Motor Neuroplastic Effects of a Novel Paired Stimulation Technology in an Incomplete Spinal Cord Injury Animal Model

Paired stimulation of the brain and spinal cord can remodel the central nervous tissue circuitry in an animal model to induce motor neuroplasticity. The effects of simultaneous stimulation vary according to the extent and severity of spinal cord injury. Therefore, our study aimed to determine the significant effects on an incomplete SCI rat brain and spinal cord through 3 min and 20 min stimulations after 4 weeks of intervention. Thirty-three Sprague Dawley rats were classified into six groups: (1) normal, (2) sham, (3) iTBS/tsDCS, (4) iTBS/ts-iTBS, (5) rTMS/tsDCS, and (6) rTMS/ts-iTBS. Paired stimulation of the brain cortex and spinal cord thoracic (T10) level was applied simultaneously for 3–20 min. The motor evoked potential (MEP) and Basso, Beattie, and Bresnahan (BBB) scores were recorded after every week of intervention for four weeks along with wheel training for 20 min. Three-minute stimulation with the iTBS/tsDCS intervention induced a significant (p < 0.050 *) increase in MEP after week 2 and week 4 treatments, while 3 min iTBS/ts-iTBS significantly improved MEP (p < 0.050 *) only after the week 3 intervention. The 20 min rTMS/ts-iTBS intervention showed a significant change only in post_5 min after week 4. The BBB score also changed significantly in all groups except for the 20 min rTMS/tsDCS intervention. iTBS/tsDCS and rTMS/ts-iTBS interventions induce neuroplasticity in an incomplete SCI animal model by significantly changing electrophysiological (MEP) and locomotion (BBB) outcomes.


Introduction
Spinal cord injuries (SCIs) alter the motor, sensory, and autonomic functioning of the spinal cord [1]. The effects of SCI depend on the involvement of the spinal cord segment [2]. The severity of the primary injury determines the grade of the patient's neurologic state, which serves as a prognostic indicator [3]. The majority of SCI patients 1.5 mA; tDCS: 1 mA) was studied in stroke patients utilizing upper extremity functional evaluation, and a significant improvement was noted in Jebsen-Taylor hand function test (JTT) and finger-to-nose test (FNT) scores [32]. In another recent study, it was shown that 20 min of treadmill activity coupled with rTMS (20 Hz, 1800 pulses) on the brain and tsDCS (2.5 mA, 1200 s) on the spinal cord altered corticospinal excitability and increased spinal motor output for up to 30 min in healthy adults [33]. Additionally, after 40 min of paired stimulation (0.1 Hz), one study found a significant decrease in intracortical inhibition and an increase in the facilitation of intracortical and primary motor neurons, which resulted in a reduction in the motor threshold and soleus H-reflex in healthy individuals [34]. One study reported the safety of paired stimulation (iTBS/tsDCS) applied for 30-60 min for up to 4 weeks in an animal brain model without observing any adverse effect on brain tissue biomarkers or histology [35].
Based on the available evidence, this newly investigated paired stimulation approach needs a deeper understanding of its effects due to a lack of focused studies in the literature. Some studies have reported the effectiveness of iTBS/tsDCS or rTMS/tsDCS waveforms in clinical and animal settings [17,32,35,36]. However, no studies have examined different combinations of rTMS, iTBS, and tsDCS applications, particularly for short and long durations, on neuroplasticity after SCI. For this purpose, we tested four different combinations of magnetic and electric stimulation waveforms in an animal model. Therefore, our study aimed to determine the significant effects on an incomplete SCI rat brain and spinal cord through 3 min and 20 min stimulations after 4 weeks of intervention.

