Regenerating Myofibers after an Acute Muscle Injury: What Do We Really Know about Them?

Injury to skeletal muscle through trauma, physical activity, or disease initiates a process called muscle regeneration. When injured myofibers undergo necrosis, muscle regeneration gives rise to myofibers that have myonuclei in a central position, which contrasts the normal, peripheral position of myonuclei. Myofibers with central myonuclei are called regenerating myofibers and are the hallmark feature of muscle regeneration. An important and underappreciated aspect of muscle regeneration is the maturation of regenerating myofibers into a normal sized myofiber with peripheral myonuclei. Strikingly, very little is known about processes that govern regenerating myofiber maturation after muscle injury. As knowledge of myofiber formation and maturation during embryonic, fetal, and postnatal development has served as a foundation for understanding muscle regeneration, this narrative review discusses similarities and differences in myofiber maturation during muscle development and regeneration. Specifically, we compare and contrast myonuclear positioning, myonuclear accretion, myofiber hypertrophy, and myofiber morphology during muscle development and regeneration. We also discuss regenerating myofibers in the context of different types of myofiber necrosis (complete and segmental) after muscle trauma and injurious contractions. The overall goal of the review is to provide a framework for identifying cellular and molecular processes of myofiber maturation that are unique to muscle regeneration.

Intercellular Adhesion Molecule-1 Enhances Myonuclear Transcription during Injury-Induced Muscle Regeneration.

The local inflammatory environment of injured skeletal muscle contributes to the resolution of the injury by promoting the proliferation of muscle precursor cells during the initial stage of muscle regeneration. However, little is known about the extent to which the inflammatory response influences the later stages of regeneration when newly formed (regenerating myofibers) are accumulating myonuclei and undergoing hypertrophy. Our prior work indicated that the inflammatory molecule ICAM-1 facilitates regenerating myofiber hypertrophy through a process involving myonuclear positioning and/or transcription. The present study tested the hypothesis that ICAM-1 enhances global transcription within regenerating myofibers by augmenting the transcriptional activity of myonuclei positioned in linear arrays (nuclear chains). We found that transcription in regenerating myofibers was ~2-fold higher in wild type compared with ICAM-1-/- mice at 14 and 28 days post-injury. This occurred because the transcriptional activity of individual myonuclei in nuclei chains, nuclear clusters, and a peripheral location were ~2-fold higher in wild type compared with ICAM-1-/- mice during regeneration. ICAM-1's enhancement of transcription in nuclear chains appears to be an important driver of myofiber hypertrophy as it was statistically associated with an increase in myofiber size during regeneration. Taken together, our findings indicate that ICAM-1 facilitates myofiber hypertrophy after injury by enhancing myonuclear transcription.

Positional Context of Myonuclear Transcription During Injury-Induced Muscle Regeneration.

Fundamental aspects underlying downstream processes of skeletal muscle regeneration, such as myonuclear positioning and transcription are poorly understood. This investigation begins to address deficiencies in knowledge by examining the kinetics of myonuclear accretion, positioning, and global transcription during injury-induced muscle regeneration in mice. We demonstrate that myonuclear accretion plateaus within 7 days of an injury and that the majority (∼70%) of myonuclei are centrally aligned in linear arrays (nuclear chains) throughout the course of regeneration. Relatively few myonuclei were found in a peripheral position (∼20%) or clustered (∼10%) together during regeneration. Importantly, transcriptional activity of individual myonuclei in nuclear chains was high, and greater than that of peripheral or clustered myonuclei. Transcription occurring primarily in nuclear chains elevated the collective transcriptional activity of regenerating myofibers during the later stage of regeneration. Importantly, the number of myonuclei in chains and their transcriptional activity were statistically correlated with an increase in myofiber size during regeneration. Our findings demonstrate the positional context of transcription during regeneration and highlight the importance of centralized nuclear chains in facilitating hypertrophy of regenerating myofibers after injury.

Myogenic Cell Expression of Intercellular Adhesion Molecule-1 Contributes to Muscle Regeneration after Injury.

