Background: Volumetric muscle loss (VML) injuries occur due to orthopaedic trauma or the surgical removal of skeletal muscle and result in debilitating long-term functional deficits. Current treatment strategies do not promote significant restoration of function; additionally appropriate evidenced-based practice physical therapy paradigms have yet to be established. The objective of this study was to develop and evaluate early rehabilitation paradigms of passive range of motion and electrical stimulation in isolation or combination to understand the genetic and functional response in the tissue remaining after a multi-muscle VML injury. Methods: Adult male mice underwent an ~ 20% multi-muscle VML injury to the posterior compartment (gastrocnemius, soleus, and plantaris muscle) unilaterally and were randomized to rehabilitation paradigm twice per week beginning 2 days post-injury or no treatment. Results: The most salient findings of this work are: 1) that the remaining muscle tissue after VML injury was adaptable in terms of improved muscle strength and mitigation of stiffness; but 2) not adaptable to improvements in metabolic capacity. Furthermore, biochemical (i.e., collagen content) and gene (i.e., gene arrays) assays suggest that functional adaptations may reflect changes in the biomechanical properties of the remaining tissue due to the cellular deposition of non-contractile tissue in the void left by the VML injury and/or differentiation of gene expression with early rehabilitation. Conclusions: Collectively this work provides evidence of genetic and functional plasticity in the remaining skeletal muscle with early rehabilitation approaches, which may facilitate future evidenced-based practice of early rehabilitation at the clinical level. Keywords: Electrical stimulation, Neuromusculoskeletal injury, Regenerative medicine, Orthopaedic trauma, Skeletal muscle injury, Range of motion * Correspondence: firstname.lastname@example.org; email@example.com Sarah M. Greising and Jarrod A. Call contributed equally to this work. Extremity Trauma and Regenerative Medicine, United States Army Institute of Surgical Research, Fort Sam Houston, Texas 78234, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 2 of 11 Background recruitment issues, either from loss of consciousness Volumetric muscle loss (VML) is a debilitating ortho- (e.g., prolonged comatose state), peripheral nerve dam- paedic condition that results in chronic functional defi- age, or damaged neural tracks, and therefore a potential cits and disability [1–3]. With no current surgical or rehabilitation technique to circumvent these limitations rehabilitative standard of care to address the soft tissue is intermittent electrical stimulation. Intermittent elec- loss, VML injures are left to follow the natural sequela trical stimulation will recruit the muscle fibers remaining of injury that ultimately results in the replacement of following injury via subdermal stimulation. Electrical contractile skeletal muscle with non-contractile patho- stimulation has been shown to promote motor and logic fibrotic tissue . Furthermore, functional capacity sensory reinnervation and regeneration following nerve after VML injury continues to deteriorate over time . injury , and induce hyperopic overload [20, 21]. Both As such, interventions and treatment approaches are range of motion and intermittent electrical stimulation urgently needed to ameliorate the progressive functional techniques represent rehabilitation paradigms that con- disability related to VML injury . ceivably could be conducted in a hospital setting, even Physical therapy and rehabilitation are an important while patients were non-weight bearing. component of functional improvements following all neu- This work sought to develop and evaluate two early romusculoskeletal injuries, but there is currently a dearth rehabilitation paradigms of passive range of motion and of experimental studies to support any evidenced-based electrical stimulation to understand the genetic and practice for VML injury. In part, the lack of clinical functional response in the tissue remaining following rehabilitation guidelines could be related to heterogeneous a multi-muscle VML injury. We developed the re- injury pattern and the prevalence of the multiple concomi- habilitation protocols broadly off of previous work tant injuries to VML, such as fracture . A limited [16, 18, 20, 21]. We hypothesized that early inter- number of VML injury case studies have consistently vention following VML injury would enhance the en- observed chronic functional deficits and disability that dogenous regenerative and oxidative capacity of the were not ameliorated by delayed, prolonged, intensive muscle remaining following VML injury, ultimately physical therapy [2, 8–10]. Lack of human-based experi- improving muscle function. mental studies have been addressed using small and large animal models . In fact, a small number of pre-clinical Methods studies involving rodent models have demonstrated benefi- Animals cial functional remodeling of skeletal muscle with running Adult male C5BL/6 mice (n = 96) were purchased from as a physical therapy modality [11–14]. However, quadru- Jackson Laboratories (Bar Harbor, ME). Animals were ped running may rely upon and engage muscles differently maintained on a 12 h light-dark schedule under specific than biped running, so more refined experimental designs pathogen-free