MEP Assessment after Each Week
For the normal group, pre vs. post MEP for pre-week 1 and post-weeks 1, 2, 3, and 4 changed but did not reach the significance level ( Figure 1). The sham group showed a change in pre-surgery post_30 min MEP as compared to pre_5 min without achieving a significant level (               Figure 6. rTMS/ts-iTBS (n = 5). rTMS/ts-iTBS (20 min); Pre_5 min vs. post_5, 10, 15, 20, 25, and 30 min were compared using paired sample t-test; The level of significance was set to p < 0.050 *. Table 1 compares MEP values between week 1 and week 4 for different time points, particularly pre_5 min of week 1 vs. post_5, 10, 15, 20, 25, and 30 min of week 4, to observe the effects of different stimulations. The following MEP values changed significantly in the 3 min iTBS/tsDCS group in post_25 min (p = 0.012 *) and post_30 min (p = 0.026 *). The effect size (d) is also shown in Table 1. In contrast, the MEP values for the same time points, i.e., pre_10 min and pre_5 min of week 1 vs. pre_10 min and pre_5 min of week 4 and post_5-30 min of week 1 vs. post_5-30 min of week 4, did not attain a significant level.  Figure 7 presents the results of all six groups, including MEP values before SCI induction surgery and after four weeks of stimulation intervention. All of the groups' pre-surgery MEP values were higher than after surgery, except for the 3 min iTBS/ts-iTBS and 20 min rTMS/tsDCS groups. Only post_10 min (p = 0.039 *) in the sham group and pre_5 min (p = 0.018 *) in the iTBS/tsDCS group showed significant differences from pre-surgery MEP values. The sham and iTBS/tsDCS groups' MEP values decreased after surgery and the four-week intervention, while rTMS/tsDCS and rTMS/ts-iTBS groups' MEP values increased after surgery and the stimulation intervention.

MEP before vs. after Surgery and Stimulation Intervention
The MEP values before surgery in pre_5 min vs. after surgery and four-week stimulation in post_5-30 min changed without reaching the level of significance. The 3 min iTBS/tsDCS group had significantly changed MEP values after the four-week intervention in post_10 min (p = 0.003 *) and post_15 min (p = 0.002 *), with an increasing trend ( Table 2).  The MEP values before surgery in pre_5 min vs. after surgery and four-week stimulation in post_5-30 min changed without reaching the level of significance. The 3 min iTBS/tsDCS group had significantly changed MEP values after the four-week intervention in post_10 min (p = 0.003 *) and post_15 min (p = 0.002 *), with an increasing trend ( Table   Figure 7. Before Surgery, MEP values before SCI surgery; After Surgery, MEP values after surgery and four-week stimulation intervention. Paired sample t-test was used for comparison of before vs. after surgery and stimulation intervention; the level of significance was set to p < 0.050 *. The one-way ANOVA results for MEP time and group factors are presented in Tables 3 and 4. There was no significant difference observed for the time factor, but the group factor showed significant changes in all weeks. Table 3. MEP comparison using one-way ANOVA with time factor (n = 24).  Group factor comprises normal, sham, iTBS/tsDCS, iTBS/ts-iTBS, rTMS/tsDCS, and rTMS/ts-iTBS; df, degree of freedom. The level of significance was set to p < 0.050 *, p < 0.001 **, and p < 0.0001 ***.

Basso, Beattie, and Bresnahan (BBB) Locomotor Scale Score
The BBB score changed in all six groups. It changed significantly during week 1 in all groups except for the normal, iTBS/tsDCS, and rTMS/tsDCS groups. During week 2 and week 3, the BBB score improved significantly in all groups except for the normal and rTMS/tsDCS groups. In post-week 4, only the sham and rTMS/ts-iTBS groups reached the level of significance. The change in BBB score is plotted in Figure 8. The week 1 vs. week 4 BBB scores are presented in Figure 9. It only showed a significant change in the rTMS/ts-iTBS group (p = 0.002 *); other groups' BBB scores changed as compared to week 1 but did not reach the level of significance. The group factor comparison of the BBB score is presented in Table 5 with the effect size.

Basso, Beattie, and Bresnahan (BBB) Locomotor Scale Score
The BBB score changed in all six groups. It changed significantly during week 1 in all groups except for the normal, iTBS/tsDCS, and rTMS/tsDCS groups. During week 2 and week 3, the BBB score improved significantly in all groups except for the normal and rTMS/tsDCS groups. In post-week 4, only the sham and rTMS/ts-iTBS groups reached the level of significance. The change in BBB score is plotted in Figure 8. The week 1 vs. week 4 BBB scores are presented in Figure 9. It only showed a significant change in the rTMS/ts-iTBS group (p = 0.002 *); other groups' BBB scores changed as compared to week 1 but did not reach the level of significance. The group factor comparison of the BBB score is presented in Table 5 with the effect size.

