1. Assessment of SCI-associated damage by MRI and H&E staining
MRI and H&E staining were performed to determine whether the compression protocol used in this study resulted in damage to the posterior and lateral spinal cord. MRI analysis identified the compression materials at sites that were either directly posterior or lateral to the spinal cord where they elicited P-SCI or L-SCI, respectively (Fig. 2A). H&E-stained sections of spinal cord tissue from rats in the sham group revealed normal histology. By contrast, H&E-stained spinal cord tissue from rats subjected to compression protocols exhibited vesicular degeneration and cavitation within the compressed regions (Fig. 2B).
2. Voluntary exercise has no impact on the recovery of motor function after SCI
Motor recovery was evaluated weekly beginning at 1 wpi using BBB scoring. Rats with SCIs exhibited significant deficits in hindlimb motor function compared with rats that underwent sham procedures at all time-points evaluated in this study (Fig. 2C, two-way ANOVA, p < 0.0001). Voluntary exercise had no impact on motor recovery (Fig. 2C). These results are consistent with the patterns of tissue sparing observed in the spinal cord lesions of rats that did or did not participate in voluntary exercise (see below). Although the sizes of the spared epicenters of these lesions were significantly different in rats with P-SCI versus L-SCI, no differences in the recovery of locomotor function were observed.
3. Voluntary exercise suppresses mechanical hypersensitivity that develops in response to SCI
To determine whether rats develop NP after compressive SCI and whether voluntary exercise results in reduced pain behaviors, rats were tested for mechanical and thermal sensitivity both before the injury and weekly thereafter.
Our findings revealed that rats with compressive SCIs developed both mechanical hypersensitivity and hyposensitivity in the contralateral hind paw at 1 wpi (Fig. 3A and 3B). Rats with P-SCI developed mechanical hypersensitivity and hyposensitivity in the ipsilateral hind paw at 1 wpi, while those with L-SCI developed these responses at 2 wpi.
Stimulation of the contralateral hind paw using 2g and 4g von Frey filaments revealed that rats with P-SCI or L-SCI exhibited a significantly higher overall response rate compared to their sham-treated counterparts (two-way ANOVA, p < 0.0001). No differences were detected between the responses of rats with P-SCI versus L-SCI. However, the response rate of rats with P-SCI or L-SCI that participated in voluntary exercise was lower than that observed in rats with SCI that did not exercise from 2 through 4 wpi (Fig. 3A and 3B, oooop < 0.0001, vvvp < 0.001, vvvvp < 0.0001). ipsilateral hind paw revealed that rats with L-SCI required an additional week to reach the same response rate to 2g and 4g von Frey filaments when compared to the response rate of rats with P-SCI rats (Fig. 3D and 3E). Rats that participated in voluntary exercise after P-SCI exhibited lower levels of mechanical hypersensitivity of the ipsilateral hind paw. Voluntary exercise also suppressed the mechanical hypersensitivity that developed in rats with L-SCI.
Stimulation of the contralateral hind paw of rats with P-SCI or L-SCI using a 15g von Frey filament revealed an overall significantly lower response rate than that observed in sham-treated animals (two-way ANOVA, ****p < 0.0001, ####p < 0.0001). Voluntary exercise had no impact on the response rate to this larger diameter filament in either of these groups (Fig. 3C). Testing of the ipsilateral hind paw revealed that rats with L-SCI had a lower response rate to 15g von Frey filament compared with sham-treated animals at 2 wpi, while rats with P-SCI rats exhibited a reduced response rate after 1 wpi (Fig. 3D).
4. Mechanical hypersensitivity that develops in response to SCI is associated with aversive supraspinal responses
PEAP testing was performed at the final time point (4 wpi) to evaluate the supraspinal processing of nociception. Briefly, rats that are allowed to move freely between dark and light chambers will naturally spend more time in the dark. However, when the dark chamber is paired with painful stimuli (von Frey 2g filament), the rats may ultimately choose to spend more time on the light side. Analysis of the time spent on the dark versus light side revealed that rats with P-SCI and L-SCI spent less time in the dark chamber at 4 wpi (Fig. 3G). However, after three weeks of voluntary exercise, all rats with SCIs spent less time on the dark side compared with those that did not participate in voluntary exercise (Fig. 3G).
5. Voluntary exercise suppresses thermal hypersensitivity that develops in response to SCI
The Plantar Test (Hargreaves method) was used to examine the thermal sensitivity of the rat hind paws. Significant differences were detected between various groups (two-way ANOVA: ****p < 0.0001, ####p < 0.0001). Rats with P-SCI and L-SCI developed significant thermal hypersensitivity at 1 wpi when compared with their baseline values and to the responses of sham-treated rats. Thermal sensitivity persisted through the end of the experiment (4 wpi). All rats with SCI exhibited reductions in the withdrawal latency at the contralateral hind paw of ~8–9 s from baseline values at 1 wpi. By contrast, the withdrawal latency decreased only by ~3–4 s in the ipsilateral hind paw of rats with L-SCI. Interestingly, voluntary exercise had a positive impact on the thermal hypersensitivity that developed in response to SCI (Fig. 4). The withdrawal latency increased by ~3–4 s in the contralateral hind paws of rats with SCI that participated in voluntary exercise compared to the sham-treated group and rats with SCI that did not participate in voluntary exercise. However, none of the response rates of rats with SCI ultimately returned to baseline levels (Fig. 4A). Voluntary exercise increased the withdrawal latency in the ipsilateral hind paw by 3–4 s in rats with P-SCI rats and suppressed the more severe thermal hypersensitivity exhibited by L-SCI rats (Fig. 4B).