conditions with ad libitum food and water are necessary to validate actively engaging VML injured in a vivarium accredited by the American Association muscle as part of a rehabilitation approach. for the Accreditation of Laboratory Animal Care. Upon Understanding of early rehabilitative interventions in arrival, all mice were given at least 1 week to acclimate VML patients has yet to be fully established. Early to the facility prior to any experimentation. All protocols mobilization and rehabilitation initiated in the hospital and animal care guidelines were approved by the Institu- setting for various other clinical conditions results in tional Animal Care and Use Committee at the United shorter admission times and improved function . States Army Institute of Surgical Research (A16–036) or Passive range of motion exercise are non-weight bearing the University of Georgia (A2017 08–004), in compli- rehabilitation techniques that do not rely on functionally ance with National Institute of Health Guidelines. All innervated muscle fibers. The tolerance and effectiveness components were conducted in compliance with the of range of motion rehabilitation to assist in recovery from Animal Welfare Act, the Implementing Animal Welfare conditions ranging from contraction-induced muscle in- Regulations and in accordance with the principles of the jury  to rotator cuff repair  has been documented. Guide for the Care and Use of Laboratory Animals. It is expected that passive movement may mitigate the At ~ 12.5 weeks of age mice underwent a VML injury muscle stiffness following VML injury, but the approach to the posterior compartment of the hindlimb and were has not been tested. Interestingly, in Duchenne muscular randomized to various treatment groups. Specific groups dystrophy, a condition presenting with pathologic fibrosis, received no-treatment (VML), rehabilitation interven- muscle weakness and stiffness, muscle activation regimens tions of range of motion exercise (ROM) or range of in combination with range of motion therapy are report- motion and intermittent electrical stimulation (ROM-E). edly more effective in improving limb endurance and All rehabilitation bouts were conducted two times per function compared to range of motion therapy alone week, beginning 2 days post-injury for up to 14 days clinically . VML injuries may present with motor unit post-injury for the acute study or 4 months for the Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 3 of 11 chronic study. A subset of uninjured (naïve, no surgical foot of the left limb was stabilized at 90 (neutral intervention) control mice was used for various analyses position) and the foot was secured to a foot plate throughout the project. Tissue harvest was conducted ~ attached to a servomotor (300B-LR, Aurora Scientific, 24 h following the final rehabilitation bout while mice Aurora, Ontario, Canada). Under computer control, the were deeply anesthetized with isoflurane (1.5–2.0%) and servomotor passively rotated the ankle 40° through mice were euthanized with an injection of Fatal Plus dorsi- and plantar-flexion, specifically 20° from neutral (150 mg/kg; intra-cardiac) while still under anesthesia. for both directions. Continuous range of motion was Following VML surgery all mice recovered promptly and conducted for 30 min with each set taking 5 s, followed displayed only slight limitations in mobility. No unex- by a 5 s rest period at neutral for the range of motion pected deaths or adverse outcomes were noted in any alone groups. For groups that received combined range of group across the 4 months evaluated. motion and intermittent electrical stimulation, stimulation occurred during rest phases of the range of motion proto- Volumetric muscle loss (VML) surgery col. Stimulation was elicited using platinum-iridium (Pt-Ir) While anesthetized (isoflurane 1.5–2.0%) a surgical VML needle electrodes positioned percutaneously on either side was created on the middle third of the posterior of the sciatic nerve (S48 and SIU5, Grass Technologies, compartment using a surgical approach modified from West Warwick, RI, USA). Progressive stimulation parame- previous work . All mice received a pre-surgical (~ ters were utilized to promote continuous adaptation and 30 min prior) administration of buprenorphine-SR were as follows: 30 Hz, 50% duty cycle (immediate (1.2 mg/kg; s.c.) for pain management. Briefly, a post-injury to 1 month); 45 Hz, 25% duty cycle (1 month posterior-lateral incision was made through the skin to to 2 months); and 80 Hz, 12.5% duty cycle (2 months to reveal the gastrocnemius muscle. Blunt and specific 4 months). These parameters were selected based on the dissection of the skin, fascia, and hamstring muscle was following rationale: 1) 30, 45, and 80 Hz represent the used to reveal the posterior aspect of the gastrocnemius linear phase of the torque-frequency relationship and muscle. Blunt dissection was used to isolate the muscle reflect ~ 25, 50, and 75% peak-isometric torques in compartment off the dorsal aspect of the tibia and a uninjured muscle [23, 24]; and 2) a reduced duty cycle for small metal plate was inserted below the deep soleus higher-frequencies contractions minimized potential for muscle but above the tibia and a punch biopsy (4 mm, fatigue. All rehabilitation occurred twice per week approximately 20% volume loss of muscle; see Table 1) throughout the study period, specifically for the acute