Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition

Summary

Contralateral silent period (cSP) assessment is a promising biomarker to index cortical excitability and treatment response. We demonstrate a protocol to assess cSP intended for studying M1 corticospinal inhibition of upper and lower limbs.

Abstract

Contralateral silent period (cSP) is a period of suppression in the background electrical muscle activity captured by electromyography (EMG) after a motor evoked potential (MEP). To obtain this, an MEP is elicited by a suprathreshold transcranial magnetic stimulation (TMS) pulse delivered to the primary motor cortex (M1) of the target muscle selected, while the participant provides a standardized voluntary target muscle contraction. The cSP is a result of inhibitory mechanisms that occur after the MEP; it provides a broad temporal assessment of spinal inhibition in its initial ~50 ms, and cortical inhibition after. Researchers have tried to better understand the neurobiological mechanism behind the cSP to validate it as a potential diagnostic, surrogate, and predictive biomarker for different neuropsychiatric diseases. Therefore, this article describes a method to measure M1 cSP of lower and upper limbs, including a selection of target muscle, electrode placement, coil positioning, method of measuring voluntary contraction stimulation, intensity setup, and data analysis to obtain a representative result. It has the educational objective of giving a visual guideline in performing a feasible, reliable, and reproducible cSP protocol for lower and upper limbs and discussing practical challenges of this technique.

Introduction

The silent period (SP) is a period of electromyographic (EMG) silence that follows a motor-evoked potential (MEP) induced by transcranial magnetic stimulation (TMS) applied during sustained muscle contraction. The suprathreshold TMS pulse can either be applied to the contralateral or ipsilateral primary motor cortex (M1) of the target muscle from which the EMG activity is being recorded yielding two phenomena: contralateral silent period (cSP) and ipsilateral silent period (iSP).

Even though iSP and cSP share similar features, they may reflect slightly different components. The first is thought to reflect transcallosal inhibition and thus be entirely of cortical origin^1,2. Conversely, cSP is investigated as a possible surrogate of corticospinal inhibition, most likely mediated by gamma-aminobutyric acid (GABA) B receptors within M1^3,4,5.

Supporting the role of cSP in GABA-mediated pathways, previous works have found an increase in cSP duration after oral administration of GABA-enhancing components^5,6,7,8. Still, spinal processes are also involved in altering its duration. The earlier phase (<50 ms) of the cSP is associated with decreased H-reflex values³-a reflex that is a product of peripheral neurocircuitry and that quantifies the excitability of spinal neurons⁹. Spinal processing is thought to be mediated through the activation of Renshaw cells, motoneuron after-hyperpolarization, and postsynaptic inhibition by spinal interneurons^{10,11,12,13,14}.

Despite spinal contribution, cSP results mainly from the activation of cortical inhibitory neurons, which are responsible for generating the later part of the cSP (50-200 ms)^{3,10,13,15,16}. In that respect, the early part of cSP duration has been associated with spinal inhibition mechanisms, whereas long cSPs request larger cortical inhibitory mechanisms^3,13,17,18.

Therefore, cSP is a promising biomarker candidate for corticospinal maladaptation due to neurological disorders, whereas more significant cSP durations potentially reflect an increase in corticospinal inhibition and vice versa^5,11. Accordingly, previous works have found an association between cSP duration and pathologies such as dystonia, Parkinson's Disease, chronic pain, stroke, and other neurodegenerative and psychiatric conditions^19,20,21,22. To illustrate, in a knee osteoarthritis cohort, a higher intracortical inhibition (as indexed by cSP) was associated with younger age, greater cartilage degeneration, and less cognitive performance in the Montreal cognitive assessment scale²³. Moreover, cSP changes could also longitudinally index treatment response and motor recovery^{24,25,26,27,28,29,30}.

As promising as the role of cSP in the neuropsychiatry field is, a challenging aspect of its assessment is that it can be too sensitive to protocol variations. For instance, the cSP duration (~100-300 ms)¹¹ is distinguishable between upper and lower limbs. Salerno et al. found an average cSP duration of 121.2 ms (± 32.5) for the first dorsal interosseous muscle (FDI) and 75.5 ms (± 21) for the tibialis anterior muscle (TA), in a sample of fibromyalgia patients³¹. Thus, the literature conveys a myriad of divergences in the parameters used to elicit cSPs, which in turn jeopardizes comparability across studies and delays the translation to clinical practice. Within a similar population, protocols have been heterogeneous regarding the suprathreshold TMS pulse setting used to stimulate M1 and the target muscle, for example. On top of that, researchers have failed to properly report the parameters used in their protocols.

Therefore, the goal is to provide a visual guideline on how to apply a feasible, reliable, and easily reproducible cSP protocol for evaluating M1 corticospinal excitability of upper and lower limbs and to discuss the practical methodological challenges of that procedure. Also, to help illustrate the reasoning for the choice of parameters, we conducted a non-exhaustive literature review on Pubmed/MEDLINE to identify published papers on cSP in chronic pain and rehabilitation populations, using the search term: Rehabilitation (Mesh) or rehabilitation or chronic pain or stroke and terms such as transcranial magnetic stimulation and single pulse or cortical silent period. No inclusion criteria were defined for the extraction, and pooled results are displayed in Table 1 for illustrative purposes only.

