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. 2016 Mar:277:46-57.
doi: 10.1016/j.expneurol.2015.12.008. Epub 2015 Dec 18.

Combined motor cortex and spinal cord neuromodulation promotes corticospinal system functional and structural plasticity and motor function after injury

Weiguo Song  1 , Alzahraa Amer  1 , Daniel Ryan  1 , John H Martin  2
Affiliations

Combined motor cortex and spinal cord neuromodulation promotes corticospinal system functional and structural plasticity and motor function after injury

Weiguo Song et al. Exp Neurol. 2016 Mar.

Abstract

An important strategy for promoting voluntary movements after motor system injury is to harness activity-dependent corticospinal tract (CST) plasticity. We combine forelimb motor cortex (M1) activation with co-activation of its cervical spinal targets in rats to promote CST sprouting and skilled limb movement after pyramidal tract lesion (PTX). We used a two-step experimental design in which we first established the optimal combined stimulation protocol in intact rats and then used the optimal protocol in injured animals to promote CST repair and motor recovery. M1 was activated epidurally using an electrical analog of intermittent theta burst stimulation (iTBS). The cervical spinal cord was co-activated by trans-spinal direct current stimulation (tsDCS) that was targeted to the cervical enlargement, simulated from finite element method. In intact rats, forelimb motor evoked potentials (MEPs) were strongly facilitated during iTBS and for 10 min after cessation of stimulation. Cathodal, not anodal, tsDCS alone facilitated MEPs and also produced a facilitatory aftereffect that peaked at 10 min. Combined iTBS and cathodal tsDCS (c-tsDCS) produced further MEP enhancement during stimulation, but without further aftereffect enhancement. Correlations between forelimb M1 local field potentials and forelimb electromyogram (EMG) during locomotion increased after electrical iTBS alone and further increased with combined stimulation (iTBS+c-tsDCS). This optimized combined stimulation was then used to promote function after PTX because it enhanced functional connections between M1 and spinal circuits and greater M1 engagement in muscle contraction than either stimulation alone. Daily application of combined M1 iTBS on the intact side and c-tsDCS after PTX (10 days, 27 min/day) significantly restored skilled movements during horizontal ladder walking. Stimulation produced a 5.4-fold increase in spared ipsilateral CST terminations. Combined neuromodulation achieves optimal motor recovery and substantial CST outgrowth with only 27 min of daily stimulation compared with 6h, as in our prior study, making it a potential therapy for humans with spinal cord injury.

Keywords: Activity-dependent plasticity; Axon sprouting; Brain stimulation; Corticospinal tract; Motor cortex; Pyramidal tract lesion; Theta burst stimulation.