was performed through the middle third of the muscle study on days 2, 6, 9, and 13 days post-surgery. During compartment. Any bleeding was stopped with light pres- rehabilitation sessions, ideal electrode placement and sure. Following the surgical injury the skin incision was current (mAmps) were validate by a series of sub-maximal closed with simple interrupted suture (6–0 Silk). In all 20 Hz stimulations. These sub-maximal active torque cases the left limb underwent the VML injury and the (ROM-E group only), as well as passive torques (both contralateral was used as an injured intra-animal control ROM and ROM-E groups) about the ankle joint was eval- for biochemical and gene expression analysis. uated post-hoc as an assessment of ongoing adaptation to the rehabilitation strategy (see Fig. 1). Rehabilitation All rehabilitation sessions were conducted while the Muscle function mouse was anesthetized (isoflurane 1.5–2.0%) and body In vivo maximal isometric torque of the ankle plantarflex- temperature was maintained. At each bout the knee and ors (gastrocnemius, soleus, and plantaris muscle) was assessed as previously described [23–25] and was deter- mined at the terminal time point. Briefly, mice were anes- Table 1 Multi-muscle volumetric muscle loss injury thetized using 2% isoflurane in oxygen, and then the left n VML defect Injured: uninjured Force deficit hindlimb was depilated and aseptically prepared, the foot mass (mg) gastrocnemius from control (%) muscle mass placed in a foot-plate attached to a servomotor (Model 3 days 4 18.6 ± 1.3 0.94 ± 0.09 – 300C-LR; Aurora Scientific, Aurora, Ontario, Canada), and Pt-Ir needle electrodes (Grass Technologies, West War- 7 days 4 18.8 ± 1.0 0.88 ± 0.08 – wick, RI, USA) were inserted percutaneously on either side 14 days 4 19.1 ± 1.3 0.66 ± 0.02* – of the nerve. To avoid recruitment of the anterior crural 1 month 6 20.2 ± 0.5 0.77 ± 0.05 - 62.5 ± 3.2† muscles responsible for dorsiflexion, the common perineal 2 months 6 18.2 ± 0.9 0.88 ± 0.06 - 61.8 ± 4.7 nerve was severed . Peak isometric torque was 4 months 6 19.8 ± 0.8 0.90 ± 0.02 - 51.0 ± 2.7 achieved by varying the current delivered to the sciatic P-value 0.592 0.029 0.043 nerve which branches to the tibial nerve thus innervating Mean ± SE; Significantly different than *3 days or †4 months post-VML the ankle plantarflexor muscles. To account for differences Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 4 of 11 isolated and gene expression was analyzed using a custom designed gene array (RT Profiler PCR Array; Qiagen) with genes related to myogenic, metabolic, fibrotic, inflammatory, and neural (Additional file 1: Table S1) response to injury per manufacture’s instruction. Data was processed with GAPDH as the endogenous control and expression was calculated relative to contra- lateral control muscle or the non-repaired VML injured muscle at the same time point, as appropriate and noted. Differentially expressed genes were analyzed with iPath- way using a fold change of 0.6 and adjusted P-value of 0.05 thresholds. Mitochondrial Immediately following dissection, portions of the medial and lateral gastrocnemius muscles from uninjured and injured limbs were dissected on a chilled aluminum block in 4 °C buffer X (7.23 mM K EGTA, 2.77 mM Ca K EGTA, 20mMimidazole,20mMtaurine,5.7 mM ATP, 14.3mMPCr,6.56mMMgCl -6H O, 50 mM k-MES) into 2 2 thin muscle fiber bundles as reported previously . Permeabilization of muscle fibers was achieved by transfer- ring fibers to a vial containing buffer X and saponin (50 μg/ml) and incubating (i.e., gentle rocking) at 4 °C for 30 min. Muscle fiber bundles were rinsed for 15 min in buffer Z (105 mM k-MES, 30 mM KCl, 10 mM KH PO , 2 4 5mMMgCl , 0.5 mg/ml BSA, 1 mM EGTA) at 4 °C. All measurements were performed using a Clark-type elec- Fig. 1 Effect of rehabilitation session number on active and passive trode (Oxygraph Plus System, Hansatech Instruments, UK) torque about the ankle joint. a Sub-maximal active torques (20 Hz) at 25 °C. Prior to each experiment, the electrode was were used to identify optimal stimulation parameters during each calibrated according to the manufacturer’sinstructionsand rehabilitation session, and there was a positive relationship between the number of rehabilitation sessions and active torque of the ankle 1mlofO infused buffer Z was added to the chamber. plantar flexor muscles. *Torque was greater at session #16 (2 Month) Muscle fiber bundles were weighed (~ 2.5 mg for all sam- compared to session #8 (1 Month) (P <0.05). # Torque was greater at ples) and added to the chamber. State 4 respiration (leak session #32 (4 Month) compared to session #8 (1 Month) (P < 0.05). respiration in the absence of ADP) was initiated by the b Passive torque about the ankle joint was assessed at 20° dorsiflexion, addition of glutamate (10 mM) and malate (5 mM). State 3 and passive torque decreased over time (session 1 compared to session 32) for ROM mice, but there was no change in passive respiration (respiration coupled to ATP synthesis) was ini- torque over time in ROM-E mice. **ROM session 32 > ROM tiated by the addition of ADP (2.5 mM) and succinate session 1 (P < 0.05) (10 mM). Cytochrome c (10 μM) was added to measure the integrity of the outer mitochondrial membrane. State 3 uncoupled respiration (respiration uncoupled from ATP in body size among mice, torques (mN●m) was normal- synthesis) was initiated by the addition of FCCP (0.5 μM). ized by body mass (kg). Mitochondrial respiration was terminated by the addition of cyanide (250 mM). Hydroxyproline Content of hyrdoxyproline in the muscle was used to determine collagen content following injury. Content was determined biochemically as previously described Statistical analysis [27, 28]. All data was analyzed using JMP (version 10.0 SAS Institute, Inc., NC). Data was analyzed separately using Gene expression a variety of ANOVAs, when appropriate Tukey HSD At the time of tissue harvest the gastrocnemius were post-hoc analysis was performed. Data are reported as excised and placed in TRIzol and snap frozen in liquid mean ± SE, unless otherwise specified and significance nitrogen and stored at -80 °C until analysis. RNA was was accepted at P < 0.05. Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 5 of 11 Results sessions and sub-maximal plantar flexor muscle torque Multi-muscle volumetric muscle loss (VML) injury about the ankle (Main Effect Time, P <0.001, Fig. 1), and VML injury in military [30, 31] and civilian populations overall torque was ~ 125% greater at the last compared to commonly involve 2 or more muscles. To date most VML the first session. There was a significant interaction injury models have been to an isolated muscle, with only a between group and time for passive torque about the limited number to multiple muscles within the quadriceps ankle joint (P = 0.034), as passive torque decreased 25% [33, 34]. Therefore, our first goal was to establish a murine with range of motion rehabilitation but was not changed multi-muscle VML injury model. Because the plantarflexor over time with combined range of motion and electrical muscles within the posterior compartment of the rodent stimulation (Fig. 1). Collectively, these inter-rehabilitation hind leg are highly recruited during normal ambulation and session analyses demonstrated on-going functional re- are weight bearing , this muscle group is ideal for modeling of the injured limb. rehabilitation studies. A full-thickness VML injury was Injured and contralateral uninjured gastrocnemius created through the plantarflexor gastrocnemius, plantaris, muscle masses were recorded to determine the long-term and soleus muscles (Table 1)atthe tibiamid-diaphyseal effect of injury and early rehabilitation on muscle atrophy level, resulting in the removal of ~ 19 mg of tissue or ~ 20% and possible hypertrophy. There was no effect of early of the combined plantarflexor muscle wet weight. The rehabilitation on injured gastrocnemius muscle mass partial tissue resection caused an ~ 50% maximal isometric relative to uninjured across time; however, independent of force loss and ~ 2 fold increase in passive torque (muscle group, the relative mass was 18% greater at 4 months stiffness) about the ankle through 4 months post-injury, compared to 1 month (Main Effect Time, P =0.025, indicating successful creation of a model that recapitulates Additional file 2: Figure S1). Body mass was not affected pathophysiological aspects of VML injury in patients [2, 9]. by early rehabilitation (Additional file 2:FigureS1). At 1, 2 or 4 months post-VML injury, peak isometric Early rehabilitation torque of the ankle plantar flexor muscles was assessed To validate early rehabilitation approaches, 2 days to determine contractile function. Independent of time, post-VML injury, mice were randomly assigned to one peak isometric plantar flexor muscle torque was greater of the following groups: VML alone (VML), passive in ROM-E mice compared to VML and ROM mice (32 range of motion (ROM), or ROM plus intermittent elec- and 21%, respectively; Main Effect Group, P < 0.001, trical stimulation (ROM-E). Range of motion rehabilita- Fig. 2 and Additional file 3: Figure S2). At 4 months, tion involved passively moving the ankle joint through VML injury represented a 51% deficit in torque (Control: − 1 − 1 40° of motion and the intent was to reduce muscle stiff- 768 ± 34 mN●mkg vs. VML: 376 ± 21 mN●mkg ; ness associated with the deposition of collagens in and see Table 1), and while ROM-E mice were stronger than around the VML injury site. Intermittent electrical VML mice, a 35% deficit remained (Control: 768 ± 34 − 1 − 1 stimulation rehabilitation involved recruitment of the mN●mkg vs. ROM-E: 496 ± 118 mN●mkg ). ankle plantar flexor muscles via sciatic nerve stimulation Collectively, rehabilitation using ROM-E gave rise to with Pt-Ir needle electrodes. The intent was to enhance functional improvements but was not able to completely strength by activating the remaining muscle after VML mitigate VML-related functional deficits. injury, during a time in which significant motoneuron Passive torque at 20° dorsiflexion (i.e., when plantar axotomy is present following injury . Rehabilitation flexor muscles are passively resisting the stretch) was strategies (twice weekly for 30 min) were continued in assessed to determine muscle stiffness. There was a different cohorts of mice for 1, 2, or 4 months strong trend for a significant interaction (P = 0.056). At post-VML (n = 6 mice/group/time). A small cohort of 4 months, VML injury resulted in over a 3-fold increase completely uninjured mice was included to observe in passive stiffness (Control: 1.5 ± 0.2 mN●m vs. VML: deficits associated with the VML injury and the relative 4.9 ± 0.4 mN●m), but early ROM rehabilitation attenu- recovery with early rehabilitation therapy (n = 8 mice). ated this