Repetitive transcranial magnetic stimulation on the modulation of cortical and spinal cord excitability in individuals with spinal cord injury

Abstract

Background:

Repetitive transcranial magnetic stimulation (rTMS) has been applied for modulating cortical excitability and treating spasticity in neurological lesions. However, it is unclear which rTMS frequency is most effective in modulating cortical and spinal excitability in incomplete spinal cord injury (SCI).

Objective:

To evaluate electrophysiological and clinical repercussions of rTMS compared to sham stimulation when applied to the primary motor cortex (M1) in individuals with incomplete SCI.

Methods:

A total of 11 subjects (35±12 years) underwent three experimental sessions of rTMS (10 Hz, 1 Hz and sham stimulation) in a randomized order at 90%intensity of the resting motor threshold and interspersed by a seven-day interval between sessions. The following outcome measures were evaluated: M1 and spinal cord excitability and spasticity in the moments before (baseline), immediately after (T0), 30 (T30) and 60 (T60) minutes after rTMS. M1 excitability was obtained through the motor evoked potential (MEP); spinal cord excitability by the Hoffman reflex (H-reflex) and homosynaptic depression (HD); and spasticity by the modified Ashworth scale (MAS).

Results:

A significant increase in cortical excitability was observed in subjects submitted to 10 Hz rTMS at the T0 moment when compared to sham stimulation (p = 0.008); this increase was also significant at T0 (p = 0.009), T30 (p = 0.005) and T60 (p = 0.005) moments when compared to the baseline condition. No significant differences were observed after the 10 Hz rTMS on spinal excitability or on spasticity. No inter-group differences were detected, or in the time after application of 1 Hz rTMS, or after sham stimulation for any of the assessed outcomes.

Conclusions:

High-frequency rTMS applied to M1 was able to promote increased cortical excitability in individuals with incomplete SCI for at least 60 minutes; however, it did not modify spinal excitability or spasticity.

Keywords

Spinal cord injury transcranial magnetic stimulation excitability spasticity physiotherapy

1 Introduction

A spinal cord injury (SCI) can partially or entirely interrupt the transmission of information from the descending or ascending pathways (Freund et al., 2011; Jurkiewicz et al., 2007; Wrathall et al., 1998; Wrigley et al., 2009). The suppression or decrease of neural pathways can generate cortical (Ellaway et al., 2007; Oudega & Perez, 2012) and spinal excitability changes (Guo & Hu, 2014; Little et al., 1999). This may lead to the onset of spasticity (Adams & Hicks, 2005; Ditunno et al., 2004; Elbasiouny et al., 2010; Kakulas, 2004). In SCI individuals, spasticity is a frequent disorder that can cause sensorimotor impairments, functional deficits, deformities, and pain (Mukherjee & Chakravarty, 2010).

Spasticity is characterized by a velocity-dependent increase in tonic stretch reflexes due to a hyperexcitability of spinal reflexes resulting from the loss of inhibitory influences descending from the spinal cord (Adams & Hicks, 2005; Mayer, 1997). Besides, individuals with SCI present alterations in cortical excitability (Ellaway et al., 2007; Oudega & Perez, 2012) due to the neuroplastic changes following the decrease or suppression of corticospinal pathways (Guo & Hu, 2014; Little et al., 1999).

Among the therapeutic approaches for spasticity management, repetitive transcranial magnetic stimulation (rTMS) has gained attention in recent years. rTMS has been used as a non-invasive strategy to modulate the central nervous system excitability and corticospinal networks (Groppa et al., 2012; Hallett, 2000; Lefaucheur et al., 2014; Nardone et al., 2014; Tazoe & Perez, 2015). Increasing evidence suggests that the modulation of corticospinal excitability via high-frequency rTMS reduces spasticity, improves motor control, and increases functional gains in individuals with incomplete SCI (Benito et al., 2012; Kumru et al., 2010).

To the best of our knowledge, no studies have investigated which rTMS frequency is the most effective in inducing modifications in cortical and spinal excitability, nor the clinical implication of these modifications in individuals with incomplete SCI. The subsequent effects of the use of rTMS depend (among other factors) on the selected stimulation frequency. In general, high-frequency rTMS facilities cortical excitability, while low-frequency rTMS has inhibitory effects (dos Santos et al., 2019; Groppa et al., 2012).

