Transcranial direct current stimulation in individuals with spinal cord injury: Assessment of autonomic nervous system activity

Abstract

Background: We hypothesized in this study that transcranial direct current stimulation (tDCS) of primary motor cortex could exert top-down modulation over subcortical systems associated with autonomic control and thus be useful to revert some of the dysfunctional changes found in the autonomic nervous system (ANS) of subjects with spinal cord injuries (SCI).

Objective: To explore the acute effect of tDCS on ANS indexed by Heart Rate Variability (HRV) in individuals with SCI and analyze whether this effect depends on the gender, degree, level and time of injury.

Methods: In this randomized, placebo-controlled, crossover, double-blinded study, 18 adults with SCI (32.9±7.9 years old) were included; the intervention consisted of a single 12-minute session of active tDCS (anodal, 2 mA) and a control session of sham tDCS applied over Cz (bihemispheric motor cortex). HRV was calculated using spectral analysis. Low-frequency (LF), high-frequency (HF), and LF/HF ratio variables were evaluated before, during, and post tDCS.

Results: A two-way repeated measures ANOVA showed that after active (anodal) stimulation, LF/HF ratio was significantly increased (P = 0.013). There was a trend for an interaction between time and stimulation for both LF and HF (P = 0.052). Paired exploratory t-tests reported effects on the difference of time [post–pre] between stimulation conditions for LF (P = 0.052), HF (P = 0.052) and LF/HF (P = 0.003).

Conclusion: Anodal tDCS of the motor cortex modulated ANS activity in individuals with SCI independent of gender, type and time of lesion. These changes were in the direction of normalization of ANS parameters, thus confirming our initial hypothesis that an enhancement of cortical excitability by tDCS could at least partially restore some of the dysfunctional activity in the ANS system of subjects with SCI.

Keywords

Transcranial direct current stimulation autonomic nervous system spinal cord injury heart rate variability

1 Introduction

Spinal cord injury patients usually present alterations of the autonomic nervous system control. This is believed to be due to a lack of afference from centers that originate in the heart as well as peripheral blood vessels and other target organs (Hou & Rabchevsky, 2014). The lack of afference results in changes in cortical and subcortical regulation that eventually leads to a down regulation of the sympathetic and parasympathetic nervous system (Fregni et al., 2006; Hou & Rabchevsky, 2014; Schestatsky, Simis, Freeman, Pascual-Leone, & Fregni, 2013; Yoon et al., 2013). In fact, catecholamine concentrations decrease, leading to less daily variations in heart rate and a decrease in the LF component (Bunten, Warner, Brunnemann, & Segal, 1998).

One of the methods to overcome the ANS dysfunction is through activation of cortical and subcortical centers via top-down regulation. In recent years, techniques of non-invasive brain stimulation, such as transcranial Direct Current Stimulation (tDCS), have been used in different conditions including chronic pain, stroke, spasticity and physical exercise with the goal to restore dysfunctional mechanisms by increasing cortical reorganization. TDCS has been frequently applied over the primary motor cortex (M1), a major target area that can influence autonomic control due to its functional connections with the ventro-lateral medulla. This brainstem region is critically involved in the control of blood pressure and the regulation of plasma catecholamine levels (Viltart et al., 2003); therefore, it can also modulate ANS activity (Clancy, Johnson, Raw, Deuchars, & Deuchars, 2014; Montenegro et al., 2011; Okano et al., 2013; Schestatsky et al., 2013).

In this regard, M1 stimulation may be a useful strategy to restore ANS activity in cases of nervous system lesions. In fact this study may also confirm the notion that M1 is a major target to restore neural activity as it has been shown for chronic neuropathic pain (Bolognini et al., 2015; Marques Filho et al., 2016; Moreno-Duarte et al., 2014; Soler et al., 2010). Additionally, tDCS of M1 has also been effective to increase the reorganization of pain-related neural circuits and thus decreasing pain in those with spinal cord injury (Fregni et al., 2006; Fregni et al., 2006). Furthermore, a recent PET study has shown that tDCS over M1 leads to an increase in metabolic activity in the medulla; thus confirming that tDCS can indeed induce distant changes and be an important tool to induce neural activity reorganization (Yoon et al., 2013).