effect. Independent of time, passive torque of the plantar flexor muscles following ROM and ROM-E Functional response to early rehabilitation rehabilitation were less compared to VML mice (− 52% To determine if early rehabilitation approaches were bene- and − 32%, respectively), and ROM resulted in 29% less ficial, functional responses were analyzed at each rehabili- passive torque compared to ROM-E (Main Effect Time, tation bout. First, in both the ROM and ROM-E groups, P < 0.001, Fig. 2). passive torque about the ankle joint was recorded and Collagen content of the gastrocnemius muscles was analyzed during each therapy session. Additionally at each measured since passive stiffness was greater with VML session, sub-maximal active torque about the ankle joint injury. There was a significant effect of injury, independ- was evaluated in the ROM-E group only. There was a ent of time, as total collagen content was ~ 2-fold positive association between the number of rehabilitation greater in injured limbs of VML, ROM, & ROM-E mice Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 6 of 11 Fig. 2 Effect of VML injury and rehabilitation on study endpoint active and passive torque about the ankle joint. a Peak isometric torque of the ankle plantar flexor muscles was greater following ROM-E rehabilitation compared to VML-alone and ROM rehabilitation, independent of time. − 1 Control = 768 ± 34 mN●mkg . b Passive torque of the ankle plantar flexor muscles at 20° dorsiflexion was greatest in VML-along compared to ROM and ROM-E rehabilitation, and lowest following ROM rehabilitation. Control = 1.5 ± 0.2 mN●m compared to uninjured limbs (P < 0.001, Fig. 3). While for permeabilized fibers isolated adjacent to the injury site there were noted improvements in passive torque about were compared to fibers from the contralateral uninjured the ankle within the muscle following ROM rehabilita- limb. There was no significant interaction or main effects tion the collagen deposition and expected fibrotic depos- (P ≥ 0.112, Fig. 4). However, independent of time and ition remained unchanged. group, oxygen consumption rates were 25% greater in fibers from completely uninjured mice compared to mice Oxidative response to early rehabilitation that had a unilateral VML injury (Control: 5054 ± To determine the metabolic function of the remaining 233 nmol/min/g vs. VML-Injured: 4225 ± 87.39 nmol/ muscle tissue after VML injury, oxygen consumption rates min/g, P <0.001, Fig. 4). This signals a previously Fig. 3 Effect of VML injury and rehabilitation on gastrocnemius muscle collagen content. Gastrocnemius muscle collagen content was greater in uninjured compared to contralateral uninjured control limbs, independent of time and rehabilitation group (P < 0.001). Control = 6.21 ± 0.59 μg collagen per mg muscle wet weight Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 7 of 11 Fig. 4 Effect of VML injury and rehabilitation on mitochondrial function of permeabilized muscle fibers. a There was no effect of time, group, or VML injury on oxygen consumption rates of permeabilized muscle fibers for VML-injured muscles. b Oxygen consumption rates of permeabilized muscle fibers was significantly greater in completely uninjured mice (Controls) compared to VML-injured mice (both injured and contralateral uninjured limbs pooled) undescribed impairment with VML injury, which may ROM-E groups were collapsed into ‘early rehabilitation’ reflect metabolic maladaptation to changes in muscle (Fig. 5). Significant differences in gene expression were recruitment that was resistant to rehabilitative approaches observed at 3 days post-injury between VML only and explored in this study. early rehabilitation groups (ROM & ROM-E). There was a significant down-regulation in mostly inflammatory gene Acute genetic response to volumetric muscle loss injury expression with rehabilitation at 3 days post-injury com- To investigate cellular mechanisms of the VML injury pared to other time points. In particular, inflammatory pathophysiology, additional cohorts of injured mice were (Il33, Tnf, Il4, CxCr3, CxC1, Ccl5, Ccl2) and metabolic allocated to VML with no repair or early rehabilitation (Akt2, Prkaal, Pparfcla, Sk2a4) genes were down-regulated (ROM or ROM-E) for 3, 7, or 14 days following injury and at 3 days post-injury. This suggests that early rehabilita- transcriptional changes in inflammatory, neurogenic, tion may mitigate the acute maladaptive response to VML fibrotic, myogenic, and metabolic genes were assessed injury, which may be related to the chronic improvements (Additional file 1: Table S1). In the VML only group, there in muscle function and passive stiffness. were 30 genes differentially regulated in the injured limb compared to the uninjured limb, independent of time Discussion (i.e., 3, 7, 14 days). Most all genes probed were signifi- Current regenerative medicine and rehabilitation tech- cantly up-regulated over control tissue, notably only niques for VML injured patients have not shown signifi- Mstn, Slc2a4, and Ppargc1a displayed down-regulation cant restoration of muscle strength or limb function . (Fig. 5 and Additional file 4: Table S2). Several of these A major limiting factor to rehabilitation is the remaining genes demonstrated transient changes in differential regu- muscle tissue following injury, and its adaptability and/ lation, as many myogenic, metabolic, and inflammatory or capacity to recover from