Thus, the objective of the present study was to evaluate the electrophysiological (i.e., cortical and spinal excitability) and clinical (i.e., spasticity) effects of a single high or low frequency rTMS session. rTMS was applied over the cortical representation area of the lower limbs in individuals with chronic incomplete SCI. Notably, the study has a relevant clinical contribution as the reduced spinal excitability probably depends on cortical inhibitory control, the effect of cortical modulation may create changes in the descending corticospinal inhibitory pathways. These changes may lead to a decrease in spinal cord excitability and spasticity in individuals with incomplete SCI.

2 Methods

A crossover, sham-controlled, randomized, and triple-blind study was performed (NCT03014999). The experimental procedures followed the Declaration of Helsinki (1964) and all participants signed informed consent before the start of the study.

2.1 Participants

Eleven volunteers with SCI were recruited through print, digital, and social media ads. Between June 2016 and April 2017, data collection took place in the Laboratory of Applied Neuroscience of Universidade Federal de Pernambuco. Individuals included in the study were: (i) subjects with a clinical diagnosis provided by a neurologist of incomplete thoracolumbar SCI (i.e., below the T1 level) according to the international standardization for neurological SCI classification (ISCSCI - International Standards for Neurological Classification of Spinal Cord Injury) (Kirshblum et al., 2011), due a traumatic etiology; (ii) in chronic phase of recovery (injury time > 8 months) (McDonald & Sadowsky, 2002); (iii) aged between 18–55 years; (iv) with a C or D degree of sensory-motor impairment according to American Spinal Injury Association Impairment Scale (AIS) (Kirshblum et al., 2011); (v) who were not regular walkers, but who also did not have a severe deficit for independent walking. The exclusion criteria were: (i) subjects with progressive degenerative disease, vestibular or visual disorders, significant cognitive deficit, or other neurological and/or orthopedic pathologies; (ii) contraindication for rTMS (Rossi et al., 2009); (iii) who had knee flexor muscle contracture > 20°; (iv) with severe skin ulcers; (v) reporting cardiac dysfunction, angina or hemodynamic disorders; or (vi) those who were taking medication affecting cortical and spinal cord excitability during the study.

2.2 Procedures

2.2.1 Experimental session

Subjects who met the eligibility criteria and agreed to participate in the study were submitted to three rTMS sessions: (i) high-frequency rTMS (10 Hz); (ii) low-frequency rTMS (1 Hz) and rTMS sham. Excitability of the CNS (spinal and cortical) and spasticity was measured before the stimulation (baseline), immediately after (T0), 30 (T30), and 60 (T60) minutes after application of rTMS. Sessions were separated by a minimum interval of seven days (washout period) to ensure no carryover effects (Wang et al., 2013).

2.2.2 Randomization, blinding and sample size

The order of the three sessions was randomized and counterbalanced among the participants using a random sequence table (www.randomization.com). A researcher not involved in the experimental procedures did the randomization. Allocation concealment was ensured using sealed and opaque envelopes.

Participants and researchers were blind to the rTMS stimulation application order. Lastly, statistical analyses were run by researchers unaware of the three different types of interventions.

A post-hoc power analysis revealed that a sample of 11 participants per group, at an alpha level of 0.05 for a two-sided test, with an observed effect size d = 1.32, achieved a power (β) of 0.80 (G*Power 3.1). The effect size was calculated as the cortical excitability (MEP) difference between the low (1.02±0.22) and high frequency (1.28±0.17) rTMS sessions.

2.3 Outcome measures

2.3.1 Evaluation of cortical excitability (primary outcome)

The excitability of the left primary motor cortex was assessed by single-pulse transcranial magnetic stimulation (sTMS). Subjects remained seated in their wheelchair and were instructed to relax in a comfortable position. A 70 mm diameter figure-eight-shaped coil was manually held over the hotspot —the region with the most intense motor evoked potential (MEP) response —of the right first dorsal interosseous (FDI) muscle. The anterior edge of the coil was directed towards the anterior region of the skull, forming a 45-degree angle with the sagittal plane. The electromyographic response was recorded by surface electrodes positioned on the muscular belly of the FDI.