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Figures

Figure 1
Figure 1
A. Schematic of experimental setup. A bipolar epidural cortical stimulating electrode (see inset) was implanted over the fore limb representation of caudal M1. Recording electrodes (inset; single dot) were implanted between the two stimulating electrodes. tsDCS was delivered through patch electrodes with cathode placed dorsally over the C4 to T1 vertebrae and the anode placed over chest. EMG activity was recorded from the ECR muscle contralaterally. The corticospinal system on the left side of the figure (dotted) highlights that after PTX all CST projections from one hemisphere are eliminated. For the affected contralateral side, the only innervation is the spared ipsilateral CST. B. PKCγ staining marks CST axons in the dorsal column. The micrograph is from a representative PTX animal showing unilateral PKCγ loss due to the lesion.
Figure 2
Figure 2
Experimental protocols. A. Protocol 1 was used to test optimal ISIs for producing facilitation. Cathodal (−) or anodal (+) tsDCS, represented as either the downward or upward directed trapezoids, was applied during the period of phasic M1 stimulation. B. Protocol 2 was used to test the effect of patterned M1 electrical stimulation (iTBS) and the effect of concurrent tsDCS. C. Protocol 3 was used in awake animals during locomotion to test the affect of iTBS, with and without c-tsDCS, on cortico-muscular coupling (R-square value of LFP-EMG relationship). This protocol is similar to Protocol 2 except that 5 epochs of iTBSs (3000 pulses total) were delivered within 1630 second. D. The timeline of protocol 4, which was used to test the effect of the combined stimulation (iTBS/c-tsDCS) on motor recovery and CST ipsilateral sprouting in the PTX subgroup. Stimulation for this protocol is the same used in Protocol 3.
Figure 3
Figure 3
MEP facilitation and effect of tsDCS on MEP facilitation. A. Effect of 3-pulse electrical stimulation of M1 on EMG output (Protocol 1). Average MEP response ± SE to the first, second, and third of the 3 stimuli within the burst at different ISIs. Data are normalized to the response to a 3 ms ISI. The response evoked by the 50ms ISI was significantly larger (measured as the peak-to-peak) than other ISIs (12 sessions from 6 rats; *p<0.05, signrank; error bar: mean + S.E). B. Polarity-dependent effect of tsDCS on the average (+S.E) peak to peak amplitude of evoked MEP (Protocol 2). (*p<0.05, signrank) Inset shows a representative average of the MEP amplitude/waveform without tsDCS (black), with anodal tsDCS (green), and c-tsDCS (red). C. To eliminate the contribution of facilitation due to the multipulse M1 stimulation, each average MEP amplitude was divided by the response without tsDCS. (Data in A and B are from 12 sessions from 6 rats; *p<0.05, signrank; error bar: mean + S.E). D. Aftereffect of c-tsDCS and a-tsDCS. Data are presented for anesthetized (darker lines) and awake animals (lighter lines). (anesthetized: n=9 sessions in 6 rats; awake: n= 6 sessions in 2 rats; signrank, * p<0.05).
Figure 4
Figure 4
Facilitation of iTBS is augmented by c-tsDCS (Protocol 2). In this figure black traces, plot lines, and data points are for iTBS only and red, iTBS+c-tsDCS. A. Average of each stimulus presentation during the 2 second iTBS stimulation period (left), aligned with the onset of stimulation. The vertical tic marks below the averages on the left correspond to the triple-pulse stimuli (ISI 50 ms) evoking each set of MEPs. These averages show the changes that occur in MEP amplitude during the 2 second duration of the burst, for the entire period of stimulation (192 sec). The traces to the right are single averages of the 3-pulse stimulation for the entire 192 sec period. B. Scatter plot of MEP integrated rectified EMG response evoked by the 3 stimuli for each burst. We summed the measurements for each stimulus to obtain a combined index of EMG for each triple-pulse stimulus. This graph quantifies the changes in MEP amplitude during the 2 second stimulation period. Linear regression data are indicated in the figure (iTBS only, p<0.001; iTBS+c-tsDCS; p<0.001). C. Amplitude of MEPs to each triple-pulse stimulus during the 192 seconds of iTBS for 4 different rats. The dark gray bars (bottom of part C) represent each 2 second burst (i.e., as in A) and the gaps represent the 8 second interval without stimulation. D. Bar graphs showing changes in MEP rectified and integrated MEP (D1) and the change in slope of linear fit for each 2 second stimulus period shown in part C (D2) (ttest; *p<0.05, ttest). E. Aftereffects of iTBS and modulation by cathodal tsDC under anesthetized condition. Mean change (+S.E) in MEP peak-to-peak amplitude during the 30 minute period following a single epoch of iTBS. Values are relative to MEP amplitude during the pre-stimulation period. (iTBS: n=5 sessions in 4 rats; c-tsDC: n= 9 sessions in 6 rat; iTBS+c-tsDC: n=3 sessions in 3 rats; * p<0.05; signrank).
Figure 5
Figure 5
Regression of local field potential (LFP) in M1 to EMG in ECR muscle during overground locomotion (Protocol 3). A. Effect of iTBS alone and in combination with c-tsDCS on the computed R-squared value of the regression between LFP and EMG. B. R-squared values normalized to the no stimulation condition. C. Correlation between MEP amplitude and R-squared value for each testing day. The correlation coefficient between MEP and R-squared value changes was significantly different among different conditions (bootstrapping, n=1000 times). (*p<0.05, signrank).
Figure 6
Figure 6
Behavioral performance during ladder crossing (Protocol 4). A. Ladder error scores for the impaired forelimb. Unilateral PTX significantly imparied ladder crossing performance of the forelimb contralateral to the lesion, which is ipsilateral to the M1 stimulation electrode. Althougth there was no significant improvement in the ladder score during the ten days of treatment sessions, the combined stimulation significantly improved the performance beginning on day 24 days. B. Ladder error scores of the unimpaired forelimb. No significant differences were found on the unimpared forelimb immediately following the unilateral PTX for both groups (<day 24), although stepping errors were significantly better for the stimulated rats then non-stimulated control rats for the last testing session (day 31). Error bars are mean ± S.E (n=6 for stimulated; n=5 for control). (*p<0.05, ttest).
Figure 7
Figure 7
M1 movement threshold changes after PTX (Protocol 4). A. Movement thresholds (minimal current to produce a movement before each therapeutic stimulation period) for representative stimulated and non-stimulated rats. The movement threshold decreased in the stimulated rat while increase in the non-stimulated control rat. B. Normalized movement thresholds. To correct for variability across animals, the movement threshold was normalizd to the second testing session (evaluation was not made on the first day because of possible residual effects of anesthesia during surgery). The normalized threshold for the stimulated group decreased and increased for the non-stimulated group. There were significant differences beginning on day 4. Error bars are mean ± S.E (n=6 for stimulated; n=3 for control). (*p<0.05, ttest).
Figure 8
Figure 8
Ipsilateral CST axon terminal ourgrowth (Protocol 4). A, B. Regional axon density maps plotted using the same color scales from the group mean data for injury only (A) and injury + stimulation (B) show that the combined stimulation promoted axonal outgrowth. Outgrowth was not uniformaly distributed within the ipsilateral gray matter. Calibration: 500 μm. Color scale bars are in arbitrary units and are the same for A and B. C, E. The dorsoventral (C) and mediolateral (E) projections of the 2-D heatmaps. Band represents ± 95% confidence interval. Calibration: 1×10−’4mm axon/pixel. D. Significance map, based on t-statistic values for each pixel. Calibration: 500 μm. F. Total axon length was significantly higher (5.4-fold) in the stimulated group than the non-stimulated control group. Error bars and line are mean ± S.E (n=6 for stimulated; n=6 for control). (*p<0.05, ttest).
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