Therefore, we conducted a double-blind, crossover, randomized, sham controlled study to test the effects of one session of anodal tDCS over bihemispheric M1 to evaluate changes in HRV and its components (LH, HF and LH/FH ratio). Our hypothesis is that motor cortex stimulation will be able to restore defective pathways (deafferentation of the sympathetic sensorial pathways) via top-down modulation, thus allowing the modulation of the autonomic nervous system in SCI patients.

2 Material and methods

2.1 Study design

The clinical trial consisted of a randomized, sham-controlled, crossover, double-blinded study.

2.2 Subjects

All participants that fulfilled the following inclusion/exclusion criteria were included in the study: 1) Spinal cord injury (complete and incomplete); 2) Lesion with at least 12 months since onset; 3) Age between 20 and 50 years; 4) Absence of brain injury; 5) Absence of diabetes; 6) Absence of heart disease or a high risk for cardiovascular disease; and 7) Absence of any metal objects in the head.

The sample was composed of 18 adults (16 males; 32.9±7.9 years old and 62.0±14.0 kg). All subjects presented chronic SCI (16.5±11.8 years since injury). Among them, seven had incomplete injury at the T12-L1 level, eight showed complete injury at the T11-T12 level, and three presented a complete injury at the T4-T6 level. All individuals were right-handed as determined by the Edinburgh Handedness Inventory (Oldfield, 1971). The baseline characteristics of the subjects are shown in Table 1.

In addition, each volunteer was instructed to avoid eating three hours prior to the beginning of the test, forgo performing vigorous physical activity, abstain from consuming alcohol and food containing caffeine, drugs or nicotine, and refrain from engaging in mental activities that may cause increased levels of stress during a period of 24 hours before the intervention session. The intervention visit was conducted in a facility that had a constant air temperature, humidity and acoustic insulation.

This study was approved by ethics in Research Committee of the Federal University of Rio Grande do Norte, Brazil (File Number 750.887). All participants were briefed on the procedures and risks before signing the corresponding informed consent.

2.3 Experimental procedures

During the first visit, each participant was assessed with a demographics questionnaire that included questions regarding the time, type, and degree of SCI injury. Additionally, the subject’s physical measurements and a personal history pertaining to the presence of chronic degenerative diseases were collected. Subjects were then randomized to either sham or active tDCS. In order to avoid carry-over effects, there was a wash-out period of 72 hours between each of the 2 tDCS stimulation sessions (Nitsche & Paulus, 2001). Moreover, the stimulation periods took place at the same time of the day for each subject (anodal and sham tDCS) to avoid the effects of the circadian cycle.

Each session lasted 32 minutes. The session was divided in three phases: 1) A ten minute pre-stimulation resting period; 2) Twelve minutes of either sham or active tDCS; 3) A ten minute post-stimulation resting period. During the final five minutes of both resting periods and the entire stimulation session, the subject’s HR was being recorded.

2.4 tDCS procedures

The electric current was applied through a portable tDCS device using an anode with an area of 36 cm² (9×4) and a cathode with an area of 35 cm² (7×5).

The international EEG 10–20 system was utilized to define the electrodes positioning in the active and sham tDCS stimulation. The center of the anodal electrode was placed over Cz (central zero) area, therefore 4.5 cm of the sponge was located over the left hemisphere and the remaining 4.5 cm over the right hemisphere. This montage allowed for bilateral stimulation of M1. The reference electrode was placed over the occipital protuberance in order to avoid inhibitory effects on the supra-orbital contralateral area (Fp2) (Clancy et al., 2014) (Fig. 1), which could potentially modify the results of the HRV. A constant current of 2 mA was applied for 12 minutes; the ramping up and ramping down procedures were adopted for the first 30 s (gradual increase of the applied current until reaching 2 mA) and the last 30 s (gradual reduction of the applied current until reaching 0 mA) respectively, for a total time of 12 min.