Protocol

This protocol involves research on human subjects and is in alliance with institutional and ethical guidelines of local ethical committees and the Declaration of Helsinki. Informed consent was obtained from subjects for using their data in the study.

1. Pre-experimental procedures

1. Screening of the subject. Screen the subject for intracranial implants, epilepsy, history of seizures, and pregnancy. Use questionnaire guidelines to ensure compliance with up-to-date safety precautions³².
   1. The delivery of electromagnetic pulses with TMS is contraindicated for individuals with intracranial implants of ferromagnetic material, such as shrapnel, aneurysm clips, or fragments from welding. Take precautions with individuals at increased likelihood of seizures.
   2. TMS assessment poses no fetal risk for pregnant women who are advised to have a conservative stance when dealing with this population. It is safe to apply TMS in pediatric populations, proceed cautiously in certain developmental stages (i.e., closure of the fontanelle, maturation of cortical excitability, and growth of the external auditory canal)³³.
2. Preparation of materials. For this procedure, other than the TMS and EMG devices, have at your disposal a swim cap, alcohol pads (with the preparation of 70% isopropyl alcohol), conductive gel, and a computer turned on with the EMG software setup and a dynamometer appropriate for the investigated muscle (see Table of materials).
   ​NOTE: Swim caps have the advantage of being the cheapest and most accessible option that still allows for reliable and reproducible TMS assessments without causing the discomfort of marking the subjects' head.

2. Appropriate instructions to the patients

1. Explain the basic steps of the procedure and how much time it will take.
2. Instruct the participant to remain awake but not to perform cognitive activities that require extra attention and/or focus (e.g., mathematical calculations, meditation, etc.) and anticipate that they might experience hand/jaw twitching or plausible side effects. Such events might seem unexpected for an inexperienced subject and thus jeopardize the procedure.
   ​NOTE: Single- and paired-pulse TMS have only been associated with mild, transient, adverse events, including headache, local pain, neck pain, toothache, and paresthesia. Seizures are rare, and no other serious adverse events have been associated³³. For extra safety, it is recommended to offer earplugs, due to the possibility of harmful sounds, and bite-blocks for possible masseter contraction³⁴.

