Nogo receptor deletion and multimodal exercise improve distinct aspects of recovery in cervical spinal cord injury

Abstract

We tested the ability of two plasticity-promoting approaches to enhance recovery in a mouse model of incomplete spinal cord injury (SCI). Genetically, we reduced myelin-mediated inhibition of neural plasticity through Nogo66-receptor (NgR) gene deletion. Behaviorally, we utilized a novel multimodal exercise training paradigm. Adult mice of wild-type or NgR-null genotype were subjected to partial lateral hemisection (LHx) at C3-C4 with the intent of producing anatomically and functionally mild deficits. Exercise training or control treatment proceeded for 14 weeks. Behavioral outcomes were assessed prior to tract tracing and histological analysis. Genotype and training exerted differing effects on performance; training improved performance on a test related to the training regimen (task-specific benefit), whereas genotype also improved performance on more generalized behaviors (task-non-specific benefit). There were no significant histological differences across genotype or training assignment with regard to lesion size or axonal tract staining. Thus either NgR gene deletion or exercise training benefits mice with mild cervical spinal injury. In this lesion model, the effects of NgR deletion and training were not synergistic for the tasks assessed. Further work is required to optimize the interaction between pharmacological and physical interventions for SCI.

FIG. 1.

Experimental outline. A total of 78 mice underwent preoperative acclimation, baseline evaluation, and surgery. A total of 14 mice dropped out over the course of the experiment (11 deaths and 3 exclusions described in the results section). Partial lateral hemisection (LHx) mice were randomized to trained or untrained groups after the 1-week evaluations, then underwent training and serial evaluation during weeks 2–14. Corticospinal tracts were anterogradely labeled with dextran amines prior to perfusion and histological analysis (WT, wild-type; NgR^–/–, NogoReceptor1-null).

FIG. 2.

Partial lateral hemisection results in consistent lesions with minimal long-term effect on corticospinal tract (CST) or serotonergic tracts. (A) The extent of a typical partial lateral hemisection (LHx) depicted in gray. (B) Representative horizontal sections of the lesion site taken at the levels of the lines shown in A (D, dorsal; M, medial; V, ventral). Horizontal sections were stained for glial fibrillary acidic protein. Lesion site is denoted with arrowhead (scale bar = 500 μm). (C) The extent of lesioned tissue in LHx mice, expressed as a percentage of the hemicord lesioned over the dorsal, medial, and ventral planes, was similar across genotype and training assignment. Numbers of mice per group: sham (Sham, 3); WT-untrained (WT-U, 10); WT-trained (WT-T, 14); NgR^–/–-untrained (NgR^–/–-U,15); NgR^–/–-trained (NgR^–/–-T,12). (D) Representative lumbar transverse section stained for PKC-γ, indicating that the CST was not affected by partial LHx (scale bar = 100 μm). (E) Representative lumbar cord transverse section stained for 5-hydroxytryptamine (5-HT). Serotonergic innervation was not significantly reduced on the lesioned side of the cord 14 weeks after LHx (scale bar = 100 μm; WT, wild-type; NgR^–/–, NogoReceptor1-null).

FIG. 3.

Mild deficits in open-field ambulation, treadmill locomotion, and overall composite scores were not significantly affected by genotype or training over time. (A) Scores on the Basso mouse scale (BMS). (B) Scores on the BMS-Forelimb Score. (C) Persistence on the accelerating treadmill. (D) Composite scores calculated from performance in the open-field, rotarod, treadmill, grip-strength, and inclined grid tests as described in the methods section. Among injured mice, there were no significant differences across genotype or training. Numbers of mice per group: sham (Sham, 3); WT-untrained (WT-U, 15); WT-trained (WT-T, 14); NgR^–/–-untrained (NgR^–/–-U,16); NgR^–/–-trained (NgR^–/–-T,16; SCI, spinal cord injury; NgR, NogoReceptor1). Color image is available online at www.liebertonline.com/neu.

FIG. 4.

Training improved performance on the inclined grid. Footfall errors were recorded over four traverses up an inclined grid. (A) Left forelimb error points. (B) Left hindlimb error points. (C) Right error points. Training significantly reduced hindlimb errors. NgR^–/– genotype also significantly reduced errors by the left hindlimb. There was a trend towards a positive interaction of training and NgR^–/– genotype to increase the rate of recovery of impaired limbs at 8 weeks (WT-U, WT-untrained; WT-T, WT-trained; NgR^–/–-U, NgR^–/–-untrained; NgR^–/–-T, NgR^–/–-trained; NgR, NogoReceptor1). Color image is available online at www.liebertonline.com/neu.

FIG. 5.

NgR deletion improved performance in rotarod endurance and grip strength. (A) Mean latency to falling off an accelerating rotarod. (B) Grip strength of the injured left forelimb expressed as a percentage of the uninjured right forelimb. Compared to untrained WT mice, untrained NgR^–/– mice performed significantly better on the rotarod at 1 and 13 weeks post-SCI, as well as displaying improved strength of the injured left forepaw at 13 weeks post-SCI (WT-U, WT-untrained; WT-T, WT-trained; NgR^–/–-U, NgR^–/–-untrained; NgR^–/–-T, NgR^–/–-trained; NgR, NogoReceptor1; SCI, spinal cord injury; WT, wild-type). Color image is available online at www.liebertonline.com/neu.