The cortical excitability of the participants was assessed through the MEP. The resting motor threshold (rMT) was defined using the Motor Threshold Assessment Tool (MTAT 2.0 software) ( http://www.clinicalresearcher.org/software.htm ). The output of the magnetic stimulator was subsequently programmed to provide 130%of the rMT and 20 electromyographic records generated an average MEP value.

2.3.2 Evaluation of spinal excitability (primary outcome)

In order to measure spinal excitability, participants were positioned in ventral decubitus with 30° knee flexion. The electrical stimuli (Neuromep-8, Russia) were applied to the posterior tibial nerve in the popliteal fossa of the right lower limb (Phadke et al., 2010). Surface electromyography was recorded by two self-adhesive Ag-AgCl electrodes (1.0 cm in diameter) with an inter-electrode distance of 2 cm. The electrodes were positioned on the medial portion of the right soleus muscle at a distance of 5 and 7 cm from the medial head of the gastrocnemius muscle. A grounded electrode was positioned over the gastrocnemius midline at 10 cm from the recording electrodes. The electromyographic records were collected at a bandwidth of 5–10000 Hz, sampling rate of 2000 Hz, and impedance below 3 kΩ.

The spinal excitability was evaluated following two electrometric measurements: H-reflex threshold (H-T), maximum H-reflex (H-max), and maximum M-wave (M-max), at every 12 seconds; single and rectangular pulses lasting 1 ms were released into the popliteal fossa. The electric current intensity applied to the posterior tibial nerve was gradually increased at 1mA intensity until the highest amplitude of H-reflex was achieved (Phadke et al., 2010).

For homosynaptic depression (HD), pairs of electrical pulses were released using the same setting, intensity and duration of H-max. Electrical pulses were delivered at intervals of 150, 200, 250, and 300 ms (Panizza et al., 1995), repeated in random order for five times, and with a minimum interval of 12 seconds. HD is a measure used to evaluate bone marrow recovery and plasticity. HD is decreased in individuals with interruption of medullary pathways, which causes higher conditioned stimulus responses. These changes are related to decreased GABAergic inhibition and excitation of the monoaminergic pathways (serotonin and noradrenaline) in the interneurons spinal network (Jankowska, 2001).

2.3.3 Spasticity evaluation (secondary outcome)

The modified Ashworth scale (MAS) (Bohannon & Smith, 1987) was used to measure the spasticity of the lower limb muscle groups bilaterally (knee flexors, dors- and plantar-flexors). MAS ranges from 0 (normal tonus) to 4 (deformities in flexion or extension). Passive movement of the limb to be evaluated is performed, and the instant when resistance to passive movement occurs is observed (Craven & Morris, 2010). The same evaluator performed the MAS assessment.

2.4 Interventions

2.4.1 Repetitive transcranial magnetic stimulation - rTMS

Low-frequency stimulation was released at 1 Hz and 1500 pulses. This protocol was chosen because a previous study showed it was effective in reducing the spasticity of post-stroke individuals (Barros Galvão et al., 2014). In high-frequency sessions, 4-second trains were delivered at a frequency of 10 Hz (40 pulses/train) with an interval of 28 seconds between trains (total of 1800 pulses). These parameters were adapted from a recent study (Benito et al., 2012) that reported gains in motor function, decreased lower limb spasticity, and improved gait in subjects with incomplete SCI after 15 stimulation sessions.

For both stimulation protocols, a figure-eight-shaped coil was manually positioned with the center held over Cz (according to the international 10/20 system for EEG), on the cortical representation area of lower limbs. The output intensity was equivalent to 90%of the rMT. Two coils were used for sham rTMS: one connected to the stimulator and positioned behind the individual (away from the scalp) to generate the sound characteristic of the stimulation, and another uncoupled from the equipment, positioned on the individual’s scalp. The parameters of sham rTMS were the same of high-frequency sessions. A MagStim Super Rapid magnetic stimulator was used (Magstim Company, Whitland, Wales, UK).