For the sham stimulation, the electrodes were placed at the same positions as for the active montage. However, the tDCS device was turned off after the ramp procedure (Gandiga, Hummel, & Cohen, 2006).

2.5 LF, HF, and LF/HF ratio

The HRV was recorded by a HR monitor (Polar^® RS800CX, Polar Electro Oy Inc., Kempele, Finland) at a sampling frequency of 1000 Hz. The use of this HR monitor for measurements of the HRV at rest was validated in previous studies (Gamelin, Berthoin, & Bosquet, 2006; Gamelin, Baquet, Berthoin, & Bosquet, 2008; Porto & Junqueira, 2009; Vanderlei, Silva, Pastre, Azevedo, & Godoy, 2008); additionally, it is both inexpensive and a non-invasive technique that allows the assessor to collect multiple measurements, while simultaneously monitoring the patient.

The result of the RR interval was uploaded to a computer with the Polar ProTrainer 5 software, which subsequently filtered out artifacts. In the frequency domain, spectral power was calculated to obtain: (1) the low-frequency band (LF, 0.04–0.15 Hz); (2) the high-frequency band (HF, 0.15–0.40 Hz); (3) and the LF/HF ratio.

The LF/HF ratio, HF, and LF were calculated by the Kubios HRV software using the final 5 minutes of both 10 minute resting periods (before and after tDCS) and the entire 12 minutes of tDCS. The LF and HF components were also presented in normalized units (LFnu and HFnu). Only frequency domains were used, since it has been shown that these components would be more accurate at evaluating HRV at rest in short-term measurements than the alternatives (Hartikainen, Tahvanainen, & Kuusela, 1998).

2.6 Statistical analysis

The normality and homogeneity of data variance was tested by the Shapiro-Wilk and Levene tests respectively. Data for LF/HF ratio was logarithmically transformed due to the high variability among individuals. The data was presented as mean±standard deviation. The effect size of the means was calculated by the Cohen’s d. The level of significance was set at P < 0.05. A two-way ANOVA for repeated measures was performed using stimulation condition comparisons (active and sham tDCS) and time (pre, during, and post OR pre and post) as main factors. Based on this test, we analyzed the effects of these factors on HRV by confirming if the main effects of time and condition and their interaction were significant changing the HRV. Post hoc Bonferroni was applied to pairwise comparison in case of a significant ANOVA. Post-hoc analyses were conducted using a paired t-test of difference between post and pre (Δ= post – pre) between conditions (anodal vs. sham tDCS). The hypothesis of sphericity was verified by Mauchly test and, if violated, the degrees of freedom were fixed by the Greenhouse-Geisser estimates. The effect size of the variance was calculated using the partial eta squared (η²_p).

Moreover, to explore our findings we analyzed the effects of injury type by utilizing a univariate ANOVA with stimulation condition (active and sham tDCS) and type of injury (incomplete injury T12-L1, complete injury T11-T12 and complete injury T4-T6) as fixed factors, and difference of time (Δ= post – pre) as the dependent variable. Similarly, in order to analyze the effects of time of injury an ANOVA model was applied using stimulation condition comparisons (active and sham tDCS) and time of injury (≤2 years, >2 years to ≤ 20 years, and >20 years) as between-subjects factors, and difference of time (Δ= post – pre) as a within-subjects factor.

To analyze the effects of gender, an ANOVA model was applied using stimulation condition comparisons (active and sham tDCS) and sex (male and woman) as between-subjects factors, and difference of time (Δ= post – pre) as a within-subjects factor. Statistical procedures were performed using the SPSS for Win/v.22.0 (Statistical Package for Social Sciences, Chicago, IL, USA).

3 Results

No adverse events were reported by participants during the application of either active or sham tDCS. Nineteen individuals were initially enrolled in the study. One individual dropped out from the study after the first session with sham tDCS (Fig. 2) and was excluded from the final analysis.

Two-way repeated measures ANOVA showed significant interaction effects between time (pre and post) and stimulation condition in some HRV measures. Regarding the LF/HF ratio, there was a significant difference in the time×condition interaction (F(1, 17) = 7.68, P = 0.013, η²_p = 0.311). For the LF, we observed a trend towards significance in the time×condition interaction (F (1, 17) = 4.39, P = 0.052, η²_p = 0.205). Regarding the HF, we observed a trend towards significance in the time×condition interaction (F (1, 17) = 4.39, P = 0.052, η²_p = 0.205) (Table 2).

