The effect of autologous repair and voluntary wheel running on force recovery in a rat model of volumetric muscle loss

Abstract

New findings: What is the central question of this study? Following large traumatic loss of muscle tissue (volumetric muscle loss; VML), permanent functional and cosmetic deficits present themselves and regenerative therapies alone have not been able to generate a robust regenerative response: how does the addition of rehabilitative therapies affects the regenerative response? What is the main finding and its importance? Using exercise along with autologous muscle repair, we demonstrated accelerated muscle force recovery response post-VML. The accentuated force recovery 2 weeks post-VML would allow patients to return home sooner than allowed with current therapies.

Abstract: Skeletal muscle can regenerate from damage but is overwhelmed with extreme tissue loss, known as volumetric muscle loss (VML). Patients suffering from VML do not fully recover force output in the affected limb. Recent studies show that replacement tissue (i.e., autograph) into the VML defect site plus physical activity show promise for optimizing force recovery post-VML. The purpose of this study was to measure the effects of autologous repair and voluntary wheel running on force recovery post-VML. Thirty-two male Sprague-Dawley rats had 20% of their left tibialis anterior (LTA) excised then replaced and sutured into the intact muscle (autologous repair). The right tibialis anterior (RTA) acted as the contralateral control. Sixteen rats were given free access to a running wheel (Wheel) whereas the other 16 remained in a cage with the running wheel locked (Sed). At 2 and 8 weeks post-VML, the LTA underwent force testing; then the muscle was removed and morphological and gene expression analysis was conducted. At 2 weeks post-injury, normalized LTA force was 58% greater in the Wheel group compared to the Sed group. At 8 weeks post-VML, LTA force was similar between the Wheel and Sed groups but was still lower than the uninjured RTA. Gene expression analysis at 2 weeks post-VML showed the wheel groups had lower mRNA content of interleukin (IL)-1β, IL-6 and tumour necrosis factor α compared to the Sed group. Overall, voluntary wheel running promoted early force recovery, but was not sufficient to fully restore force. The accentuated early force recovery is possibly due to a more pro-regenerative microenvironment.

Keywords: VML; exercise; inflammation; regenerative medicine; rehabilitative medicine.

FIGURE 1

Schematic representation of the experimental design

FIGURE 2

In vivo isometric peak torque measurements of the LTA at 2 and 8 weeks post-VML. (a) Peak isometric torque of the LTA normalized to body mass 2 and 8 weeks post-VML in sedentary (Sed) and voluntary wheel running (Wheel) animals. (b) Peak isometric torque of the injured LTA normalized to the uninjured RTA at 2 and 8 weeks post-VML in sedentary (Sed) and voluntary wheel running (Wheel) animals. Dashed line is a reference point for uninjured right tibialis anterior (RTA). Data are means ± SD. †Difference between Control or VML limbs; ‡difference between sedentary or wheel running groups. P < 0.05. n = 3–8 per group

FIGURE 3

mRNA abundance, normalized to 18S, of cell cycle regulators. (a) IGF-1 mRNA abundance 2 weeks post-VML; (b) IGF-1 mRNA abundance 8 weeks post-VML; (c) IGF-1 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (d) Myostatin mRNA abundance 2 weeks post-VML; (e) Myostatin mRNA abundance 8 weeks post-VML; (f) Myostatin mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (g) CyclinD1 mRNA abundance 2 weeks post-VML; H) CyclinD1 mRNA abundance 8 weeks post-VML; (i) CyclinD1 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML). Data are means ± SD. n = 3–8 per group. ME, main effect; †difference between Control or VML limbs; ‡difference between sedentary or wheel running groups; P < 0.05

FIGURE 4

mRNA abundance, normalized to 18S, of satellite cell markers. (a) Pax7 mRNA abundance 2 weeks post-VML; (b) Pax7 mRNA abundance 8 weeks post-VML; (c) Pax7 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (d) MyoD mRNA abundance 2 weeks post-VML; (e) MyoD mRNA abundance 8 weeks post-VML; (f) MyoD mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (g) Myogenin mRNA abundance 2 weeks post-VML; (h) Myogenin mRNA abundance 8 weeks post-VML; (i) Myogenin mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML; (j) Ki67 mRNA abundance 2 weeks post-VML; (k) Ki67 mRNA abundance 8 weeks post-VML; (l) Ki67 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML). Data are means ± SD. n = 3–8 per group. ME, main effect; †difference between Control or VML limbs; ‡difference within Sedentary or Wheel groups; P < 0.05

FIGURE 5

mRNA abundance, normalized to 18S, of macrophage markers. (a) CD68 mRNA abundance 2 weeks post-VML; (b) CD68 mRNA abundance 8 weeks post-VML; (c) CD68 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (d) Arginase 1 mRNA abundance 2 weeks post-VML; (e) Arginase mRNA abundance 8 weeks post-VML; (f) Arginase 1 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML). Data are means ± SD. n = 3–8 per group. ME, main effect; †difference between Control or VML limbs; ‡difference within Sedentary or Wheel groups; P < 0.05

FIGURE 6

mRNA abundance, normalized to 18S, of inflammatory response markers. (a) IL-1β mRNA abundance 2 weeks post-VML; (b) IL-1β mRNA abundance 8 weeks post-VML; (c) IL-1β mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (d) TNF-α mRNA abundance 2 weeks post-VML; (e) TNF-α mRNA abundance 8 weeks post-VML; (f) TNF-α mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML); (g) IL-6 mRNA abundance 2 weeks post-VML; (h) IL-6 mRNA abundance 8 weeks post-VML; (i) IL-6 mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML; (j) IFNγ mRNA abundance 2 weeks post-VML; (k) IFNγ mRNA abundance 8 weeks post-VML; (l) IFNγ mRNA abundance control comparison (control limb of sedentary group at 2 and 8 weeks post-VML). Data are means ± SD. n = 3–8 per group. ME, main effect; †difference within Control or VML limbs; ‡difference within Sedentary or Wheel groups; P < 0.05