Forced exercise as a rehabilitation strategy after unilateral cervical spinal cord contusion injury

Abstract

Evaluation of locomotor training after spinal cord injury (SCI) has primarily focused on hind limb recovery, with evidence of functional and molecular changes in response to exercise. Since trauma at a cervical (C) level is common in human SCI, we used a unilateral C4 contusion injury model in rats to determine whether forced exercise (Ex) would affect spinal cord biochemistry, anatomy, and recovery of fore and hind limb function. SCI was created with the Infinite Horizon spinal cord impactor device at C4 with a force of 200 Kdyne and a mean displacement of 1600-1800 microm in adult female Sprague-Dawley rats that had been acclimated to a motorized exercise wheel apparatus. Five days post-operatively, the treated group began Ex on the wheel for 20 min per day, 5 days per week for 8 weeks. Wheel speed was increased daily according to the abilities of each animal up to 14 m/min. Control rats were handled daily but were not exposed to Ex. In one set of animals experiencing 5 days of Ex, there was a moderate increase in brain-derived neurotrophic factor (BDNF) and heat shock protein-27 (HSP-27) levels in the lesion epicenter and surrounding tissue. Long-term (8 weeks) survival groups were exposed to weekly behavioral tests to assess qualitative aspects of fore limb and hind limb locomotion (fore limb scale, FLS and BBB [Basso, Beattie, and Bresnahan locomotor rating scale]), as well as sensorimotor (grid) and motor (grip) skills. Biweekly assessment of performance during wheel walking examined gross and fine motor skills. The FLS indicated a significant benefit of Ex during weeks 2-4. The BBB test showed no change with Ex at the end of the 8-week period, however hind limb grid performance was improved during weeks 2-4. Lesion size was not affected by Ex, but the presence of phagocytic and reactive glial cells was reduced with Ex as an intervention. These results suggest that Ex alone can influence the evolution of the injury and transiently improve fore and hind limb function during weeks 2-4 following a cervical SCI.

FIG. 1.

Western blot analysis of spinal cord tissue extracts following contusion injury and 5 days of forced exercise (Ex). (A) Representative images of nitrocellulose replicas probed with antibodies to heat shock protein–70 (HSP-70), HSP-27, and brain-derived neurotrophic factor (BDNF) from two groups of animals, spinal cord injury (SCI) alone (animals 1 and 2), and SCI + Ex (animals 1 and 2). (B) Quantitative analysis of immunoblots. The optical densities of immunoreactive bands were normalized to actin, and expressed as a ratio to the animal group with injury only (arbitrary units [AU]). Each bar represents combined values from three areas: epicenter (EPI), caudal (C) and rostral (R) to the injury for each animal (mean ± SD).

FIG. 2.

Locomotor assessment of fore limbs and hind limbs after forced exercise (Ex). (A) Fore limb locomotor score (FLS) over the 8 week period. Both ipsilateral (spinal cord injury [SCI] alone, square; SCI + Ex, diamond) and contralateral fore limbs (triangle) were evaluated (mean ± SEM). Testing began 3 days post-SCI, and the effects of Ex were evident by week 3. A significant difference between SCI and SCI + Ex occurred during weeks 3 and 4 (*p < 0.05). Week 5 data displays a slight decrease in the mean FLS performance that was due to the inactivity of a few animals for that week only. (B) Recovery was grouped into two phases: early (weeks 2–4) and late (weeks 5–8). The FLS during the early phase of recovery was significantly higher with Ex (*p < 0.05) compared to SCI alone (mean ± SEM). (C) Basso, Beattie, and Bresnahan locomotor rating scale (BBB) assessment over the 8-week time period (mean ± SEM). Injured hind limbs from each group SCI alone (square) and SCI + Ex (diamond) along with the contralateral uninjured hind limb (triangle) were examined. No significant difference in hind limb function was found when animals were subjected to force Ex.

FIG. 3.

Grip strength measurements. (A) Percentage of baseline grip strength measurements of spinal cord injury (SCI) alone (square), and SCI + forced exercise (Ex; diamond) display a decrease in grip strength immediately after injury, followed by an increase in force over the remaining 8 weeks (mean ± SEM). Grip strength measurements are from the ipsilateral fore limb only. (B) Separating recovery into early (weeks 2–4) and late (weeks 5–8) phases showed no significant improvement with exercise.

FIG. 4.

Grid walk performance of the affected fore limb and ipsilateral hind limb. (A) Average percentage of correct foot placements on the grid by the right fore limb (mean ± SEM). The 8-week period displays an initial deficit during 1 week after spinal cord injury (SCI) in both groups to ∼75% of baseline. Throughout the remaining weeks both groups perform in a similar manner reaching ∼80% by 8 weeks. (B) The percentage of correct foot placements completed by the ipsilateral hind limb. SCI alone (square) made significantly fewer correct placements compare to the SCI + forced exercise (Ex; diamond) over the 8-week period (mean ± SEM). The Ex group made significantly fewer mis-steps during weeks 3, 4, 6, and 7 (*p < 0.05). The largest separation between groups occurred at week 3 implying accelerated spontaneous recovery with Ex. Week 5 data displays a decrease but the variability is made evident by the large SEM indicating percentage of placements were not much different. (C) Mean percentage of correct foot placements made by the ipsilateral hind limb when split in to two phases of recovery, with the Ex group performing significantly more correct foot placements early after injury (mean ± SEM).

FIG. 5.

Wheel assessment. Biweekly evaluation of wheel walking performance. Three parameters were observed: successful steps, fine motor skill (grip ability) and foot placement. A significant difference was found in both groups 1 week after spinal cord injury (SCI) compared to baseline performance (*p < 0.05). A significant difference was not found between groups, but there was a trend towards accelerated recovery with forced exercise (Ex) at week 1. Beginning at week 3 and continuing for the duration of the study, both groups displayed similar performance in wheel walking (mean ± SEM).

FIG. 6.

Anatomical and histological evaluation of lesion Site after 8 weeks of forced exercise (Ex). (A) Images from the epicenter with a Nissl-myelin stain. Amount of spared white and gray matter were comparable with or without Ex. (B) Representative images caudal to the lesion site labeled with glial fibrillary acidic protein (GFAP). A graph representing the area of reactive astrogliosis located rostral, at the epicenter and caudal to the lesion site (mean ± SD). A significant decrease in the density of reactive astrogliosis caudal to the lesion site is shown. (C) Representative images caudal to the lesion site with positive labeling of macrophages. The graph displays the area of tissue positive for macrophages caudal to the lesion site, which shows a trend towards decreased phagocytic presence after 8 weeks of Ex (mean ± SD).