When conducting a pairwise comparison (before minus after and comparing active vs. sham condition) for all patients (n = 18), we detected significant changes in HRV measures (Fig. 3). For the LF, we observed a trend towards significance (t(17) = –2.09, P = 0.052, d = –0.70), showing that the anodal tDCS session resulted in an increase of the LF heart rate. As expected, the HF results trended towards significance (t(17) = 2.09, P = 0.052, d = 0.70), which supported that anodal tDCS could decrease the HF. Finally, the LF/HF ratio significantly increased as a result of a single anodal tDCS session (t(17) = –3.46, P = 0.003, d = –0.69) (Fig. 3).

In the exploratory analysis we evaluated the effects of type and time since injury in all the HRV components (LF, HF and LF/HF ratio). To further clarify our findings, we analyzed the effects of injury type in an ANOVA model using stimulation condition comparisons, type of injury (incomplete injury T12-L1; complete injury T11-T12; and complete injury T4-T6), and difference of time (Δ= post – pre) as variables. For the LF, we observed no significant difference in the condition × type of lesion interaction (F(2, 30) = 1.97, P = 0.157, η²_p = 0.116). Similarly, there was no significant difference in the condition × type of lesion interaction for HF (F(2, 30) = 1.97, P = 0.157, η²_p = 0.116). Finally, there was no significant difference in the condition × type of lesion interaction for the LF/HF ratio (F(2, 30) = 0.22, P = 0.807, η²_p = 0.014)(Table 2).

Likewise, to analyze the effects of time of injury we used the same model. For the LF, we observed no significant difference in the condition × time of lesion interaction (F(2, 30) = 0.84, P = 0.444, η²_p = 0.053). Regarding the HF, there was no significant difference in the condition × time of lesion interaction (F(2, 30) = 0.84, P = 0.444, η²_p = 0.053). Concerning the LF/HF ratio, there was no significant difference in the condition × time of lesion interaction (F(2, 30) = 0.27, P = 0.762, η²_p = 0.018).

We analyzed the effects of gender in an ANOVA model using stimulation condition comparisons, gender (male vs. woman), and difference of time (Δ= post – pre) as variables. For the LF, we observed no significant difference in the condition × gender interaction (F(1, 32) = 1.55, P = 0.223, η²_p = 0.046). Regarding the HF, there was no significant difference in the condition × sex interaction (F(1, 32) = 1.55, P = 0.223, η²_p = 0.046). Concerning the LF/HF ratio, there was no significant difference in the condition × sex interaction (F(1, 32) = 1.47, P = 0.234, η²_p = 0.044).

4 Discussion

In this double-blind, crossover, randomized, sham controlled study we showed that anodal tDCS (12 minutes) was able to significantly modulate the HRV components. Our data is one of the first evidence that shows that a single session of tDCS can affect autonomic activity in SCI patients as indexed by changes in HRV. Moreover, our analysis showed that the response was not dependent on the gender, degree, level and time of injury; though these results should be considered as exploratory.

4.1 tDCS effects on ANS

Recently, most of the results regarding the effects of tDCS on autonomic nervous control evaluated ANS aspects as secondary outcomes/components while primarily evaluating safety measurements (Schestatsky et al., 2013). These studies support the hypothesis that tDCS is able to exert some effects over the cardiovascular system through mechanisms of top-down modulation in healthy subjects; however, these findings are dependent on the stimulation time and montage applied (Clancy et al., 2014; Montenegro et al., 2011; Vandermeeren, Jamart, & Ossemann, 2010).