2.5 Data processing and analysis

Descriptive statistic was used to report the data. Mean±standard deviation was used for continuous variables, while the median and interquartile range was used for discrete variables. Percentage frequencies were calculated for the nominal variables. All analyses were performed according to normalized data in relation to the baseline values. The Shapiro-Wilk test was used to investigate the normal distribution of the data.

The mean peak-to-peak amplitude of 20 MEP trials was calculated at each time recording. The maximum amplitude of the H-reflex was normalized by the maximum amplitude of the M wave (H-max/M-max) to reduce variations between individuals. For the HD, peak-to-peak amplitude mean of conditioned (H1) and unconditioned (H2) H-reflex was calculated for intervals (150 ms, 200 ms, 250 ms, and 300 ms) and expressed as a percentage of the ratio for unconditioned (H2) to the conditioned (H1) stimuli (H2/H1×100).

Concerning MAS analysis, subjects were individually classified into two groups according to MAS changes: (i) a decrease in at least one point of the scale (considered the minimal clinically important difference (MCID) for MAS (Shaw et al., 2010)); (ii) or no decrease occurred. Next, a frequency analysis was performed using the chi-square test for each evaluation time after the intervention (i.e., T0, T30, and T60).

The Friedman test was used for the MEP analysis, and after the Wilcoxon test and all other data were analyzed using the repeated measures ANOVA (3x4) test to compare the means between sessions (3) and times (4). The Mauchly sphericity test was also applied and the Greenhouse-Geisser correction was adopted if needed. Paired t-tests were subsequently performed with Bonferroni correction when appropriate.

Statistical analysis was performed using SPSS software version 20.0. The level of significance was set at p < 0.05.

3 Results

One hundred thirty-eight individuals with SCI were contacted and invited to participate in the study. Of these, 103 were excluded for several reasons, e.g., not being able to attend the research site, diagnosis of complete SCI, or injury at the cervical level. Thus, thirty-five individuals met the criteria to be screened, but only eleven volunteers (8 men and 3 women; mean age: 35±12 years) met the eligibility criteria and composed the sample (Fig. 1). The clinical characteristics of study participants are depicted in Table 1.

Fig. 1

Study flow diagram. TMS: transcranial magnetic stimulation. ASIA: American Spinal Injury Association.

Table 1

Clinical characteristics of patients at baseline

N	Age (years)	Gender	Injury level	ASIA	Time of injury (years)	Weight (Kg)	Height (cm)	Medication	Use of bracing
1	26	M	T10	D	3	58	176	Muscle relaxant/antispasmodic	Yes
2	33	M	T4-T5	C	6	77	178	Benzodiazepine/Others	Yes
3	40	M	T10-T12	C	3	85	176	Muscle relaxant/antispasmodic	Yes
4	24	M	T8	C	7	59	170	Muscle relaxant/antispasmodic	Yes
5	24	F	T2-T3	C	3	52	159	Muscle relaxant/antispasmodic	Yes
6	33	F	T-11	C	9	48	169	Muscle relaxant/antispasmodic/antihistamine	No
7	48	M	T7-T8	D	3	103	178	Benzodiazepine/ antihypertensive	Yes
8	52	M	T8-T11	D	4	56	148	Muscle relaxant/antispasmodic/Others	Yes
9	54	F	T12-L1	D	6	62	155	Muscle relaxant/antispasmodic/Others	Yes
10	18	M	T9	C	5	55	174	Muscle relaxant/antispasmodic	Yes
11	33	M	T1	C	3	50	163	Do not use	No
N	11	–	–		11	11	11	11	11
Mean	35	–	–		4.73	64.09	167.82	–	–
SD	12.12	–	–		2.05	17.14	10.22	–	–

All patients well-tolerated stimulation, and there was no drop-out in the study. There were a few temporary adverse effects reported such as neck pain and mild drowsiness. A significant increase in cortical excitability was only observed in subjects submitted to 10 Hz rTMS immediately after stimulation when compared to sham stimulation (F(11) = 25.421; p = 0.008). This increase was also significant at times T0 (Z = –2.599; p = 0.009), T30 (Z = –2.803; p = 0.005) and T60 (Z = –2.803; p = 0.005) when compared to the baseline condition (Fig. 2).