3. Experimental procedures (Figure 1)

1. Select the muscle for positioning the electrodes.
   1. Ask the subject to put their hand over the table, in a prone position. Select the FDI muscle, localized between the first and second metacarpal osseous. To identify the FDI, ask the subject to abduct their index finger against resistance, keeping the rest of the hand still and laying on the table, while you are palpating the area.
   2. Expose the selected area. Use a disposable razor to shave the area to improve electrode contact with the skin, if necessary, and clean the area with alcohol pads to remove skin oils and other factors that could increase impedance. Certify that there is free skin to ensure contact with the electrode.
      NOTE: If evaluating lower limb activity, use the TA muscle for electrode placement. It is localized on the lateral side of the tibia and lies near the superficies of the skin. It can be identified by ankle dorsiflexion.
2. Place the surface EMG electrodes
   1. With the area exposed and cleaned, apply the conductive gel to each electrode of the channel to ensure good impedance.
   2. Place the negative electrode on the belly of the FDI muscle (the center or the most prominent bulge of the muscle belly) and the positive on the distal interphalangeal joint, with an inter-electrode distance of at least 1.5 cm. Place the reference electrode (neutral) on the wrist, over the ulnar styloid process.
      NOTE: The presence of motor endpoints, muscle tendons, or other active muscles can impact the stability of the recordings, so it is important to avoid these locations³⁵. For the TA muscle, the electrodes should be placed at one-third on the line that connects the tip of the fibula and the tip of the medial malleolus. Provide a 20 mm distance between each electrode's pole and place the reference electrode in the ankle.
3. Determine the required muscle contraction force
   1. Use a digital pinch dynamometer and a quadrangular pyramid support to minimize mechanical distortions and elevate the sensitivity for minimal contractions.
   2. Place the dynamometer between the first and second fingers with the help of the pyramidal support. Ensure that the third, fourth, and fifth fingers lay still on the table, while the 1^st and 2^nd generate the forces of the pinching movement.
   3. With the fixed position, ask the participant to press the dynamometer with the first finger and the side of the pyramid with the index finger, squeezing the dynamometer-pyramid system with their maximum force and creating a strong contraction of the FDI muscle.
   4. Using that value as reference, determine the 20% of maximum force. The participant must practice maintaining the target at 20% of sustained contraction. Allow for variations from 15%-25% of MVC.
      NOTE: Alternatively, in case of unavailability of a dynamometer that can catch the isolated muscle activity being investigated, use EMG feedback to standardize force. The recording software will measure the maximum peak-to-peak amplitude that corresponds to the subject's maximum force, and using that value as reference, will determine the 20% MVC. Subjects can receive visual and/or auditory clues of when 20% is achieved.
4. Identification of the initial location for hotspot searching
   1. Put a swim cap on the subject's head. All the reference points will be marked on it.
   2. Measure the sagittal circumference of the head from the nasion (the point between the forehead and the nose) to the inion (the most prominent point in the occipital region). Divide that value by two and mark that middle spot on the head.
   3. Mark the location of the patient's nasion, inion, the helix of both right and left external ears, and right and left supraorbital ridge. This is to certify that the cap has not slipped during the procedure, and/or that in future experiments it will be equally positioned on the patient's head.
   4. As described above, measure the tragus-to-tragus distance and add a mark halfway. Mark the intersection between them, a point identified as the vertex (Cz).
   5. From the vertex, move 5 cm laterally in parallel to the midsagittal line, on the contralateral side to the selected muscle. This mark approximately identifies the (M1), on the same coronal level as the hand motor cortex. Use this as the first spot to initiate the search for the hotspot.
   6. The hotspot is the area of the motor cortex where the lowest motor threshold is detectable. Set up a low intensity (e.g., 30% of maximum stimulator output [MSO]) and initiate the search by delivering multiple pulses to the first spot.
   7. Pursue with small intensity increments until identifying the lowest stimulus that detects an EMG-indexed response (i.e., MEP). For the delivery of the stimuli, angle the figure-of-eight coil at 45° in relation to the midsagittal line with the handle pointed toward the posterior of the patient.
   8. To ensure that the best spot was identified, move around the first spot and test the subsequent ~3 MEPs at 1 cm anterior, 1 cm lateral, 1 cm medial, and 1 cm posterior to it. Repeat this procedure as many times as needed for a consistent response; stick to the spot that elicits the largest MEP³⁶.
   9. Once the hotspot is found, mark that spot in the patient's head (swim cap). Use this location during this experiment and the potential follow-up visits. Be cautious as not to cause discomfort to the subject due to extra pressure. Use both hands to support the coil on the subject's head.
5. Determine resting motor threshold (RMT)
   1. Estimate the motor threshold as the minimum intensity required to promote an MEP of a minimal detectable amplitude (usually at least 50-100 µV).
   2. To determine the motor threshold, apply ten consecutive stimuli at the hotspot and select the lowest intensity that produced an MEP with a peak-to-peak amplitude of at least 50 µV on the target muscle, in 50% of the trials.
      NOTE: This protocol can be done with the target muscle at rest (resting motor threshold [RMT]) or during active contraction (active motor threshold [AMT]). Both can further be used as references for the suprathreshold TMS pulses. The acquisition of the AMT is more prone to variability because it relies on the standardization of MVC, which can be an issue for longitudinal studies with multiple assessments.
6. CSP protocol
   1. Deliver suprathreshold stimuli to elicit MEPs during tonic voluntary contraction of the target muscle.
   2. Deliver 10 stimuli with the stimulation intensity (SI) of 120% of the RMT with 10 s period in between them. During the application of the stimuli, ask the patient to maintain 20% of the maximum motor contraction of the target muscle, as practiced with the dynamometer.
   3. To ensure capturing the whole SP, certify that the EMG time window is long enough to capture up to 400 ms of EMG activity. Not infrequently - depending on the disease being studied - subjects might require higher SIs for a successful cSP to be obtained.

Results

After following the step-by-step procedure, the delivery of a suprathreshold TMS pulse (120% of the RMT) will elicit an observable MEP in the EMG recording of the target muscle, and a subsequent period of background EMG activity suppression of approximately 150 ms to 300 ms (Figure 2). From that EMG pattern, it's possible to calculate the cSP metrics. The most reported outcomes are the duration (in the range of ms) of the relative and absolute SP. The relative SP is measured from the MEP...

Discussion

The default SI to elicit MEP and SPs can vary according to the population. Intensities as low as 80% RMT have been shown to elicit cSP in healthy individuals³⁹, still studies on both healthy and diseased populations have used intensities as high as 150% RMT^49,50,51. Although this source of heterogeneity can be inherent to the nature of the target population, it should not be neglected as different SIs hav...

Materials

Name	Company	Catalog Number	Comments
Alcohol pads	Medline		Preparation with 70% isopropyl alcohol
Conductive gel	Weaver and Company		Used on the electrode
Echo Pinch	JTECH medical	0902A302	Digital dynamometer.
Mega-EMG	Soterix Medical	NS006201	Digital multiple channel EMG with built in software.
MEGA-TMS coil	Soterix Medical	NS063201	8 shaped TMS coil
Mega-TMS stimulator	Soterix Medical	6990061	Single Pulse TMS
Neuro-MEP.NET	Soterix Medical		EMG software used to analyse the muscles eletrical activity.
Swim cap	Kiefer