TDCS is a reliable technique proven to be efficacious in increasing excitability in cortical and subcortical levels (Brunoni et al., 2013; Callan, Falcone, Wada, & Parasuraman, 2016; Flood, Waddington, & Cathcart, 2016; Krone et al., 2016). Given these effects, it has been hypothesized that tDCS can therefore be useful to induce neural reorganization especially in cases of neural deafferentation such as it is seen in spinal cord injury. As aforementioned, there is relatively extensive literature showing tDCS effects in SCI (Fregni, Boggio et al., 2006; Jankowska, Kaczmarek, Bolzoni, & Hammar, 2016; Murray et al., 2015; Ngernyam et al., 2015; Silva, 2015; Soler et al., 2010; Yamaguchi et al., 2016).

Given our primary hypothesis that tDCS is able to at least partially reestablish neural reorganization, it is conceivable that tDCS effects on ANS would be larger in a dysfunctional system such as seen in SCI.

4.2 Motor cortex stimulation and ANS control

To date, the role of the LF component, HF component and the LF/HF ratio indexing the sympathetic (SNS) and parasympathetic nervous system (PSNS) activity are not fully understood, as previous studies have yielded mixed results (Montano et al., 1994; Mukai & Hayano, 1995; Reyes del Paso, Langewitz, Mulder, Roon, & Duschek, 2013). However numerous clinical trials in healthy subjects reported that an increase in the LF component reflects the activity of the SNS (Montano et al., 1994; Mukai & Hayano, 1995; Rimoldi et al., 1992). Thus some aspects of ANS activity can be useful to understand modulation of ANS system and test our hypothesis of cortical modulation of M1 to restore neural reorganization and affect ANS activity.

Given that the lack of afference present in patients with spinal cord injury is believed to be a crucial factor in the inadequate control of the ANS, tDCS of M1 could restore this imbalance and thus affect LF/HF ratio as well as in the LF and HF components. It is therefore important to discuss the neurocircuits that are potentially involved in the restoration of these deafferented pathways. We believe that three main circuits are involved: (1) Cortico-Thalamic and (2) Thalamo-hypothalamic (3) Thalamus/hypothalamus-Brainstem-ANS. For the Cortico-Thalamic pathway, current evidence from several pain studies reported the presence of neuronal projections from the sensory motor cortex to the Paraventricular Nucleus (PVT) and the Mediodorsal Nucleus (MD) in the thalamus (Cortico-thalamic control) (Cummiford et al., 2016; Fang, Stepniewska, & Kaas, 2006; Polanía, Paulus, & Nitsche, 2012). Indeed tDCS over M1 has shown to change thalamic activity as shown by neuroimaging studies (Goto et al., 2008; Peyron et al., 1999; Polanía et al., 2012) and evidence by EEG studies (Alshelh et al., 2016; Fuggetta & Noh, 2013; Jensen et al., 2013; Jensen et al., 2014; Moreno-Duarte et al., 2014). Regarding the Thalamo-Hypothalamic circuit, numerous studies discussed the presence of projections that travel from thalamic regions (PVT and MD) to the paraventricular nucleus of the hypothalamus (PVN) (Groenewegen, 1988; Moga, Weis, & Moore, 1995; Ray & Price, 1992; Xu, Zheng, & Patel, 2013). For the Thalamus/hypothalamus-brainstem network, literature findings showed neural projections from either the thalamic nucleus (PVT and MD) as well as hypothalamic nucleus (PVN) to areas in the brainstem including the Nucleus Tractus Solitarius (NTS), Dorsal Motor Nucleus of the Vagus (DMV), Rostral Ventrolateral Medulla (RVLM); as well as the Rostral Ventral Medulla (RVM), and Raphei Nucleus (thalamo/hypothalamo-brainstem control) (Chen & Su, 1990; Cornwall & Phillipson, 1988; Krout & Loewy, 2000; Otake, Reis, & Ruggiero, 1994; Ray & Price, 1992; Xu et al., 2013). Projections from those zones in the brainstem are connected with the intermedio-lateral column in the spinal cord that will eventually transmit the stimuli to the corresponding neurons of either the sympathetic or parasympathetic nervous system (Brainstem-ANS control) (Accorsi-Mendonca, Castania, Bonagamba, Machado, & Leao, 2011; Buijs, 2012; Clark, Hasser, Kunze, Katz, & Kline, 2011; Sclocco et al., 2016; Sequeira, Viltart, Ba-M’Hamed, & Poulain, 2000; Wehrwein, Orer, & Barman, 2016). In this regard, the primary motor cortex may be an effective entry port to modify ANS activity restoring the deafferentation and correcting the dysfunctional control of the ANS system