Fig. 2

Cortical excitability before and after repetitive transcranial magnetic stimulation (rTMS) sessions. Mean and standard error are shown for each time. Filled symbols show significant difference in relation to the baseline, whereas asterisk displays a significant difference between the 10 Hz rTMS group and the sham group. Hz: Hertz; min minutes. MEP: motor evoked potential.

For spinal excitability, no significant differences were found between the sessions [F(3, 24) = 1.50, p = 0.24] or in the comparison over time [F(2, 16) =0.46, p = 0.64] H-max/M-max, and for HD at 150 ms (Stimulation: [F(2, 12) = 1.45, p = 0.27]; Time: [F(3, 18) = 0.30, p = 0.82)], 200 ms (Stimulation: [F(2, 18) = 0.66, p = 0.53]; Time: [F(3, 27) = 0.40, p = 0.76]), 250 ms (Stimulation: [F(2, 18) = 1.15; p = 0.40]; Time: [F(3, 27) = 0.99; p = 0.41]) and 300 ms intervals (Stimulation: [F(2, 16) = 0.77; p =0.48]; Time: [F(3, 24) = 0.58; p = 0.92]) (Table 2).

Table 2

Baseline-standardized values for excitability spinal

	10 Hz rTMS	1Hz rTMS	Sham rTMS
H-max/M-max (mV)
T0	0.95±0.17	0.98±0.15	1.05±0.19
T30	1.01±0.19	1.01±0.16	1.05±0.18
T60	0.96±0.17	0.99±0.17	1.08±0.15
HD (mV)
ISI 150 ms
T0	0.90±0.30	0.92±0.31	1.07±0.29
T30	0.70±0.39	1.09±0.21	1.05±0.33
T60	0.86±0.37	1.01±0.20	1.03±0.27
ISI 200 ms
T0	1.05±0.40	1.08±0.28	0.88±0.31
T30	0.95±0.38	1.04±0.13	0.96±0.24
T60	0.95±0.26	1.13±0.37	0.96±0.20
ISI 250 ms
T0	0.95±0.19	1.17±0.33	0.90±0.28
T30	0.94±0.19	0.98±0.15	0.94±0.22
T60	1.04±0.37	1.06±0.20	0.86±0.28
ISI 300 ms
T0	0.96±0.19	0.99±0.15	0.95±0.27
T30	0.88±0.28	1.01±0.29	1.07±0.35
T60	0.92±0.26	1.09±0.26	1.02±0.14

ISI: interstimulus interval; H-max/M-max: maximal amplitude of the Hoffman reflex normalized by the maximal amplitude of the wave M; HD: homosynaptic depression; rTMS: repetitive transcranial magnetic stimulation; T0: immediately after stimulation; T30: evaluation 30 minutes after stimulation; T60: evaluation 60 minutes after stimulation.

No significant reduction was observed regarding lower limb spasticity after the rTMS in any session (Table 3).

Table 3

Minimal clinically important difference (MCID) range of the 11 volunteers for the modified Ashworth scale for each muscle grouping of the right and left lower limbs