injury. Unfortunately, in genes were significantly up-regulated at 3 in comparison VML-injured patients, rehabilitation often begins after to both 7 and 14 days post-injury. A small number of significant fibrosis has occurred in the muscle unit, fibrotic and neurogenic genes (Mmp9, Col3a1, Fbxo32, contributing to low functional improvements . To Tgfbr3, and Nrg1) were significantly up-regulated at 3 in overcome this timing limitation we investigated tech- comparison to only14 days. This supports a VML-related niques (i.e., passive range of motion and electrical stimu- regulation of inflammatory genes which had at least a lation) that can begin early after VML injury. The most 4-fold increase at 3 days compared to both the 7 and salient findings were that 1) early initiation of passive 14 day time points, although notably inflammation was range of motion therapy attenuated injury-induced still significantly elevated at the later time points. elevations of muscle stiffness, but did not improve active muscle function (Fig. 2), early co-delivery of neural elec- Acute genetic response to rehabilitation trical stimulation with passive range of motion therapy To determine how early rehabilitation approaches may 2) improved active muscle function, but did not attenu- alter the gene expression pattern associated with VML ate rising muscle stiffness (Fig. 2), and 3) abrogated the injury, gene expression of the gastrocnemius muscle (24 h capacity of passive range of motion therapy to prevent following the final rehabilitation bout) from mice that injury induced elevations of muscle stiffness. underwent rehabilitation (ROM & ROM-E) were com- Skeletal muscle fibrosis is known to impede muscle pared to non-repaired VML muscle at the same time healing and regeneration, alter the microenvironment points (3, 7, or 14 days post-VML). Both the ROM and of the muscle, and causes destruction of muscle Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 8 of 11 Fig. 5 Custom designed (inflammatory, neurogenic, fibrotic, myogenic, and metabolic, see Additional file 1: Table S1) PCR array analysis presents a significant change in regulation following VML injury and early rehabilitation. a The response to VML injury was assessed at 3, 7 and 14 days post-VML compared to contralateral control muscle. b The response to early rehabilitation was compared to non-repaired VML at the same time point (3, 7, or 14 days post-VML), treatment groups were collapsed. The dotted horizontal and longitudinal axes indicate the lower thresholds for statistical (P < 0.05) and biological significance (2 fold change) of expression, respectively. c Specific fold changes for genes that were significantly regulated due to rehabilitation are presented. (One way ANOVA; significantly different than *3 days or †14 days post-VML) architecture . It is possible that the overwhelming stiffness. The observation of reduced stiffness after fibrotic response after injury may be limiting rehabili- range of motion therapy partially supports this tation and/or further impacting the remaining muscle hypothesis; however, we did not detect predictable as it is left to follow the natural sequela of injury. In differences among rehabilitation groups in terms of pathologies such as Duchenne muscular dystrophy total collagen content in the injured limb (Fig. 4). This and cerebral palsy it has been proposed that the discrepancy is similar to prior observations of discon- organization and structure of the fibrotic deposition nect between collagen crosslinking characteristics with may have a greater role in functional impairments tissue stiffness  and supports further investigation than the total amount of collagen . Because fi- of range of motion therapy impact on collagen type, brotic tissue fills the void left by VML injury [4, 37], organization, or structure of collagen. The notable we initially hypothesized that early range of motion positive impact of reduced passive muscle stiffness therapy, alone or in combination with electrical stimu- with range of motion therapy alone does stand to have lation, would attenuated fibrotic tissue deposition and translatable benefits for this patient population, in Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 9 of 11 which even modest improvements to daily activities inflammatory response, especially at 3 days post-injury may have significant impact on patient quality of life. and future work should investigate how early rehabilita- Skeletal muscle metabolic capacity is highly plastic and tion may impact any systemic inflammation related to under most conditions has the capability to regenerate VML injury. after injury. Specifically, during the normal regenerative It stands to reason that VML-injured animals are ex- processes, such as occurring after traumatic myotoxic pected to be less physically active compared to uninjured injury, mitochondrial biogenesis accompanies muscle controls, which could produce a lower basal metabolic recovery from injury and is likely necessary to meet the capacity. A current limitation for the field is an under- energy demands of muscle repair [41, 42]. Injuries such standing of the physical or metabolic activity of patients as VML present a non-recoverable injury, in which the with VML injury. Importantly though, VML-injured ro- muscle has limited regenerative potential and loses the dents are able to elevate physical activity as evidenced by ability to recover . Mitochondrial