This study investigated intercellular adhesion molecule-1 (ICAM-1), a membrane protein that mediates cell-to-cell adhesion and communication, as a mechanism through which the inflammatory response facilitates muscle regeneration after injury. Toxin-induced muscle injury to tibialis anterior muscles of wild-type mice caused ICAM-1 to be expressed by a population of satellite cells/myoblasts and myofibers. Myogenic cell expression of ICAM-1 contributed to the restoration of muscle structure after injury, as regenerating myofibers were more abundant and myofiber size was larger for wild-type compared with Icam1-/- mice during 28 days of recovery. Contrastingly, restoration of muscle function after injury was similar between the genotypes. ICAM-1 facilitated the restoration of muscle structure after injury through mechanisms involving the regulation of myofiber branching, protein synthesis, and the organization of nuclei within myofibers after myogenic cell fusion. These findings provide support for a paradigm in which ICAM-1 expressed by myogenic cells after muscle injury augments their adhesive and fusogenic properties, which, in turn, facilitates regenerative and hypertrophic processes that restore structure to injured muscle.

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury.

Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. STATEMENT OF SIGNIFICANCE: COX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model. The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Expression of intercellular adhesion molecule-1 by myofibers in mdx mice.

INTRODUCTION: We investigated the extent to which intercellular adhesion molecule-1 (ICAM-1), a critical protein of the inflammatory response, is expressed in skeletal muscles of mdx mice (a murine model of Duchenne muscular dystrophy). METHODS: Muscles were collected from control and mdx mice at 2-24 weeks of age and analyzed for ICAM-1 expression by means of Western blot and immunofluorescence. RESULTS: Western blot revealed higher expression of ICAM-1 in mdx compared with control muscles through 24 weeks of age. In contrast to control muscles, ICAM-1 was expressed on the membrane of damaged, regenerating, and normal myofibers of mdx mice. CD11b+ myeloid cells also expressed ICAM-1 in mdx muscles, and CD11b+ cells were closely associated with the membrane of myofibers expressing ICAM-1. CONCLUSIONS: These findings support a paradigm in which ICAM-1 and its localization to myofibers in muscles of mdx mice contributes to the dystrophic pathology.

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle.

Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

We previously reported that leukocyte specific ß2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for ß2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a ß2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and ß2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with ß2 integrin expressing myeloid cells.

Do inflammatory cells influence skeletal muscle hypertrophy?

Most research on muscle hypertrophy has focused on the responses of muscle cells to mechanical loading; however, a number of studies also suggest that inflammatory cells may influence muscle hypertrophy. Neutrophils and macrophages accumulate in skeletal muscle following increased mechanical loading, and we have demonstrated that macrophages are essential for hypertrophy following synergist ablation. Whether neutrophils are required remains to be determined. Non-steroidal anti-inflammatory drugs impair adaptive responses of skeletal muscle in both human and animal experiments suggesting that the routine use of such drugs could impair muscle performance. Much remains to be learned about the role of inflammatory cells in muscle hypertrophy, including the molecular signals involved in calling neutrophils and macrophages to skeletal muscle as well as those that regulate their function in muscle. In addition, although we have demonstrated that macrophages produce growth promoting factors during muscle hypertrophy, the full range of functional activities involved in muscle hypertrophy remains to be determined. Further investigation should provide insight into the intriguing hypothesis that inflammatory cells play integral roles in regulating muscle hypertrophy.

Cytokines derived from cultured skeletal muscle cells after mechanical strain promote neutrophil chemotaxis in vitro.

We tested the hypothesis that cytokines derived from differentiated skeletal muscle cells in culture induce neutrophil chemotaxis after mechanical strain. Flexible-bottom plates with cultured human muscle cells attached were exposed to mechanical strain regimens (ST) of 0, 10, 30, 50, or 70 kPa of negative pressure. Conditioned media were tested for the ability to induce chemotaxis of human blood neutrophils in vitro and for a marker of muscle cell injury (lactate dehydrogenase). Conditioned media promoted neutrophil chemotaxis in a manner that was related both to the degree of strain and to the magnitude of muscle cell injury (ST 70 > ST 50 > ST 30). Protein profiling using a multiplex cytokine assay revealed that mechanical strain increased the presence of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, monocyte chemotactic protein (MCP)-1, and IL-6 in conditioned media. We also detected 14 other cytokines in conditioned media from control cultures that did not respond to mechanical strain. Neutralization of IL-8 and GM-CSF completely inhibited the chemotactic response for ST 30 and ST 50 and reduced the chemotactic response for ST 70 by 40% and 47%, respectively. Neutralization of MCP-1 or IL-6 did not reduce chemotaxis after ST 70. This study enhances our understanding of the immunobiology of skeletal muscle by revealing that skeletal muscle cell-derived IL-8 and GM-CSF promote neutrophil chemotaxis after injurious mechanical strain.

Beta2-integrins contribute to skeletal muscle hypertrophy in mice.

We tested the contribution of beta(2)-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common beta-subunit of beta(2)-integrins (CD18(-/-)), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18(-/-) mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in beta(2)-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18(-/-) mice. In conclusion, our findings indicate that beta(2)-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling.

