Physical rehabilitation improves muscle function following volumetric muscle loss injury

Abstract

Background: Given the clinical practice of prescribing physical rehabilitation for the treatment of VML injuries, the present study examined the functional and histomorphological adaptations in the volumetric muscle loss (VML) injured muscle to physical rehabilitation.

Methods: Tibialis anterior muscle VML injury was created in Lewis rats (n = 32), and were randomly assigned to either sedentary (SED) or physical rehabilitation (RUN) group. After 1 week, RUN rats were given unlimited access to voluntary running wheels either 1 or 7 weeks (2 or 8 weeks post-injury). At 2 weeks post-injury, TA muscles were harvested for molecular analyses. At 8 weeks post-injury, the rats underwent in vivo function testing. The explanted tissue was analyzed using histological and immunofluorescence procedures.

Results: The primary findings of the study are that physical rehabilitation in the form of voluntary wheel running promotes ~ 17% improvement in maximal isometric torque, and a ~ 13% increase in weight of the injured muscle, but it did so without significant morphological adaptations (e.g., no hypertrophy and hyperplasia). Wheel running up-regulated metabolic genes (SIRT-1, PGC-1α) only in the uninjured muscles, and a greater deposition of fibrous tissue in the defect area of the injured muscle preceded by an up-regulation of pro-fibrotic genes (Collagen I, TGF-β1). Therefore, it is plausible that the wheel running related functional improvements were due to improved force transmission and not muscle regeneration.

Conclusions: This is the first study to demonstrate improvement in functional performance of non-repaired VML injured muscle with physical rehabilitation in the form of voluntary wheel running. This study provides information for the first time on the basic changes in the VML injured muscle with physical rehabilitation, which may aid in the development of appropriate physical rehabilitation regimen(s).

Keywords: Function; Muscle; Rehabilitation; Running; Trauma.

Figure 1

Wheel running animals gained less weight throughout the study. A subset of the animals was given access to voluntary running wheels one week post-injury and was allowed to run for 7 weeks (A). At the end of 7 weeks, the animals in the RUN group were significantly (~10%) lighter than animals from the SED group (B). * ≠ SED; p < 0.05.

Figure 2

Physical rehabilitation in the form of voluntary wheel running improves in vivo tibialis anterior muscle torque. Maximal isometric torque (@ 150 Hz) of the tibialis anterior muscle was assessed in vivo following distal extensor digitorum longus muscle (EDL) tenotomy (see Methods). Average maximal isometric torque normalized to body weight is shown for the uninjured and injured muscle for the SED and RUN groups. Values are mean ± SEM. Sample size is listed in Table  3. * ≠ uninjured (contralateral); § ≠ sedentary injured; p < 0.05. All VML responses, regardless of group, were lesser than uninjured contralateral values.

Figure 3

Physical rehabilitation in the form of voluntary wheel running mitigates force imbalance developed as a result of VML injury. Maximal isometric torque prior to tenotomy of the EDL was normalized to the maximal isometric torque after tenotomy of the EDL. Values are mean ± SEM. Sample size is listed in Table  3. * ≠ uninjured (contralateral); § ≠ sedentary injured; p < 0.05.

Figure 4

Physical rehabilitation in the form of voluntary wheel running does not result in morphological adaptations (fiber cross-sectional area).100× non-overlapping images from the injured muscle were analyzed for fiber cross-sectional area (CSA) measurements (A). From these measurements, the fiber cross-sectional area CSA frequency distribution was obtained for the uninjured (B) and injured muscle (C) Values are mean ± SEM. n = 6 muscles/group; p < 0.05.

Figure 5

Physical rehabilitation in the form of voluntary wheel running prevents collapsing of muscle fibers. The muscle fibers collapse around the injury site in the SED group (A), while they enclose the fibrotic scar in the RUN group (B). In either group, the area immediately adjacent to defect has disorganized muscle fibers. Scale bar = 100 μm.

Figure 6

Physical rehabilitation in the form of voluntary wheel running exacerbates chronic injury in the injured muscle. Uninjured contralateral (not shown) and injured muscle of the SED (A) and RUN (B) groups were analyzed for the presence of centrally located nuclei (white arrows) (Scale bar = 100 μm). Inset images are high magnification (200×) images in the injured muscle (Scale bar = 50 μm). Physical rehabilitation significantly increased the presence of CLN in the injured muscle (C). Values are mean ± SEM. n = 6 /group; * denotes ≠ uninjured (contralateral); § denotes ≠ sedentary injured; p < 0.05.

Figure 7

Physical rehabilitation in the form of wheel running does not exacerbate injury related intramuscular collagen content. Uninjured contralateral (A,C) and injured muscle (B,D) of SED and RUN groups, respectively were analyzed for intramuscular collagen content (E). Scale bar = 100 μm. Only tissue within the injured muscle (not in the defect area) was included for analysis. Values are mean ± SEM. n = 3-6 muscles/group; * denotes ≠ uninjured (contralateral); £ denotes ≠ sedentary uninjured; p < 0.05.

Figure 8

Physical rehabilitation in the form of voluntary wheel running causes the development of a fibrotic scar in the defect area of the injured muscle. Whole TA muscle cross-sections of the injured muscle of the SED (A) and RUN (B) groups are presented. White dashed line illustrates the formation fibrotic scar in the injured muscle of the RUN group (B). White dashed boxes indicate the approximate region where images were taken in the defect area of the SED (C) and RUN (D) groups. No muscle regeneration was observed in either group.

Figure 9

Gene expression of myogenic and fibrotic markers is up regulated, while metabolic markers are down regulated in the injured muscle. TA muscles from SED and RUN (one week of running) injured muscles were harvested two weeks post-injury. Tissue samples comprised of defect area and the remaining muscle were assayed for gene expression of A) Embryonic heavy chain myosin (eMHC), B) Collagen I (Col I), C) Transforming growth factor-β1 (TGF-β1), D) Silent mating type information regulation 2 homolog-1 (SIRT-1) and E) Peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α). Note: All gene expression data was normalized to SED uninjured. Values are mean ± SEM. n = 3-5 muscles/group; # denotes ≠ injured; p < 0.05.