	10 Hz rTMS			1 Hz rTMS			Sham rTMS
		T0	T30	T60	T0	T30	T60	T0	T30	T60	^* Chi-square
RLL
Hip flexors	YES	18.18%	36.36%	18.18%	9.09%	9.09%	18.18%	0%	0%	9.09%	(X² = 2.27;
	NO	81.82%	63.64%	81.82%	90.01%	90.01%	81.82%	100%	100%	90.01%	p = 0.32)
Hip extensors	YES	9.09%	18.18%	9.09%	0 %	0%	0%	0%	9.09%	21.27%	(X² = 1.06;
	NO	90.01%	81.82%	90.01 %	100%	100%	100%	100%	90.01%	72.73%	p = 0.59)
Knee flexors	YES	0%	18.18%	0%	18.18%	9.09%	9.09%	18.18%	21.27%	21.27%	(X² = 4.25;
	NO	100%	81.82%	100%	81.82%	90.01%	90.01%	81.82%	72.73%	72.73%	p = 0.12)
Knee extensors	YES	9.09%	21.27%	9.09%	18.18%	9.09%	36.36%	18.18%	18.18%	21.27%	(X² = 0.57;
	NO	90.01%	72.73%	90.01 %	81.82%	90.01%	63.64%	81.82%	81.82%	72.73%	p = 0.75)
Plantar flexors	YES	9.09%	9.09%	21.27%	18.18%	21.27%	21.27%	9.09%	9.09%	9.09%	(X² = 1.89;
	NO	90.01%	90.01%	72.73%	81.82%	72.73%	72.73%	90.01%	90.01%	90.01%	p = 0.39)
Dorsiflexors	YES	0%	0%	0%	0%	0%	0%	9.09%	9.09%	9.09%	(X² = 2.06;
	NO	100%	100%	100%	100%	100%	100%	90.01%	90.01%	90.01%	p = 0.36)
LLL
Hip flexors	YES	21.27%	21.27%	18.18%	9.09%	18.18%	21.27%	9.09%	9.09%	9.09%	(X² = 1.89;
	NO	72.73%	72.73%	81.82%	90.01%	81.82%	72.73%	90.01%	90.01%	90.01%	p = 0.39)
Hip extensors	YES	36.36%	36.36%	36.36%	18.18%	18.18%	18.18%	18.18%	9.09%	9.09%	(X² = 1.22;
	NO	63.64%	63.64%	63.64%	81.82%	81.82%	81.82%	81.82%	90.01%	90.01%	p = 0.32)
Knee flexors	YES	21.27%	36.36%	36.36%	18.18%	9.09%	9.09%	9.09%	9.09%	0%	(X² = 1.22;
	NO	72.73%	63.64%	63.64%	81.82%	90.01%	90.01%	90.01%	90.01%	100%	p = 0.48)
Knee extensors	YES	21.27%	18.18%	21.27%	9.09%	21.27%	36.36%	21.27%	45.45%	21.27%	(X² = 1.45;
	NO	72.73%	81.82%	72.73%	90.01%	72.73%	63.64%	72.73%	55.55%	72.73%	p = 0.48)
Plantar flexors	YES	9.09%	21.27%	36.36%	9.09%	9.09%	18.18%	18.18%	21.27%	21.27%	(X² = 0.56;
	NO	90.01%	72.73%	63.64%	90.01%	90.01%	81.82%	81.82%	72.73%	72.73%	p = 0.75)
Dorsiflexors	YES	0%	0%	0%	9.09%	9.09%	9.09%	0%	0%	0%	(X² = 2.06;
	NO	100%	100%	100%	90.01%	90.01%	90.01%	100%	100%	100%	p = 0.36)
Adductors (R and L)	YES	0%	9.09%	9.09%	0%	0%	0%	0%	0%	9.09%	(X² = 1.88;
	NO	100%	90.01%	90.01%	100%	100%	100%	100%	100%	90.01%	p = 0.99)

rTMS: repetitive transcranial magnetic stimulation; T0: immediately after stimulation; T30: evaluation 30 minutes after stimulation; T60: evaluation 60 minutes after stimulation. RLL: right lower limb. LLL: left lower limb. R: right. L: left. ^*Data regarding analysis immediately after rTMS.

4 Discussion

The main result of this study was the increased cortical excitability after a session of high-frequency rTMS compared to the control group in individuals with SCI. Compared to the baseline condition, this increase in cortical excitability in the high-frequency group lasted for at least 60 minutes after the stimulation. No significant cortical changes were observed after low-frequency rTMS. In addition, no significant reduction in spinal cord excitability and spasticity of individuals after both rTMS conditions.