Tumor necrosis factor-alpha promotes the accumulation of neutrophils and macrophages in skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has been associated with cachexia and is known to regulate multiple inflammatory cell (neutrophil and macrophage) responses. We tested the hypothesis that neutrophils and macrophages accumulate in the extensor digitorum longus (EDL) and soleus muscles of mice after chronic TNF-alpha administration. Murine recombinant TNF-alpha (approximately 100 microg x kg(-1) x day(-1)) in vehicle solution or vehicle solution alone (sham) was administered to C57BL/6 mice for 7 days via osmotic minipumps. In EDL muscles from TNF-alpha-treated mice, neutrophil and macrophage concentrations were elevated seven- and threefold, respectively, compared with sham mice. Neutrophil and macrophage concentrations were also elevated five- and twofold, respectively, in solei of TNF-alpha- relative to sham-treated mice. Treatment with TNF-alpha elevated ubiquitin content by approximately 25% relative to sham values for both the EDL and soleus muscles; however, these elevations were not statistically significant. No differences were observed between TNF-alpha- and sham-treated mice in body weight, food consumption, muscle mass, myofiber cross-sectional area, carbonyl groups, total protein content, or relative abundance of myosin heavy chain protein. Furthermore, no overt signs of muscle injury or regeneration were observed in muscles from TNF-alpha-treated mice in either the EDL or soleus muscles. These observations suggest that 7 days of TNF-alpha administration promote muscle inflammation as indicated by the accumulation of neutrophils and macrophages without overt signs of atrophy, injury, or regeneration.

Neutrophils contribute to muscle injury and impair its resolution after lengthening contractions in mice.

We tested the hypotheses that: (1) neutrophil accumulation after contraction-induced muscle injury is dependent on the beta(2) integrin CD18, (2) neutrophils contribute to muscle injury and oxidative damage after contraction-induced muscle injury, and (3) neutrophils aid the resolution of contraction-induced muscle injury. These hypotheses were tested by exposing extensor digitorum longus (EDL) muscles of mice deficient in CD18 (CD18(-/-); Itgb2(tm1Bay)) and of wild type mice (C57BL/6) to in situ lengthening contractions and by quantifying markers of muscle inflammation, injury, oxidative damage and regeneration/repair. Neutrophil concentrations were significantly elevated in wild type mice at 6 h and 3 days post-lengthening contractions; however, neutrophils remained at control levels at these time points in CD18-/- mice. These data indicate that CD18 is required for neutrophil accumulation after contraction-induced muscle injury. Histological and functional (isometric force deficit) signs of muscle injury and total carbonyl content, a marker of oxidative damage, were significantly higher in wild type relative to CD18-/- mice 3 days after lengthening contractions. These data show that neutrophils exacerbate contraction-induced muscle injury. After statistically controlling for differences in the force deficit at 3 days, wild type mice also demonstrated a higher force deficit at 7 days, a lower percentage of myofibres expressing embryonic myosin heavy chain at 3 and 7 days, and a smaller cross sectional area of central nucleated myofibres at 14 days relative to CD18-/- mice. These observations suggest that neutrophils impair the restoration of muscle structure and function after injury. In conclusion, neutrophil accumulation after contraction-induced muscle injury is dependent on CD18. Furthermore, neutrophils appear to contribute to muscle injury and impair some of the events associated with the resolution of contraction-induced muscle injury.

Mechanical loading and injury induce human myotubes to release neutrophil chemoattractants.

The purpose of this study was to 1) test the hypothesis that skeletal muscle cells (myotubes) after mechanical loading and/or injury are a source of soluble factors that promote neutrophil chemotaxis and superoxide anion (O(2)(-).) production and 2) determine whether mechanical loading and/or injury causes myotubes to release cytokines that are known to influence neutrophil responses [tumor necrosis factor-alpha (TNF-alpha), IL-8, and transforming growth factor-beta1 (TGF-beta1)]. Human myotubes were grown in culture and exposed to either a cyclic strain (0, 5, 10, 20, or 30% strain) or a scrape injury protocol. Protocols of 5, 10, and 20% strain did not cause injury, whereas 30% strain and scrape injury caused a modest and a high degree of injury, respectively. Conditioned media from strained myotubes promoted chemotaxis of human blood neutrophils and primed them for O(2)(-). production in a manner that was dependent on a threshold of strain and independent from injury. Neutrophil chemotaxis, but not priming, progressively increased with higher magnitudes of strain. Conditioned media only from scrape-injured myotubes increased O(2)(-). production from neutrophils. Concentrations of IL-8 and total TGF-beta1 in conditioned media were reduced by mechanical loading, whereas TNF-alpha and active TGF-beta1 concentrations were unaffected. In conclusion, skeletal muscle cells after mechanical loading and injury are an important source of soluble factors that differentially influence neutrophil chemotaxis and the stages of neutrophil-derived reactive oxygen species production. Neutrophil responses elicited by mechanical loading, however, did not parallel changes in the release of IL-8, TGF-beta1, or TNF-alpha from skeletal muscle cells.

