Effect of different pulse numbers of transcranial magnetic stimulation on motor cortex excitability: Single‐blind, randomized cross‐over design

Abstract Aims We aimed to investigate the effect of different pulse numbers of high‐frequency repetitive transcranial magnetic stimulation (rTMS) over the motor cortex on cortical excitability in healthy participants. Methods Fifteen healthy participants received 600 and 1200 pulses of 5‐Hz rTMS on separate days in a random order. Stimulation (duration, 2 seconds and interval, 1 seconds) was delivered over the left primary motor cortex for the hand, at 90% of resting motor threshold (rMT). The rMT and motor evoked potential (MEP) were measured before stimulation, and at 0 and 30 minutes after rTMS. Results No significant differences were observed between the two conditions for MEP (P = .919) or rMT (P = .266). Compared with baseline, MEP was increased significantly at 0 (P < .001) and 30 minutes (P < .001) after stimulation. After stimulation, rMT was decreased at 0 minute for the 600 and 1200 pulse conditions (P < .001), but had recovered by 30 minutes (P = .073). Conclusion Subthreshold 5‐Hz rTMS increased motor cortex excitability in healthy humans. However, the number of pulses may exhibit a ceiling effect in that beyond a certain point, that is, increasing the number of pulses may exhibit no further increase in cortical excitability.


| INTRODUC TI ON
Transcranial magnetic stimulation (TMS) techniques can stimulate the cortical nerves noninvasively. The current generated by TMS can change the cell membrane potential of cortical nerves, causing a series of physiological and biochemical effects. Since it was first successfully applicated by Barker in 1985, 1 TMS has been used successfully in the diagnosis and treatment of several neurological diseases. [2][3][4] Transcranial magnetic stimulation could evaluate the excitability of corticospinal tract using the motor evoked potential (MEP) and resting motor threshold (rMT) parameters. Increase in MEP amplitude or decrease in rMT indicates improvement in excitability. 5,6 Repetitive TMS (rTMS) is the application of recurrent TMS pulses to continuously and repetitively stimulates the cortex. Certain parameters of rTMS modulate cortical excitability. rTMS frequency is the main factor that influences cortical activity. High-frequency rTMS (≥5 Hz) increases cortical excitability, while low frequencies rTMS (≤1 Hz) decreases cortical excitability. [7][8][9] Studies indicate that the number of pulses may also affect the regulation of brain excitability. [10][11][12] Currently, however, there is no consensus on the optimal number of rTMS pulses required to achieve cortical excitability. Previous studies have used 150 to more than 2000 pulses for rTMS. [13][14][15] Different rTMS pulse numbers might affect the changes in MEP amplitude and the effect duration.
Increasing the pulse number within a limited pulse range, reportedly, has greater effects on excitability. 8,10,16 However, it remains unknown whether a ceiling effect exists for increasing the numbers of rTMS pulses.
In this study, we investigated whether increasing the number of rTMS pulses could induce greater effects on cortical excitability. We compared the effect of 600 and 1200 pulses of 5-Hz rTMS over the primary motor area responsible for hand control in healthy participants.

| Participants
Fifteen healthy college students were included in this study; seven were men and eight were women (Table 1)

| Study design
The study followed a single-blind, randomized cross-over design.
All subjects received 600 and 1200 pulses of high-frequency rTMS (CCY-IA Wuhan Yiruide Co., Ltd.). The sequence of the 600 or 1200 pulses was randomly allocated according to computer-generated numbers. The sequence number of each participant was enclosed in an envelope until the completion of rTMS.

| Parameter settings for rTMS
Conditions for the application of 600 pulses were as follows: Frequency, 5 Hz; intensity, 90% rMT; pulse number, 600; pulse sequence, 60; sequence duration, 2 seconds; sequence interval, 1 seconds; and stimulation time, 3 minutes. The stimulation sites comprised specific areas of the left primary motor cortex.
Conditions for the application of 1200 pulses were as follows: Frequency, 5 Hz; intensity, 90% rMT; pulse number, 1200 pulses; pulse sequence, 120; sequence duration lasted 2 seconds; sequence interval, 1 seconds; and stimulation time, 6 minutes. The stimulation site comprised the left primary motor cortex area.
To avoid residual effects, the interval between the two stimuli conditions was longer than 24 hours. To avoid sequential effects, each participant received two stimuli in a randomized order (Table 1). The experimental duration was relatively fixed. The participants were asked to avoid consuming drugs or drinks that might affect their brain activity and to maintain a relatively regular schedule during the experiment period. The rMT was measured before stimulation, immediately after rTMS (0 minute), and 30 minutes after stimulation. To avoid tester bias, the rMT test before stimulation, and at 0 and 30 minutes after stimulation were performed by different testers. The second tester was blinded to the rTMS frequency.
The experiment was conducted in a quiet and comfortable environment. The participants were relaxed and sat on a chair with armrests on both sides. A pillow was placed over their thighs to relax their hands. The TMS coil's central point was fixed over the hotspot.
The participants were instructed not to move their head to maintain a constant coil position during stimulation.