Discussion
In the present study, the effects of paired stimulation on the SCI rat brain and spinal cord were evaluated. In this study, we applied four different paired stimulations on the motor cortex and T10 spinal cord simultaneously in an incomplete SCI animal model. Threeminute stimulation with the iTBS/tsDCS intervention induced a significant (p < 0.050 *) increase in MEP after week 2 and week 4 treatments, while 3 min iTBS/ts-iTBS significantly improved MEP (p < 0.050 *) only after the week 3 intervention. The 20 min rTMS/ts-iTBS intervention showed a significant change only in post_5 min after week 4.
In our study, iTBS/tsDCS stimulation for 3 min significantly changed MEP in post_5, 10, 15, 20, and 25 min in week 2 and post_25 and 30 min in week 4, respectively (Figure 3). According to a prior study on healthy rats, MEP can be continually increased for up to 30 min following a single session of iTBS [37]. A study on an SCI animal model reported the effects of iTBS stimulation on mild and moderate SCI groups' neuroplasticity, with enhanced MEPs from baseline after week 1 and week 4 stimulation compared to the severe SCI group [38], which is consistent with our current study results. In our current study, pre-vs. post-week 1 and week 4 also showed a significant increase in post_25 min (p = 0.012 *) and post_30 min (p = 0.026 *) MEPs (Table 1). In another study conducted on SCI subjects, the iTBS/tsDCS waveform produced a significant increase in MEP (p < 0.050 *) [17]. The safety of the iTBS/tDCS waveform ranging from 2.5-4.5 mA/cm 2 was previously tested in an animal model without any adverse effects on brain tissue biomarkers or scalp tissue histology [35]. A study on stroke patients demonstrated a significant improvement (p < 0.050 *) in upper-limb recovery without any side effects after 20 min of 1 mA tDCS and 1.5 mA iTBS application on the primary motor cortex [32].
The 3 min iTBS/ts-iTBS waveform showed a significant change in MEP only in week 3 in post_5, 25, and 30 min. However, the MEP was reduced after the stimulation intervention, inducing inhibitory effects through paired stimulation. A similar effect was observed for the 20 min rTMS/ts-iTBS waveform in post_5 min after week 4. Despite the use of various combinations of iTBS, tsDCS, and rTMS in 3-20 min interventions for four weeks along with wheel training, we did not obtain a lot of significant results for these waveforms. However, iTBS/tsDCS and rTMS/ts-iTBS enhanced cortical excitability through increased MEPs in the SCI animal model. As mentioned in the available literature, paired stimulation can result in instant neuroplasticity in healthy human and animal populations [39]. They reported increased MEP and a decreased spinal threshold for up to 40 min after the rat's brain and spinal cord had experienced recurrent paired associative stimulation (PAS) for 5-10 min [39].
The changes in BBB scores are presented in Figures 8 and 9. All of the interventions after week 1 to week 4 significantly improved the BBB scores, except for 3 min iTBS/tsDCS and iTBS/ts-iTBS in post-week 4 and for 20 min rTMS/tsDCS in all weeks of the intervention. After surgery, the BBB score was recorded in the range of 3-10 out of 22 in all groups. A greater improvement was noted in the 3 min iTBS/tsDCS group from 0.00 ± 0.00 to 5.00 ± 5.40 (p = 0.010 *) after week 2 and to 10.00 ± 9.19 (p = 0.340) after week 4. For the 3 min iTBS/ts-iTBS group, week 3 showed an improvement in the BBB score from 5.38 ± 7.11 to 10.13 ± 7.60 (p = 0.064), and for the 20 min rTMS/ts-iTBS group, it improved from 3.20 ± 5.54 to 7.80 ± 5.53 (p = 0.006 *) after week 2 and to 9.80 ± 5.16 (p = 0.008 *) after week 4. Differentiating the effect of these stimulation waveforms is very difficult because the sham group also significantly improved its BBB score. In addition, another study previously mentioned that iTBS does not significantly improve BBB scores in mild or moderate SCI animal models [38].

Strengths of Study
Specific brain or spinal cord stimulations such as rTMS, iTBS, tsDCS, etc., have been previously reported in SCI subjects, but our study explored simultaneous brain and spinal cord stimulations in the form of paired stimulation in an incomplete SCI animal model to investigate the effects on cerebral and spinal cord plasticity through the change in MEP peak-to-peak amplitude and BBB score. In our study, we anesthetized rats before stimulation to record their MEPs before and after the stimulation intervention. We also administered TMS at 120% of RMT for MEP recording, and the stimulation intensity used was 80% of RMT, which is higher than in previous studies. The assessment of MEP through lower-body muscles can also reveal the extent and severity of SCI, which is the key component of clinical rehabilitation.