6. Voluntary exercise resulted in improved somatosensory evoked potentials in rats with SCI
Somatosensory evoked potentials (SEPs) were examined in order to evaluate the functionality of ascending sensory and proprioception pathways after SCI (Fig. 5). As shown in Fig. 5A, categories of SEP responses that have been modeled in rats include N and P points, which refer to the upward (N; negative) and downward (P; positive) peaks of the far-field cortical potential. Onset latency was measured as the time delay before N and the amplitude was measured as the voltage differences between N and P. Rats with L-SCI exhibited significant decreases in amplitude and prolonged latency at the side that was ipsilateral to the compression compared to the sham-treated controls (two-way ANOVA, **p < 0.01, ****p < 0.0001). Greater deterioration of SEPs was observed on the ipsilateral versus the contralateral side in response to the compression lesion in rats with L-SCI. By contrast, similar deterioration was observed on both sides of the lesion in rats with P-SCI.
Rats with L-SCI showed no significant differences in onset latency on the side that was contralateral to the lesion when compared to the responses of sham rats. Participation in voluntary exercise resulted in significant improvements in SEP amplitudes on both sides of the lesion in rats with L-SCI (two-way ANOVA, #p < 0.05, ####p < 0.0001). After voluntary exercise, the differences between the ipsilateral and contralateral SEP amplitudes were not significant, although the SEP latency detected on the side that was ipsilateral to the lesion remained significantly longer than responses detected on the contralateral side.
Rats with P-SCI exhibited similar amplitude decreases and prolongation of onset latency on both sides of the compression lesion. Participation in voluntary exercise resulted in improvements in the amplitude but had minimal impact on SEP latency of rats with P-SCI.
7. Voluntary exercise does not result in modification of the structural changes that result from a compressive SCI
To exclude the possibility that differences in lesion size might influence the changes in pain sensitivity secondary to SCI and examine the impact of voluntary exercise on processes that promote neuroprotection, white matter sparing was evaluated in Eriochrome cyanine R (EC)-stained coronal sections of the rat spinal cord (Fig. 6A) The extent of white matter sparing was quantified at the lesion epicenter (Fig. 6B). We found no significant differences in white matter sparing at the lesion epicenters when comparing spinal cord tissue from rats with SCI (either P-SCI or L-SCI) that have or have not participated in voluntary exercise (Fig. 6B). Interestingly, spinal cord tissues from injured rats exhibit a comparatively large percentage of white matter and complete loss of gray matter at the lesion epicenters compared to sham-treated controls (one-way ANOVA, ***p < 0.001). Tissues from rats with L-SCI exhibit a smaller fraction of white matter loss at the lesion epicenter compared with tissues from rats with P-SCI (one-way ANOVA, ***p < 0.001).
8. SCI-induced increases in calcitonin gene-related peptide (CGRP) expression are suppressed by voluntary exercise
Calcitonin gene-related peptide (CGRP) is a neuropeptide that may be expressed in response to peptidergic C and Aδ innervation and that has been detected at high levels in the skin, blood, cerebrospinal fluid, and spinal cord tissue in association with a variety of chronic pain conditions. Therefore, we quantified CGRP expression bilaterally in lumbar laminae I and II by using qPCR and IHC (Fig.7).
We detected a significant increase in CGRP expression was significantly increased in laminae I and II of the lumbar spinal cord in rats with P-SCI or L-SCI. Both groups of rats with SCI exhibited significant 1.7-fold increases in CGRP expression compared to sham-treated controls (one-way ANOVA, ***p < 0.001). No significant differences were detected between rats with P-SCI vs. L-SCI. Interestingly, voluntary exercise resulted in significant reductions in CGRP expression in lumbar laminae I and II in rats with SCI. By contrast, no differences in CGRP expression were detected in sham vs. sham+V controls.
9. SCI-induced increases in microglial activation are suppressed by voluntary exercise
CGRP is a mediator of microglial activation and may promote nociceptive signaling in the dorsal horn . Based on our detection of CGRP overexpression in the lumbar spinal cords of rats with P-SCI and L-SCI, we proceeded to evaluate microglial activation in the lumbar dorsal horn in experiments designed to detect and quantify expression of ionized calcium-binding adapter molecule 1 (Iba-1).
We found significantly increased expression of Iba-1 bilaterally in the lumbar spinal cord of rats with P-SCI compared to sham-treated controls (Fig. 8). Participation in voluntary exercise suppressed Iba-1 overexpression in these rats (one-way ANOVA, ***p < 0.001). By contrast, Iba-1 expression was significantly increased in the ipsilateral lumbar dorsal horn, but not in the contralateral lumbar dorsal horn of rats with L-SCI. Voluntary exercise also suppressed Iba-1 overexpression in the ipsilateral lumbar dorsal horn in rats with L-SCI. Similar to our findings for CGRP expression, we detected no statistically significant differences in Iba-1 expression in the lumbar spinal cord when comparing results from the sham vs. the sham+V groups..