Previous studies with healthy subjects also observed that a single high-frequency rTMS session could promote increased activation of the motor cortex in isolation (Arai et al., 2007; Peinemann et al., 2004) and association with moderate exercise (Albuquerque et al. 2018). However, although low-frequency rTMS induced a reduction in cortical excitability in many studies (Hoogendam et al. 2010), a single 1Hz-rTMS failed to decrease the cortical excitability in individuals with SCI of the present study. The lower number of stimuli in low-frequency protocol (1500 pulses), compared to high-frequency (1800 pulses), may in part explain the absence of MEP reduction. Furthermore, it is acknowledged that the aftereffects of such protocols depend highly on the number of pulses, as shown by Pascual-Leone et al. (1994).

The effects of rTMS are not restricted to the stimulated region (Peinemann et al., 2004; Valero-Cabré et al., 2001); they can also modulate other regions in which the target cortical area has functional connections. Modulation of cortical excitability through rTMS may also alter the descending cortical-spinal projections and significantly affect spinal excitability. Indeed, previous evidence showed that a single 5 Hz-rTMS session applied on the leg motor area of healthy individuals results in spinal excitability alteration, observed by a reduction in H-reflex after stimulation (Perez et al., 2005). Similarly, Berardelli et al. (1998) when applied a single supraliminal high-frequency (5 Hz) rTMS (120%of rMT) session on the left motor cortex of healthy individuals, reported a reduction of H-reflex (Berardelli et al., 1999). These two studies suggest that high-frequency rTMS can modulate transmission in specific spinal circuits through increased presynaptic inhibition of Ia afferent fibers. Such modulation may lead to changes in the corticospinal pathway in healthy individuals. In our study, a single high-frequency rTMS session applied over the cortical representation area of the lower limbs was not effective in modulating the spinal monosynaptic reflexes. Our findings corroborate Kumru et al. (2010) study, which no alteration in the H-reflex was observed even after five sessions of 20 Hz subliminal rTMS in the motor area of the legs. In the present study and Kumru et al. (2010), the stimulation frequency applied to access the corticospinal pathways damaged by SCI was below the rMT, which may have hindered the activation of the descending inhibitory pathway. In addition, differences in the study populations (SCI and healthy individuals) could explain the discrepancies.

While in the present study, rTMS did not promote a reduction in spinal excitability nor did it clinically change spasticity, high-frequency rTMS can be considered as a recommended technique for rehabilitation of individuals with incomplete SCI, since other studies adopting different parameters and with a higher number of sessions have confirmed its therapeutic effects (Benito et al., 2010; Tazoe & Perez, 2015, Nogueira et al. 2020). In this context, Belci et al. (2004) reported that 10 Hz subliminal rTMS applied for five days on the left primary motor cortex in four individuals with incomplete and chronic SCI promoted an improvement in sensory-motor functions (Belci et al., 2004). In another study, fifteen sessions were performed with subliminal 20 Hz rTMS applied on the leg motor area in 17 individuals with incomplete and subacute SCI, showing an improvement in sensory-motor functions (Benito et al., 2012).

Furthermore, no significant differences were found in the HD after any of the rTMS protocols. The HD mechanism is not yet fully elucidated; one theory is that it is a presynaptic mechanism that occurs at the synapses of Ia afferent fibers with motoneuron-α due to a transient reduction in the release of neurotransmitters from previously activated fibers (Meunier et al., 2007; Perez et al., 2005; Pierrot-Deseilligny & Mazevet, 2000). Some authors suggest that only changeling training would increase the presynaptic inhibition of the Ia afferent fibers (Meunier et al., 2007; Perez et al., 2005). The absence of HD response in individuals with incomplete SCI after an isolated application of rTMS can be attributed to the nature of this measure. Perez et al. (2005) found a significant reduction in the depression of soleus H-reflex in a study that evaluated the presynaptic control of Ia afferent fibers after acquiring a new motor skill in healthy individuals, suggesting that HD can be induced by learning a visuo-motor task. Another study assessing HD after a single session of qualified and unskilled cycling training found that although both groups presented improvements, the performance of the skilled training group was more satisfactory (Meunier et al., 2007). Thus, the protocol proposed by the present study may not have played the role of plasticity inducer, considering that this would require an association of rTMS with some motor training.