Limitations of Study
The MEP-based results confirmed the effects on neuroplasticity in the SCI animal model. However, to consider their clinical application, these stimulation waveforms need to be tested on a large sample size. MRI-based studies will be required to highlight the structural or functional changes in cortical and spinal cord circuitry, particularly for incomplete SCI, to develop concrete clinical usage.

Clinical Implications
Spontaneous recovery in terms of a significant increase in MEP amplitude and a change in the BBB score after a 3 min iTBS/tsDCS intervention for four weeks confirmed the effects of paired stimulation in an incomplete SCI animal model, which will have clinical applications in the future for incomplete SCI patients.

Selection and Care of Animals
A total of thirty-three (n = 33) adult Sprague Dawley rats obtained from BioLASCO Taiwan, Yilan, Taiwan, with weights ranging from 300 to 450 g were recruited in the study. Out of 33 rats, 9 were excluded because they died due to urine infections. The remaining twenty-four were divided into six different groups. All of the rats were placed in a clean, temperature-and humidity-controlled environment in a well-equipped animal facility as per ethical standards. They were kept in a 12-h light/12-h dark cycle with easy accessibility to pellet food and water ad libitum. The animals were acclimated for 7 to 10 days. The experimental techniques and animal use/methods were approved by the Institutional Animal Care and Use Committee of Taipei Medical University (IACUC-TMU approval no. LAC-2018-0402, 14 January 2019).

SCI Surgery
An incomplete SCI was induced using a commercial compression clip (Micro Vascular Clip, RS-6472; Roboz Surgical Instrument Co., Gaithersburg, MD, USA). Each rat was given an intraperitoneal (i.p.) injection of tiletamine-zolazepam (50 mg/kg, i.p.; zoletil, Vibac, Carros, France) and xylazine (10 mg/kg, rompun, Bayer, Leverkusen, Germany). A dorsal laminectomy between T9 and T11 was performed to expose the T10 spinal cord, and a compression clamp was utilized at the T10 level for 60 s through an applicator (Micro Clip Setting Forceps; RS 6496; Roboz Surgical Store, MD, USA). The whole surgical procedure was carried out in an aseptic environment. The rats were checked daily and given postoperative care. The bladder of each rat was squeezed two times a day until spontaneous voiding occurred, and an intramuscular injection of ampicillin (100 mg/kg, subcutaneously (s.c.), Novopharm, Toronto, Canada) was given to prevent infection for five days [38,40] (Figure 11). proval no. LAC-2018-0402, 14 January 2019).

MEP Assessment
The rat was positioned in a stereotaxic apparatus following i.p. anesthesia with tiletamine-zolazepam (50 mg/kg, i.p.; zoletil) and xylazine (10 mg/kg, rompun) [37]. To record EMG activity, unilateral monopolar uninsulated 27 G stainless steel needle electrodes (Axon Systems, Hauppauge, NY, USA) were administered into the hindlimb muscle belly of the biceps femoris. The ground electrode was placed in the tail, while the reference electrode was in a paw [41]. Using a Biopac MP-36R four-channel device (Biopac System, Goleta, CA, USA), signals were collected. All MEP stimulations were carried out through a MagVenture MagPro and Cool-40 Rat circular coil (Tonica Electronic, Farum, Denmark). To elicit hindlimb MEPs, the circular coil was set so that it was in slight contact with the scalp surface (locator coordinates AP: ±1.0 mm; ML: ±1.25 mm; bregma as the center) [42]. A detailed method was described in our previous publication [38]. In the SCI rat model, a single TMS pulse elicited MEPs to assess cortical excitability during each session. The resting motor threshold (RMT) is the lowest intensity of TMSs necessary to elicit MEPs from the bicep femoris muscle with at least a 20 μV amplitude in five consecutive trials under muscle relaxation brought on by anesthesia [41]. The reported intensity of TMS for RMT was 100% of the machine output percentage, which corresponds to the highest magnetic field strength [41]. The MEP amplitude was measured two times before intervention

