Exercise facilitates regeneration after severe nerve transection and further modulates neural plasticity

Abstract

Patients with severe traumatic peripheral nerve injury (PNI) always suffer from incomplete recovery and poor functional outcome. Physical exercise-based rehabilitation, as a non-invasive interventional strategy, has been widely acknowledged to improve PNI recovery by promoting nerve regeneration and relieving pain. However, effects of exercise on chronic plastic changes following severe traumatic PNIs have been limitedly discussed. In this study, we created a long-gap sciatic nerve transection followed by autograft bridging in rats and tested the therapeutic functions of treadmill running with low intensity and late initiation. We demonstrated that treadmill running effectively facilitated nerve regeneration and prevented muscle atrophy and thus improved sensorimotor functions and walking performance. Furthermore, exercise could reduce inflammation at the injured nerve as well as prevent the overexpression of TRPV1, a pain sensor, in primary afferent sensory neurons. In the central nervous system, we found that PNI induced transcriptive changes at the ipsilateral lumber spinal dorsal horn, and exercise could reverse the differential expression for genes involved in the Notch signaling pathway. In addition, through neural imaging techniques, we found volumetric, microstructural, metabolite, and neuronal activity changes in supraspinal regions of interest (i.e., somatosensory cortex, motor cortex, hippocampus, etc.) after the PNI, some of which could be reversed through treadmill running. In summary, treadmill running with late initiation could promote recovery from long-gap nerve transection, and while it could reverse maladaptive plasticity after the PNI, exercise may also ameliorate comorbidities, such as chronic pain, mental depression, and anxiety in the long term.

Keywords: Long-gap nerve transection; MEMRI; Nerve regeneration; Neuroplasticity; Rehabilitation; Treadmill exercise.

Fig. 1

Schematic of the long-gap sciatic nerve transection, autograft repair, treadmill running paradigm, and experimental design.

Fig. 2

Mechanical and thermal sensory functions as well as locomotor functions were determined at predetermined time points post-injury. A Withdrawal threshold of the right hind paw in response to mechanical stimuli measured by using a Von Frey test; B Latency of the right hind paw in response to heat stimuli measured with a Hargreaves test. (Sham vs Autograft: *p < 0.05, **p < 0.01, ***p < 0.001; Sham vs Exercise: #p < 0.05, ##p < 0.01; Autograft vs Exercise: &p < 0.05, n = 4 or 5); C Photographs recorded during rats walking inside a self-built rat-walking apparatus. The red circle indicates a touchdown point of the right hind paw; D Representative plots of stance traces of each paw during rat walking. The relative perpendicular distance from each paw to the green central body line was indicated with a red line; E Gait patterns indicating swing phases and stance phases for both hind paws; F–I Quantitative data analysis for the relative force (F), perpendicular distance from paw to the central body line (G), stance/swing phase (H), and swing speed (I) based on the ratio of the right (operated) to left (unoperated) hind paw. (*p < 0.05, **p < 0.01, ***p < 0.001, n = 4 or 5). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Histological and morphological analysis of ipsilateral nerve segments. A-B Longitudinal-sectional IHC staining for TNF-α (Scale bar: 200 μm) (A) and quantification of the TNF-α positive area (n = 6) (B) at right sciatic nerve segments; C Longitudinal-sectional IF staining with general nerve marker (Tubulin β-3) and myelin marker (MBP) (Scale bar: 200 μm); D-E Measurement of axonal alignment at the right nerve segments, based on IF images (D) and quantitative results (n = 5) (E); F-G Cross-sectional toluidine blue staining (Scale bar: 200 μm) (F) and TEM images (Scale bar: 10 μm) (G) of the right nerve segments; H–K Quantitative data of myelinated axon density (n = 3) (H), size of myelinated axons (I), myelin area (J), and area-based G-ratio (K) in single nerve fibers. (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Muscular wet weight and histological analysis at ipsilateral gastrocnemius muscles. A Representative photographs of gastrocnemius muscles from hindlimbs. Muscles indicated by a red circle were from injured hindlimbs; B Wet weight of gastrocnemius muscles from the right side (n = 5); C Cross-sectional H&E images of right gastrocnemius muscles (Scale bar: 100 μm); D Average size of single myofibers in right gastrocnemius muscles (n = 4 or 5); E Cross-sectional IF staining with Pax7 and laminin of right gastrocnemius muscles (Scale bar: 100 μm); F Frequency of Pax7⁺ cells in right gastrocnemius muscles (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

IF staining for plastic changes in right lumbar L4/L5 DRGs as well as in spinal cords. A Right lumbar L4/L5 DRGs double stained with tubulin β-3 and TRPV1, a pain receptor expressed on sensory neurons (Scale bar: 100 μm); B Fluorescent intensity of TRPV1 based on DRG IF images (n = 5); C Cross-sectional lumbar spinal cords double stained with tubulin β-3 and Iba-1, a microglial marker (Scale bar: 1000 μm), and Iba-1 expression at right SDHs, as indicated by white rectangular areas (Scale bar: 100 μm); D Quantitation of Iba-1 positive area at right SDHs (n = 5). (**p < 0.01, ***p < 0.001).

Fig. 6

Expression changes of total RNA at the ipsilateral SDH after a long-gap PNI and treadmill exercise by using RNA-Seq assay. a Heat map showing hierarchical clustering of DEGs compared among Sham, Autograft, and Exercise groups; b Venn diagram illustrating the number of overlapping DEGs among the three groups; c Volcano plot displaying up- and down-regulated RNAs by comparing the Autograft group to the Sham group; d-e KEGG enrichment analysis of up- and down-regulated RNAs by comparing the Autograft group to the Sham group; f Volcano plot displaying up- and down-regulated RNAs by comparing the Exercise group to the Autograft group; g-h KEGG enrichment analysis (g) and PPI network analysis (h) of 71 selected DEGs. In the PPI network plot, red nodes indicate seed genes, while purple nodes indicate genes that have known interactions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Microstructural and metabolite changes in supraspinal ROIs after the long-gap PNI and treadmill exercise detected by DTI and MRS studies, respectively. A-B Differences in ADC (A) and FA (B) at the CTX, hippocampus, thalamus, and hypothalamus of right and left hemispheres; C Calibrated concentrations of NAA, Ins, tCHO, GABA, Glu, and Gln in the CTX, hippocampus, and thalamus. (n = 4 or 5, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 8

Volumetric changes and neuronal activity in the brain after the long-gap PNI and treadmill exercise measured by T1-weighted MEMRI. a ROI analysis of the rat brain. ROIs were defined based on the SIGMA rat brain templates and atlases; b Color map of averaged T1-weighted images in each group. The color bar indicates normalized signal intensity; c-d Overall volume (c) and neuronal activity (d) of ROIs in the brain based on T1-weighted signals. (n = 4 or 5, *p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)