4.3 Neural Characteristics influencing ANS control and tDCS effects

In our exploratory analysis there were no differences regarding degree, level and time of injury. On the other hand, previous reports showed that time of injury can result in different phenotypes and responses to treatment in SCI patients. In fact, studies in acute and chronic SCI have shown differences in gray vs. white matter relationship, atrophy levels and blood flow in different brain areas (Çermik et al., 2006; Freund et al., 2011; J-M Hou et al., 2014).

Therefore, different response may be expected between participants when comparing time since injury; however, our results reported no significant effects on this regard. In this regards, it is important to point out that current evidence shows that the longer the time since injury the greater the changes generated over the motor cortex and spinal cord architecture, which could potentially modify the responses of the ANS when indexed by changes in HRV after the stimulation (Freund et al., 2013). Nevertheless, in our sample, time since injury of the enrolled group of participants was very heterogeneous, ranging from 1.5 to 44.9 years post lesion and did not show statistically significant difference; therefore, it is important to consider potential alternative explanations here such as lack of power or sample characteristics to explain this negative result in this exploratory analysis.

Another factor that may be relevant is the level of injury. Lower concentrations of catecholamines have been reported in individuals with high paraplegia and tetraplegia, whereas higher levels were seen in healthy controls and individuals with low paraplegia which showed higher levels of adrenaline (Schmid et al., 1998; Schmidt-TrucksaÉû, Lehmann, Berg, & Keul, 2000). Notably, these characteristics suggest that different degrees of injury will potentially result in different changes and alterations of the central nervous system, leading to diverse responses to tDCS. Nevertheless, our analysis showed that this factor was not significant in order to generate changes on tDCS response despite the fact of having injuries at different levels of the spinal cord.

An additional relevant factor is the different levels of GABA control activity among individuals with SCI. According to Nardone et al. (2015), SCI individuals with normal central motor conduction showed increased I-wave facilitation; and SCI individuals with abnormal central motor conduction showed decreased I-wave facilitation, when compared to a control group (Nardone et al., 2015). In addition, I-wave facilitation is a mechanism that works under control of GABA related inhibition and occurs at the level of the motor cortex. These alterations can be related with differences in motor cortical excitability in SCI individuals; however, there is no sufficient evidence to determine if those changes represent an adaptive or a maladaptive plastic response (Nardone et al., 2015). Consequently, these modifications in the brain and neural structures as well as abnormal GABAergic activity observed in subjects with chronic SCI could potentially be correlated with the increase of sympathetic response indexed by changes in HRV after the stimulation with tDCS (Clancy et al., 2014).

4.4 Future directions and conclusions

Our preliminary results revealed important points regarding the use of tDCS in individuals with SCI. Moreover, it provides additional knowledge to further understand aspects underlying tDCS mechanism in ANS modulation in SCI population (in which the lack of afference from peripheral centers may induce further dysfunction of ANS control).

In our study, we explored the acute effects exerted by tDCS to modulate HRV of SCI individuals during rest condition, testing the hypothesis that M1 modulation would exert a top-down control in a similar fashion as seen for chronic neuropathic pain.

In fact the results found in our study support the hypothesis that tDCS may exert significant changes in the ANS system and thus potentially induce reorganization in the some of the circuits affected by spinal cord injury. Although we have not tested the direct mechanisms of these effects, given the extensive literature on M1 tDCS, it is conceivable that tDCS influenced cortico-subcortical circuits.

In conclusion, this study provides evidence that a single session of anodal tDCS applied bilaterally over the motor cortex significantly modulates the ANS balance towards sympathetic dominance in adult participants with SCI. The response is not dependent of the degree, level, or time of injury nor was it dependent on gender. This is one of the first direct evidence regarding the effects of tDCS over ANS activity; therefore, revealing important information for future research applications with tDCS.

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