Downhill running in rats: influence on neutrophils, macrophages, and MyoD+ cells in skeletal muscle.

The accumulation of neutrophils and macrophages, as well as the activation of satellite cells, are early events following skeletal muscle injury. We examined the temporal relationship between changes in neutrophils, macrophages, and MyoD protein, a marker of satellite cell activation, after injurious exercise. Male rats ( n=47) performed an intermittent downhill (-16% grade) running (17 m/min) protocol and the solei were obtained at 0, 2, 6, 24, 48, or 72 h post-exercise. Neutrophils, macrophages (ED1 and ED2), and MyoD+ cells were determined in muscle cross sections using immunohistochemistry. Downhill running increased ( P

Deferoxamine reduces and nitric oxide synthase inhibition increases neutrophil-mediated myotube injury.

We tested the contribution of reactive oxygen species (ROS), reactive nitrogen species (RNS) and the beta 2 integrin CD18 to neutrophil-mediated myotube injury. Human myotubes were cultured with human neutrophils in the presence or absence of inhibitors directed against ROS, RNS, and CD18. Muscle injury was assessed by a (51)Cr release assay. The inclusion of superoxide dismutase (50-500 U/ml) in the culture medium did not affect myotube injury. A significant protective effect was provided by including catalase (600-2400 U/ml), deferoxamine (1-2 mM), or anti-CD18 antibody (10 microg/ml) in the culture medium. S-Ethylisothiourea (500-1000 microM), an inhibitor of nitric oxide synthase (NOS), significantly increased myotube injury and reduced nitric oxide (NO) in cultures consisting of only myotubes. In conclusion, neutrophil-mediated skeletal muscle injury appears to be largely dependent on CD18-mediated neutrophil adhesion and iron-dependent hydroxyl radical production. In addition, skeletal muscle NOS activity may protect skeletal muscle against the injury caused by neutrophils.

Passive stretches protect skeletal muscle of adult and old mice from lengthening contraction-induced injury.

We tested the hypothesis that a single bout of training with passive stretches or isometric contractions protects the extensor digitorum longus muscle in old mice from contraction-induced injury. Lengthening contractions produced similar decreases in force (approximately 70%-80%) and numbers of overtly injured fibers (approximately 15%-20%) in adult and old mice, but twofold greater inflammatory cell accumulation above untreated control values in old versus adult mice. For both age groups, prior training with passive stretches improved postinjury force almost twofold compared with untrained muscles and reduced injured fibers by one half. Training with passive stretches or isometric contractions reduced inflammatory cell accumulation following lengthening contractions by as much as two thirds in old mice, but not in adult mice. The data indicate that passive stretches provide some protection against contraction-induced injury in old mice, and that accumulation of inflammatory cells does not correlate strongly with force deficit and number of injured fibers.

Ibuprofen and acetaminophen: effect on muscle inflammation after eccentric exercise.

PURPOSE: We examined the influence of ibuprofen and acetaminophen on muscle neutrophil and macrophage concentrations after novel eccentric contractions. METHODS: Twenty-four males (25 +/- 3 yr) were divided into three groups that received the maximal over-the-counter dose of either ibuprofen (1200 mg x d-1), acetaminophen (4000 mg x d-1), or a placebo after eccentric contractions of the knee extensors. Biopsies from the vastus lateralis were taken before and 24 h after exercise. Inflammatory cells were quantified in muscle cross-sections using immunohistochemistry. RESULTS: Macrophage concentrations were elevated by 1.5- to 2.5-fold (P < 0.05) at 24 h postexercise relative to preexercise concentrations, whereas neutrophil concentrations were not significantly elevated. Muscle inflammatory cell concentrations were unaffected by treatment with ibuprofen or acetaminophen when compared with placebo. CONCLUSIONS: Maximal over-the-counter doses of ibuprofen or acetaminophen, when administered therapeutically, do not affect muscle concentrations of neutrophils or macrophages 24 h after a novel bout of eccentric contractions.