the institutional review board. Written informed consent was signed by all study participants.

Patients with MS.

Thirty Central-Southern Italian patients with relapsing-remitting MS (RRMS)¹⁴ were enrolled (table) by referral from the MS Center, Neurology Unit, Policlinico Tor Vergata, Rome. Data collection was performed from 2010 to 2012. Sample size was determined on the basis of a previous study.⁸

Patient eligibility was based on the following criteria: (1) clinically definite RRMS,¹⁴ (2) remitting phase of disease, (3) Expanded Disability Status Scale (EDSS) score between 2 and 7, and (4) presence of spasticity in the lower limbs.

Disease onset was defined as the first episode of focal neurologic dysfunction indicative of MS. All patients were in a remitting phase in the 3 months preceding recruitment and during the study. Remission was defined as the absence of new or recurrent neurologic symptoms not associated with fever or infection lasting for at least 24 hours.

All patients started disease-modifying drugs (DMDs) at the time of diagnosis. First-line treatment prescribed was glatiramer acetate (20 mg SC daily), interferon β-1a (44 μg SC 3 times weekly), interferon β-1a (30 μg IM), or interferon β-1b (250 μg SC every other day). Mitoxantrone (12 mg/m² IV every 3 months with a lifetime maximum of 140 mg/m²) or natalizumab (300 mg IV every 4 weeks) was prescribed as second-line treatment.

Determination of AAT repeats in the CNR1 gene.

Samples of peripheral blood from recruited patients were collected in BD Vacutainer tubes with EDTA (Beckton Dickinson, Franklin Lakes, NJ). Genomic DNA was isolated and extracted from human whole blood (200 μL) using a MagNA Pure LC DNA Isolation Kit and automated extractor (Roche Diagnostics GmbH, Mannheim, Germany) following manufacturer's instructions. One hundred fifty ng of genomic DNA was used to amplify the CNR1 region, including the AAT repeats through PCR. PCR was carried out in a 25 μl volume containing polymerase buffer, 1 mM MgCl₂, 0.2 mM of each dNTP, 0.5 pmoles of each primer (sense: 5′-CACCCCTGGGCTGTAAAATA-3′; antisense: 5′-GTTGCAGTGAGCCAAGATCA-3′), and 1.5 U Taq DNA polymerase (Invitrogen, Madison, WI). Denaturation was achieved through a first step of 5 minutes at 94°C followed by 35 cycles of 45 seconds at 95°C, annealing for 1.5 minutes at 58°C and elongation for 1 minute at 72°C, and final elongation for 7 minutes at 72°C. For sequencing analysis, 10 ng of PCR products Agencourt AMPure PCR Purification kit (Agencourt Bioscience Corporation, Beverly, MA), 0.5 pmoles of the sequencing primer (5′-ACCTCCACCCACAAATCAAA-3′), and the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) were used. Sequencing reactions initiated with 1 minute denaturation at 96°C and proceeded with 40 cycles of 10 seconds at 96°C, 5 seconds at 50°C, and 4 minutes at 60°C. Product of sequencing was purified through a CleanSEQ dye terminal removal kit (Agencourt Bioscience Corporation) and run on the Applied Biosystems 3730 DNA Analyzer Instrument (Applied Biosystems), complying with manufacturer's instructions. The number of AAT repeats was counted on the resulting electropherograms.

Disability assessment.

All patients were clinically assessed by expert neurologists who were blinded to laboratory and neurophysiology results. EDSS score and a 0–10 Numerical Rating Scale (NRS) score assessing self-reported severity of spasticity⁹ were recorded in all patients with MS before the first and immediately after the last PT session.

MRI acquisition and analysis.

3T MRI scans consisted of a dual-echo turbo spin echo (TSE), a fluid-attenuated inversion recovery (FLAIR), and precontrast and postcontrast T1-weighted spin echo images. All images were acquired in the axial orientation with 3-mm-thick contiguous slices. T2 hyperintense lesions were determined by consensus of 2 investigators on proton density–weighted short echo TSE images. Lesions were outlined through semiautomated local thresholding contouring software (Jim 4.0, Xinapse System, Leicester, UK). Confidence in lesion identification was increased using FLAIR and T2-weighted scans as a reference. Presence of lesions within the brainstem, corpus callosum, posterior fossa, periventricular, and spinal cord was recorded for each patient to evaluate lesion distribution.

Transcranial magnetic stimulation.

LTP can be measured in patients through transcranial magnetic stimulation (TMS). TMS can activate neurons in a focal region of the cerebral cortex through electromagnetic induction. Delivered repetitively on the motor cortex, TMS can induce plastic modifications of cortical excitability that can be measured by recording the motor evoked potentials (MEPs) from the muscles represented within the stimulated region. An increase or decrease of the MEP amplitude lasting after the end of repetitive TMS indicates that LTP-like or long-term depression–like plastic changes occurred, respectively.