MEP Assessment
The rat was positioned in a stereotaxic apparatus following i.p. anesthesia with tiletamine-zolazepam (50 mg/kg, i.p.; zoletil) and xylazine (10 mg/kg, rompun) [37]. To record EMG activity, unilateral monopolar uninsulated 27 G stainless steel needle electrodes (Axon Systems, Hauppauge, NY, USA) were administered into the hindlimb muscle belly of the biceps femoris. The ground electrode was placed in the tail, while the reference electrode was in a paw [41]. Using a Biopac MP-36R four-channel device (Biopac System, Goleta, CA, USA), signals were collected. All MEP stimulations were carried out through a MagVenture MagPro and Cool-40 Rat circular coil (Tonica Electronic, Farum, Denmark). To elicit hindlimb MEPs, the circular coil was set so that it was in slight contact with the scalp surface (locator coordinates AP: ±1.0 mm; ML: ±1.25 mm; bregma as the center) [42]. A detailed method was described in our previous publication [38]. In the SCI rat model, a single TMS pulse elicited MEPs to assess cortical excitability during each session. The resting motor threshold (RMT) is the lowest intensity of TMSs necessary to elicit MEPs from the bicep femoris muscle with at least a 20 µV amplitude in five consecutive trials under muscle relaxation brought on by anesthesia [41]. The reported intensity of TMS for RMT was 100% of the machine output percentage, which corresponds to the highest magnetic field strength [41]. The MEP amplitude was measured two times before intervention stimulation and six times after intervention every five minutes ( Figure 12). The recorded MEP comprised 15 single pulses at 10 s intervals with an RMT of 120%. Peak-to-peak MEP amplitude analysis was performed offline [38]. 0.5 mA on the T10 spinal cord for 20 min. (4) rTMS/ts-iTBS consisted of 20 Hz rTMS on the brain and ts-iTBS on the T10 spinal cord for 20 min. iTBS/tsDCS and iTBS/ts-iTBS were applied for 3 min 3 times a week for 4 weeks, while rTMS/tsDCS and rTMS/ts-iTBS were applied for 20 min once a week for 4 weeks. The rTMS and iTBS stimulation intensity used was 80% of RMT [37]. The sham group protocol was the same as that in the intervention group, but the coil was inverted above the rat's head [43]. Each animal in each group received 20 min of wheel training after every stimulation intervention.

Statistical Analysis
The study data were analyzed in SPSS vers. 17.0 (SPSS, Chicago, IL, USA) with the significance level set to p < 0.050 *. All data are presented in the form of mean ± standard deviation (SD) and paired sample t-test for each time factor and group, and one-way ANOVA for time and group factors was utilized for MEP and BBB scores. The figures were prepared using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Cohen's effect size (d) is reported for the MEP and BBB score change after 4 weeks of the stimulation intervention as: (1) small = 0.20, (2) medium = 0.50, and (3) large = 0.80.

Stimulation Protocol
The stimulation intervention consisted of different combinations of rTMS, iTBS, and tsDCS on the brain and spinal cord in four groups: (1) iTBS/tsDCS comprised iTBS three pulse bursts at 50 Hz repeated at 5 Hz. Each pulse contained a 2 s train, which was repeated every 10 s for 20 repetitions for 600 pulses [26] at 0.05 mA on the brain, while tsDCS was applied at 0.5 mA on the T10 spinal cord for 3 min [17,36]. (2) iTBS/ts-iTBS was applied with the same parameters described earlier for 3 min, with iTBS on the brain and ts-iTBS on the T10 level. (3) rTMS/tsDCS consisted of 20 Hz rTMS on the brain and tsDCS at 0.5 mA on the T10 spinal cord for 20 min. (4) rTMS/ts-iTBS consisted of 20 Hz rTMS on the brain and ts-iTBS on the T10 spinal cord for 20 min. iTBS/tsDCS and iTBS/ts-iTBS were applied for 3 min 3 times a week for 4 weeks, while rTMS/tsDCS and rTMS/ts-iTBS were applied for 20 min once a week for 4 weeks. The rTMS and iTBS stimulation intensity used was 80% of RMT [37]. The sham group protocol was the same as that in the intervention group, but the coil was inverted above the rat's head [43]. Each animal in each group received 20 min of wheel training after every stimulation intervention.

Statistical Analysis
The study data were analyzed in SPSS vers. 17.0 (SPSS, Chicago, IL, USA) with the significance level set to p < 0.050 *. All data are presented in the form of mean ± standard deviation (SD) and paired sample t-test for each time factor and group, and one-way ANOVA for time and group factors was utilized for MEP and BBB scores. The figures were prepared using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Cohen's effect size (d) is reported for the MEP and BBB score change after 4 weeks of the stimulation intervention as: (1) small = 0.20, (2) medium = 0.50, and (3) large = 0.80.

Conclusions
The iTBS/tsDCS and rTMS/ts-iTBS interventions induce neuroplasticity in an incomplete SCI animal model by significantly changing electrophysiological (MEP) and locomotion (BBB) outcomes.