| Evaluation parameters and assessment methods
Each participant's rMT was determined using a descending staircase method to find the lowest intensity at which five of the ten consecutive pulses applied induced the MEP amplitude greater than 50 µV. 17,18 The rMT was expressed as the percentage of TMS output intensity.
The procedure for measuring rMT was as follows: Participants' bilateral upper limb muscles were kept relaxed, and the recording electrode was placed on the first dorsal interosseous muscle. The Determination of rMT: In the relaxed state of the participants, we stimulated the hotspot with a high output intensity to induce MEP. The output intensity was reduced to identify the critical intensity, which on ten continuous stimulations could induce at least five MEP amplitudes greater than 50 µV. The output intensity was increased or decreased by 1% for fine-tuning, and the above-described process was repeated. The minimum stimulus intensity was considered as the rMT. The rMT was measured before rTMS, and at 0 and 30 minutes after rTMS.
MEP measurements were recorded from the first dorsal interosseous muscle by surface electromyography (Yiruide EE). We used TMS to stimulate the hotspot with 120% of the rMT intensity at the different time points. 16 Five measurements were obtained at each time point.

| Statistical analysis
We normalized the data of MEP and rMT at 0 and

| Changes in MEP
The results are shown in Figure 1. MEP after 30 minutes also differed statistically different from that at baseline (P < .001). No significant differences in MEP were found between the 0 and the 30 minutes time points (P = .059).

| Changes in rMT
The result was shown in Figure 2. rMT differed significantly between

| Adverse reactions
Three participants experienced slight headache during rTMS. No other adverse reactions were reported.  11 In our study, we compared the effect of 600 and 1200 pulses on corticospinal excitability. We found no effect on excitability with increase in pulse numbers. We predicted that the effect of rTMS would increase with higher pulse numbers in a certain range. However, a ceiling effect is observed after a certain threshold of pulse number has been attained.

| D ISCUSS I ON
In this study, 5-Hz rTMS had a facilitatory effect on cortical excitability in healthy adults. Previous studies have also reported excitatory effects of rTMS, but the duration of excitation was unclear. For example, in Peinemann's study, the effect of 1800 pulses of rTMS lasted less than 40 minutes. 9 Similarly, Muellbacher et al used 1-Hz rTMS at 115% rMT intensity for 15 minutes and found significant reduction in motor cortex excitability for more than 30 minutes. 17 In this study, the effect on MEP lasted at least 30 minutes. The effect on rMT did not last 30 minutes, although this may have been due to an insufficient sample size.
Presently, we used a cross-over study design and randomized stimulus order on this study. The advantages of this design were that the same subject received two different types of stimuli, which can reduce the influence of different responses on different participants.
We did the two stimulation conditions with an interval of more than 24 hours to avoid the possibility of sequential effects. Furthermore, we used a randomized order of stimulation to eliminate the interference of stimulation sequences. Finally, in this study no significant orders effect was found.
This study has several limitations. First, we only focused on the hand motor cortex area of the brain. It therefore remains unknown whether a ceiling effect exists for other brain areas. Second, the sample size was small, which might affect the efficiency of our testing. Third, the participants' age was 20-30 years, and the study included only healthy people. Fourth, we measured the rMT before measuring the MEP at each time points, which may affect the corticospinal excitability. Therefore, further experiments are needed with diagnosed patients in order to evaluate possible clinical applications.
In conclusion, 5-HZ rTMS can significantly increase the corticospinal tract excitability, but the number of pulses may have a ceiling effect.

ACK N OWLED G M ENTS
The study was supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors confirm that this article contains no conflict of interest.