Electromyographic traces were recorded from the right first dorsal interosseus muscle (FDI) with surface electrodes, amplified (Digitimer, Hertfordshire, UK), sampled at 5 kHz, 20 Hz–2 kHz bandpassed, and stored for offline analysis.

MEPs were evoked through a Magstim 200² magnetic stimulator (Magstim Company, Whitland, Wales, UK) with a 70-mm diameter figure-of-eight coil. Coil was positioned tangentially to the scalp over the left hemisphere to evoke MEPs from the right FDI (“hot spot”), the handle pointing posterolaterally at 45°.

For iTBS, a Magstim Rapid² delivered 20 cycles of 10 bursts repeated at 5 Hz and separated by 8-second pauses, with each burst composed of a triplet of TMS pulses at 50 Hz. Stimulation intensity was 80% of the active motor threshold (AMT). AMT was defined as the minimum stimulation intensity required to evoke at least 5 MEP, ∼200 µV peak-to-peak amplitude out of 10 pulses during FDI's voluntary contraction (10% of maximal). Immediately before iTBS and at 2 different time points (0 and 15 minutes) after the end of iTBS, we collected 35 MEPs evoked by a test stimulus (TS) with a stimulation intensity set to induce a stable MEP of approximately 1 mV peak-to-peak amplitude in the relaxed FDI at baseline. MEP amplitudes were averaged at each time point and normalized to the mean baseline amplitude.

Short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF)¹⁵ were tested using paired-pulse (pp) TMS with a conditioning stimulus (CS) at 80% AMT intensity, preceding the TS.¹⁵ Five conditions were randomly presented: TS alone and 4 pp conditions with CS preceding TS at 1 of 4 different interstimulus intervals (ISIs) (2, 3, 10, and 15 msec).

Short-interval intracortical facilitation (SICF) was tested using a CS at 90% of resting motor threshold (RMT) intensity. Six conditions were randomly presented: TS alone and 5 conditions with TS preceding CS at 1 of 5 different ISIs (1.5, 2.1, 2.7, 3.7, and 4.5 msec).¹⁶

Long-interval intracortical inhibition (LICI), mediated by GABA_B,¹⁷ was tested through a CS at 120% RMT intensity. Two conditions were presented in a random order: TS alone and CS preceding TS at an ISI of 100 msec. For each experimental condition 10 responses were collected. MEP changes at each ISI were expressed as percentage of the mean unconditioned MEP amplitude.

Physical therapy.

Two to 4 weeks after TMS evaluation, patients started the therapeutic exercise program at the Physical Rehabilitation Department of the University Hospital Policlinico Tor Vergata in Rome. Exercises were performed on a daily basis for 2 weeks and consisted of 1 hour on land followed by another hour in 28–30°C water in a 150-cm wide pool, as in a previous study.^8,18 The program was devised and coordinated by a physician specializing in physical medicine and rehabilitation and consisted of both passive and active therapeutic exercises specifically aimed at restoring or maintaining muscular flexibility, range of motion, balance, coordination of movements, postural passages and transfers, and ambulation. Different types of therapeutic exercises were scheduled according to the individual disability status and administered by qualified physiotherapists. The exercises consisted of (1) repetition of different movements (i.e., tips and heels, 90° flexed hips and knees) for ambulation and stair climbing; (2) repetition of crossed patterns of movements for coordination; (3) postural reactions while standing with eyes open and closed, including the use of oscillatory boards for balance; (4) strengthening lower limb antigravitary muscles (i.e., gluteus minor and major, quadriceps femoralis); and (5) low-intensity and long-duration static stretching of iliopsoas, rectus femoralis, hamstrings, triceps surae, and lumbar spinal muscles for muscular flexibility and range of motion.

During each session patients performed either 2 or 3 sets per exercise type, with each set consisting of about 15 repetitions. Intensity of exercise was graded on the level of disability. Compensative pauses of time were included in relation to each patient's tolerance to the exercise regimen, for proper restoring.

Data analysis.

As in previous studies,^13,19 patients with MS were grouped according to the number of AAT repeats of the CNR1 gene. In the short-AATn group patients had 1 or 2 alleles with ≤11 repeats of AAT triplets, and in the long-AATn group patients had 2 alleles with ≥12 repeats. Student t test, Mann–Whitney test, Fisher exact test, and repeated-measures analysis of variance (ANOVA) were used to analyze differences between groups, as appropriate. Type of DMD was entered as a covariate because of its effect as a potential confounder. Duncan post hoc correction was performed. Correlation between EDSS and TMS parameters was explored through regression analysis. Data are presented as mean ± SEM. p < 0.05 was considered significant.