Spinal cord pathways partly mediate the neural mechanisms that contribute to spasticity (Tazoe & Perez, 2015), while corticospinal activity can modulate the motoneuron activity that is projected to extra (α) and intrafusal (γ) skeletal muscle fibers (Mukherjee & Chakravarty, 2010). While previous studies observed an association of increased cortical excitability with a reduction in spasticity after applying high-frequency rTMS at the cortical level in individuals with chronic incomplete SCI (Benito et al., 2012; Kumru et al., 2010), the increased cortical excitability after high-frequency rTMS in the present study was not sufficiently capable of influencing spasticity in this population.

According to Elbasiouny (2010), upper motor neuron lesion associated with lower SCI results in a reduced tendency for developing spasticity in these individuals (Elbasiouny et al., 2010). In our study, only individuals with thoraco-lumbar injury and low spasticity degree were included, suggesting that the ability to detect significant clinical differences might be masked because the clinical improvement in these individuals is minimal, as the baseline motor impairment was mild (Bohannon & Smith, 1987). Moreover, studies regarding individuals with chronic incomplete SCI who had significant improvement in the spasticity degree after application of rTMS (Benito et al., 2012; Kumru et al., 2010) underwent more than one intervention session. Another reason might be the biochemical changes in SCI related to the expression of the GABA neurotransmitter (Wu & Sun, 2015) and of the BDNF (Brain-derived neurotrophic factor) (Lewin & Barde, 1996) which are related to the onset of spasticity and to neural plasticity, respectively. Therefore, it is possible to assume that a single rTMS session has not influenced the level of GABA neurotransmitter and BDNF. However, previous studies have demonstrated the potential of rTMS to produce cumulative plastic changes (Bäumer et al., 2003; Benito et al., 2012; Benito Penalva et al., 2010; Buhmann, 2004; Tazoe & Perez, 2015). Thus, it is supposed that a single rTMS session does not influence GABA neurotransmitter and the BNDF expression, especially after the biochemical changes resulting from SCI. Thus, in individuals with incomplete chronic SCI, rTMS effects appear to be related to both the application parameters of rTMS and the number of sessions individuals undergo.

The main limitation of this study was the limited sample size, particularly for AIS C-D injuries that may include a variable range of spinal cord involvement, making the study findings more difficult to interpret. In addition, the participants underwent to one rTMS session, which makes it difficult to acknowledge the cumulative effects that rTMS can cause in the cortical and spinal cord excitability. The absence of MEP recordings from lower limb muscles is also a limitation of our study. Lower limb MEPs may provide valuable information in quantifying the degree of functional involvement of the spinal cord and support the interpretation of the results. However, as previously pointed out (Kesar et al., 2018), the lower limb cortical representation is at greater depth from the scalp surface compared to the upper limb representation, making it difficult to evaluate with a figure-eight-shaped coil. Finally, rMT values might be influenced by anatomical (e.g., skull thickness) and physiological (e.g., stress) intrinsic individual characteristics (Chagas et al., 2018; Herbsman et al., 2009).

Further studies should investigate which mechanisms underlie the cumulative effects of several consecutive sessions and whether these effects are long-lasting and have effects for clinical improvement. It can be assumed that a protocol with more than one session would be able to modulate both spinal excitability and spasticity in individuals with incomplete SCI. In addition, a comparison of the modulation of cortical and spinal cord excitability after high or low-frequency rTMS between healthy and individuals with incomplete SCI may enhance our understanding of the potential effects of different stimulations and unfold additional and clinical information.

5 Conclusions

In summary, the 10 Hz rTMS applied to the motor cortex was able to promote increased cortical excitability in subjects with chronic incomplete SCI. However, only one session of either high- or low-frequency rTMS was unable to modify the medullary excitability and spasticity of these individuals. Study findings point out a new perspective regarding the evaluation and therapeutic intervention in incomplete SCI by modulating the descending corticospinal pathway.

Funding

The researchers were supported by the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq), Brazil. Katia Monte-Silva is supported by CNPq, Brazil (grant 311224/2019-9).

Conflict of interest

The authors declare that there are no types of conflict of interest.

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