Chronic electrical stimulation of the intact corticospinal system after unilateral injury restores skilled locomotor control and promotes spinal axon outgrowth

Abstract

Injury to the brain or spinal cord usually preserves some corticospinal (CS) connections. These residual circuits sprout spontaneously and in response to activity-based treatments. We hypothesized that augmenting activity in spared CS circuits would restore the skilled motor control lost after injury and augment outgrowth of CS terminations in the spinal cord. After selective injury of one half of the CS tract (CST) in the rat, we applied 10 d of electrical stimulation to the forelimb area of motor cortex of the spared half and tested motor performance for 30 d. Rats with injury and CST stimulation showed substantial improvements in skilled paw placement while walking over a horizontal ladder. By the end of the testing period, the walking errors of the previously impaired forelimb in rats with injury and stimulation returned to baseline, while the errors remained elevated in rats with injury only. Whereas the time to perform the task returned to normal in all animals, the pattern of errors returned to normal only in the stimulated group. Electrical stimulation also caused robust outgrowth of CST axon terminations in the ipsilateral spinal cord, the side of impairment, compared with rats with injury only. The outgrowth was directed to the normal gray matter territory of ipsilateral CST axon terminations. Thus, stimulation of spared CS circuits induced substantial axon outgrowth to the largely denervated side of the spinal cord and restored normal motor control in the previously impaired limbs.

Figures

Figure 1.

Schematic of experimental methods. All rats were subjected to unilateral pyramidotomy (“X”). The day after injury, the intact half of the CS system was electrically stimulated (STIM) daily for 10 d. We measured CS axon length in the impaired half of the spinal cord (dashed box).

Figure 2.

Motor cortex electrical stimulation improves ladder-walking performance. Rats were trained to cross a horizontal ladder with irregularly spaced rungs. Error rates were measured at baseline and every 5 d after injury to day 30. Mean error rates at each testing time are shown ±SEM (except day 5 in the impaired hindlimb of injury-only rats shows only −SEM). A, Impaired forelimb. Error rates were not different at baseline or day 5, but they differed overall (F = 35.89, p < 0.0001, repeated-measures ANOVA). By day 20, the error rates in the two groups were significantly different, and this persisted at days 25 and 30 (p < 0.01, asterisks). For the impaired hindlimb (C), there was no significant improvement with forelimb motor cortex stimulation. The performance in the unimpaired limbs (B, D) did not change with either injury or stimulation.

Figure 3.

Electrical stimulation improves all classes of forelimb paw placement errors. Rats make several types of errors while walking on a horizontal ladder (oversteps, understeps, or missed steps; see Materials and Methods), with the majority being oversteps. We compared the error rates at baseline, days 5–10, days 15–20, and days 25–30. A, Oversteps. The overall difference between the groups was highly significant (F = 12, p = 0.002, repeated-measures ANOVA). Post hoc testing revealed a significant difference at days 25–30 (p < 0.05, asterisk). B, C, The understeps (B) and missed steps (C) did not have significant differences overall with ANOVA testing, due to the small numbers of each of these error types. However, at days 25–30, oversteps were reduced by 53%, understeps by 80%, and misses by 39% compared with injury only, suggesting reduction of all error types with stimulation.

Figure 4.

Rats with injury and stimulation cross the ladder as quickly as rats with injury only. The relationship between speed and accuracy is demonstrated by plotting the forelimb error rate against the time to cross the ladder. The dots plot the error rate and time to cross for a single rat on one day of testing. We compared baseline against the time of improvement, days 20–30. The points are colored according to the legend. The mean time and error values (±SEM) were used to create the plus (+) symbols, which are colored the same as the points used to create these values. Rats with injury only and rats with injury and stimulation did not differ in the time to cross the ladder either at baseline (p = 0.22) or at days 20–30 (p = 0.1).

Figure 5.

M1 stimulation promotes CS axon outgrowth to the spinal cord on the impaired side of the rat (i.e., Fig. 1, dashed box). A, Injury-only rats. B, Injury-and-stimulation rats. The top row contains photomicrographs of C6 spinal cord cut in transverse section and stained for BDA. Inset in A1 shows the approximate location of the pictures. Midline was determined by bisecting the central canal. Magnification, 200×; scale bar, 100 μm. The gray matter ipsilateral to BDA injection is outlined and has sparse BDA-labeled axons in the representative rat with injury only (A1), while the rat with injury and M1 electrical stimulation (B1) has more BDA-labeled axons. The heat maps (middle row) demonstrate axon length as a density map colored from blue (low) to red (high). The color bar demonstrates the axon length in micrometers within each region of interest. A representative rat and the average data for rats (n = 5) in each group are pictured. Stimulation caused abundant local outgrowth, which is densest in medial lamina 7. Scale bar, 500 μm. C, Quantification of axon length within the gray matter ipsilateral to stimulation. Bars plot mean axon length for each group; dots represent individual rats. D, Dorsoventral plot of axon label. The same data used to generate the heat maps were used to create this dorsoventral plot of axon label. Stimulation caused robust outgrowth without spread beyond the dorsoventral distribution of rats with injury only. Scale bar, 500 μm.