function was less in increased voluntary wheel running distance [11–14], but VML-injured mice compared to completely uninjured the ability of the VML-injured limb or uninjured limb to (i.e., naïve) control mice, however there was no detect- positively adapt to the elevated physical activity in terms able differences in mitochondrial function between in- of metabolic capacity, balance in protein synthesis/deg- jured and contralateral uninjured muscles paired across radation, and fiber type distributions is unknown and the same animal. This finding raises several intriguing future work should begin to understand this complex questions regarding the systemic and chronic effects of relationship. Additionally, future work is needed to VML injury and possibility of low-grade systemic inflam- continue to understand the pathophysiologic state of the mation. Large-scale traumatic injuries such as burn trau- muscle remaining after VML injury with or without mas have been associated with low-grade, systemic additional rehabilitation, as there is a significant need to inflammation that is reported to negatively influence understand potential therapeutic targets that could mitochondrial function [43, 44]. Furthermore, following benefit the loss of function following VML injury. various neuromusculoskeletal injuries such as hip frac- Collectively, limited and/or lost mobility, poor metabolic ture there is noted systemic inflammation which is hy- function, and/or low-grade systemic inflammation after pothesized to contribute to lack of muscle regeneration VML injury may all contribute to development and/or . VML injury induction of acute and chronic sys- exacerbation of metabolic syndrome and cardiovascular temic inflammation presents a potential pivotal compo- disease in patients with VML injury. Therefore, identify- nent of the pathogenic response that when left untreated ing therapeutic interventions that promote muscle may worsen disability and may impede rehabilitative and health and physical activity may lessen the health burden regenerative treatment efficacy. and medical costs of VML injury. To date only a few studies have examined fibrotic and myogenic responses at 1 to 2 weeks following VML in- jury. Previous work has indicated that connective tissue Conclusions growth mediated regulation through TGF-1β family gene Many existing patients with a VML injury could benefit expression is greater at 1 week following VML and at 2 from more readily translatable strategies directed toward weeks there is induction of myogenic and inflammatory improving the remaining tissue, allowing them to engage genes [13, 46]. Unique to VML injury however is the in more daily actives, and improving quality of life. Strat- duration of inflammatory gene induction, which appears egies to improve the quality of the remaining muscle to be both heightened and prolonged following injury may also better prepare the individual to take advantage [34, 47] compared to common endogenously healing of advanced regenerative engineering approaches to injuries . This work investigated the early genetic regenerate tissue as they become available in the future. response to VML injury inflammatory, neurogenic, This work developed and evaluated early rehabilitation fibrotic, myogenic, and metabolic genes over the first 2 paradigms to understand the metabolic, genetic, and weeks post-VML. Few selected genes were functional response of the remaining tissue after a down-regulated following VML injury alone; specifically multi-muscle VML injury, in efforts to improve the Mstn, Slc2a4, and Ppargc1a downregulation occurred at muscle remaining following injury. We expect that iden- all time points through 2 weeks post-injury. Primarily tifying genetic and functional plasticity in the remaining there was a substantial up-regulation of probed genes skeletal muscle with early rehabilitation approaches may following VML injury. In particular inflammatory genes facilitate evidenced-based practice at the clinical level probed appear to most up-regulated at 3 days over 7 and following further translation. Herein we suggest that the 14 days post-VML. Notably the expression at both 7 and remaining tissue following VML injury beneficially 14 days was still greatly up-regulated from uninjured adapts to early rehabilitation, but that limitations in the muscle. Early rehabilitation appears to dampen this metabolic plasticity of the muscle still exist. Greising et al. BMC Musculoskeletal Disorders (2018) 19:173 Page 10 of 11 Additional files Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Table S1. Genes probed following VML injury and early rehabilitation. (DOCX 22 kb) Author details Additional file 2: Figure S1. Effect of VML injury and rehabilitation on Extremity Trauma and Regenerative Medicine, United States Army Institute study endpoint body mass and gastrocnemius muscle mass. a There was of Surgical Research, Fort Sam Houston, Texas 78234, USA. Department of a main effect of time, independent group, for body mass indicating mice Physical Therapy, Byrdine F. Lewis School of Nursing and Health Professions, at 4 Month post-VML injury weighed significantly more (~ 6%) than mice Georgia State University, Atlanta, GA 30302, USA. Department of Kinesiology, at 1 Month and 2 Month post-VML injury. b There was a main effect of University of Georgia, Athens, GA 30602, USA. Regenerative Bioscience time, independent of group, for injured gastrocnemius muscle mass as a Center, University of Georgia, Athens, GA 30602, USA. fraction of the contralateral uninjured control indicating mice at 4 Month post-VML injury had significantly more (~ 13%) proportional injured Received: 1 February 2018 Accepted: 16 May 2018 gastrocnemius muscle mass than mice at 1 Month. (JPG 839 kb) Additional file 3: Figure S2. Representative torque-time waveforms during peak isometric contraction from 4 Month VML, ROM, and ROM-E groups compared to completely uninjured controls. The rate of relaxation References for all terminal peak isometric contractions was evaluated. The rate of 1. Corona BT, Rivera JC, Owens JG, Wenke JC, Rathbone CR. Volumetric muscle relaxation was greater following ROM-E rehabilitation compared to VML- loss leads to permanent disability following extremity trauma. J Rehabil Res alone and ROM rehabilitation, independent of time. Control = 576 ± 34 Dev. 2015;52(7):785–92. mN●m sec − 1. (JPG 99 kb) 2. Garg K, Ward CL, Hurtgen BJ, Wilken JM, Stinner DJ, Wenke JC, Owens JG, Corona BT. Volumetric muscle loss: persistent functional deficits beyond Additional file 4: Table S2. Fold change in gene expression (vs. control) frank loss of tissue. J Orthop Res. 2015;33(1):40–6. following VML injury. (DOCX 30 kb) 3. Grogan BF, Hsu JR. Skeletal trauma research C. Volumetric muscle loss J Am Acad Orthop Surg. 2011;19(Suppl 1):S35–7. 4. Corona BT, Rivera JC, Greising SM. Inflammatory and Physiological Abbreviations consequences of debridement of fibrous tissue after volumetric muscle loss E: Intermittent electrical stimulation; Pt-Ir: Platinum-iridium; ROM: Range of injury. Clin Transl Sci. 2017; motion; VML: Volumetric muscle loss 5. Rivera JC, Corona BT. Muscle-related disability following combat injury increases with time. US Army Med Dep J. 2016:30–4. Acknowledgements 6. Greising SM, Dearth CL, Regenerative CBT. Rehabilitative medicine: a We thank Ms. Monica Jalomo, Mr. Javier Chapa, and Mr. Zach Agan for necessary synergy for functional recovery from volumetric muscle loss technical assistance in the completion of these studies. injury. Cells Tissues Organs. 2016;202(3–4):237–49. 7. Corona BT, Wenke JC, Ward CL. Pathophysiology of volumetric muscle loss injury. Cells Tissues Organs. 2016;202(3–4):180–8. Funding 8. Gentile NE, Stearns KM, Brown EH, Rubin JP, Boninger ML, Dearth CL, This research received funding from the Alliance for Regenerative Rehabilitation 3 Ambrosio F, Badylak SF. Targeted rehabilitation after extracellular matrix Research & Training (AR T) awarded to SMG and JAC, which is supported by scaffold transplantation for the treatment of volumetric muscle loss. Am J the Eunice Kennedy Shriver National Institute of Child Health and Human Phys Med Rehabil. 2014;93(11 Suppl 3):S79–87. Development (NICHD), National Institute of Neurological Disorders and Stroke 9. Mase VJ Jr, Hsu JR, Wolf SE, Wenke JC, Baer DG, Owens J, Badylak SF, (NINDS), and National Institute of Biomedical Imaging and Bioengineering Walters TJ. Clinical application of an acellular biologic scaffold for surgical (NIBIB) of the National Institutes of Health under Award Number P2CHD086843. repair of a large, traumatic quadriceps femoris muscle defect. Orthopedics. The content is solely the responsibility of the authors and does not necessarily 2010;33(7):511. represent the official views of the National Institutes of Health. 10. Sicari BM, Rubin JP, Dearth CL, Wolf MT, Ambrosio F, Boninger M, Turner NJ, Weber DJ, Simpson TW, Wyse A et al. An acellular biologic scaffold Availability of data and materials promotes skeletal muscle formation in mice and humans with volumetric The datasets used and/or analyzed during the current study are primarily muscle loss. Sci Transl Med 2014; 6(234):234ra258. presented in the current manuscript and are available from the 11. Aurora A, Garg K, Corona BT, Walters TJ. Physical rehabilitation improves corresponding author on reasonable request. muscle function following volumetric muscle loss injury. BMC Sports Sci Med Rehabil. 2014;6(1):41. 12. Aurora A, Roe JL, Corona BT, Walters TJ. An acellular biologic scaffold does Declarations not regenerate appreciable de novo muscle tissue in rat models of The opinions or assertions contained here are the private views of the volumetric muscle loss injury. Biomaterials. 2015;67:393–407. authors and are not to be construed as official or as reflecting the views of 13. Corona BT, Garg K, Ward CL, McDaniel JS, Walters TJ, Rathbone CR. the Department of the Army, the Department of Defense, or the United Autologous minced muscle grafts: a tissue engineering therapy for the States Government. volumetric loss of skeletal muscle. Am J Physiol Cell Physiol. 2013; 305(7):C761–75. Authors’ contributions 14. Quarta M, Cromie M, Chacon R, Blonigan J, Garcia V, Akimenko I, Hamer M, JAC and SMG designed the study. JAC, GLW, WMS, ASN, AMQ, and SMG Paine P, Stok M, Shrager JB, et al. Bioengineered constructs combined with performed experiments and collected data. JAC, BTC, and SMG analyzed and exercise enhance stem cell-mediated treatment of volumetric muscle loss. interpreted the data. JAC and SMG wrote the manuscript. All authors have Nat Commun. 2017;8:15613. read and approved the final version of this manuscript. 15. 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BMC Musculoskeletal Disorders – Springer Journals
Published: May 29, 2018
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