INTRODUCTION

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SKELETAL MUSCLE RESPONSES to mechanical loading are influenced by not only external load but also contraction type. Specifically, eccentric contractions elicit greater perturbations in signal transduction pathways associated with skeletal muscle hypertrophy and higher rates of protein synthesis relative to concentric contractions (2, 27, 44, 45). In addition, eccentric contractions are more likely to cause muscle dysfunction and histological abnormalities (overt muscle injury) compared with concentric contractions (1, 6, 23, 30). The overt injury after eccentric contractions is accompanied by activation of the acute inflammatory response, as indicated by elevations of neutrophils and/or macrophages in skeletal muscle (15, 33, 41, 43). Whether concentric contractions influence the concentrations of neutrophils and/or macrophages in skeletal muscle, however, is unknown.

In theory, inflammatory cells could accumulate in skeletal muscle after concentric contractions because of an increase in reactive oxygen species production and/or NF-B activation (11, 19). Both reactive oxygen species and NF-B activation can initiate inflammatory cell chemotaxis via the production of oxidatively modified proteins (32) and chemokines (12), respectively. To date, the function of neutrophils and macrophages in skeletal muscle is generally thought to be limited to the phagocytosis of injured tissue via their arsenal of free radicals and proteases (25, 31). Inflammatory cells, however, can release cytokines, chemokines, and growth factors independent of phagocytosis (4, 29). Because inflammatory cell-derived products are known to influence the transcription of redox-sensitive genes, myoblast proliferation and differentiation, muscle growth, and angiogenesis (9, 10, 39, 46), inflammatory cells may have important biological functions in skeletal muscle that are independent from the phagocytosis of tissue debris after overt injury.

The purpose of the study was to test the hypothesis that both eccentric and concentric contractions elevate muscle concentrations of neutrophils and macrophages (ED1⁺ and ED2⁺). Muscle contractions in rats were elicited via electrical stimulation of the sciatic nerve, which results in maximal contraction of all hindlimb muscles in the distal compartment (2, 20, 21, 27, 44, 45). In this model, the plantar flexors [gastrocnemius, soleus (Sol), and plantaris (Pln)] undergo concentric contractions, and the dorsiflexors [tibialis anterior (TA) and extensor digitorum longus (EDL)] undergo eccentric contractions (44, 45). Therefore, the TA, Pln, and Sol were used to determine changes in neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages after both eccentric and concentric contractions. Because the electrical muscle stimulation model used in the present study is a frequently used animal paradigm to evaluate the time course of changes in molecular, cellular, and tissue level events associated with resistance exercise (2, 20, 21, 27, 44), we also sought to characterize the cellular events more completely by quantifying the time course of changes in neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages.

	MATERIALS AND METHODS

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Animals. All procedures were approved by the Animal Care Committee at the University of Illinois at Chicago and were in accordance with Guidelines for Care and Use of Laboratory Animals. Six- to seven-week-old female Wistar rats (n = 18) (Charles River Laboratories, Wilmington, MA), weighing between 200 and 250 g at the time of death, were maintained on a constant 12:12-h light-dark cycle with food and water available ad libitum. On arrival, animals were allowed to acclimatize for 6 days before commencement of any experimental procedures. The surgical and electrical stimulation procedures were performed under anesthesia (pentobarbital sodium, 50 mg/kg ip; supplemental doses as needed). After electrode implantation, animals were allowed to recover for 5 days before initiation of the stimulation protocol (2).

Stimulation protocol. Muscle contractions were induced by a single bout of high-frequency electrical stimulation (HFES), as previously described (27). Multistrand electrodes (Medwire, Mount Vernon, NY) were implanted on both sides of the right sciatic nerve above the anatomic branching point. Tetanic contractions were delivered with the use of a Grass S5 stimulator (Grass Instruments, Quincy, MA) at 100 Hz, 6-12 V, 1-ms duration, 9-ms delay, for 10 sets of six repetitions, with each repetition lasting 3 s. A 10-s delay was given between repetitions and 1 min between sets. The stimulation protocol lasted a total time of 20 min. This stimulation protocol causes maximal activation of all distal hindlimb muscles and significantly reduces muscle glycogen levels in the TA and Sol (77.9 and 51%, respectively) (27). Training with this protocol has been reported to cause skeletal muscle hypertrophy in the dorsiflexor muscles (2, 45).

Immunohistochemistry of inflammatory cells. Sol, Pln, and TA muscles were excised at 6, 24, and 72 h after HFES, coated with optimum cutting temperature compound, frozen in melting isopentane cooled on dry ice, and stored at 80°C. Muscles from the contralateral limb served as controls. A minimum of four and a maximum of seven rats were analyzed at each time point. Cross sections (10 µm) were cut from the muscle midbelly, adhered to chromium potassium sulfate and gelatin-treated glass slides, and frozen at 20°C. Muscle sections were prepared for immunohistochemistry as previously described (7). The primary antibodies for neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages, which were incubated for 2 h at room temperature, were an anti-rat neutrophil (HIS48; 1:25; PharMingen, Franklin Lake, NJ), mouse anti-rat ED1 (1:100; Serotec; Oxford, UK), and mouse anti-rat ED2 (1:100; Serotec), respectively. The sections were then washed in PBS and incubated with either biotinylated goat anti-mouse IgM (1:200; Vector Laboratories; Burlingame, CA) (HIS48) or biotinylated horse anti-mouse IgG (1:200, Vector Laboratories) (ED1 and ED2) for 30 min. After incubation with the secondary antibody, sections were washed with PBS and incubated with horseradish peroxidase (1:1,000; Vector Laboratories). After three washes, the antibody-antigen complex was developed by using the peroxidase substrate kit 3-amino-9-ethylcarbazole (Vector Laboratories).

Histological evaluation. Muscle cross sections (10 µm) from exercise and contralateral control muscles were labeled numerically, stained with hematoxylin and eosin, and examined for evidence of muscle injury, as previously described (13). With the use of light microscopy, each muscle section was blindly classified as injured if it exhibited two or more of the following criteria: 1) invasion of myofibers with cells, 2) pale or diffuse staining cytoplasm, and 3) centrally located nuclei. Because these criteria have been shown to be significantly elevated at 3 days after eccentric contractions in mice (13), only the 72-h post-HFES time point was used for the assessment of overt muscle injury. After all muscles were examined, the numeric labeling code was revealed, and the muscles were then classified as exercise or contralateral control.

Statistics. Because the purpose of the study was to determine the effect of HFES on inflammatory cells in the individual muscles and not to compare responses between the muscles, inflammatory cells in Sol, Pln, and TA muscles were analyzed by separate two-way ANOVA tests. The Newman-Keuls post hoc test was used to determine differences between means when the observed F-ratio was significant (P  0.05). All data are represented as means ± SE.

	RESULTS

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Our results demonstrate that concentric contractions can elicit significant elevations in muscle inflammatory cell concentrations. Specifically, neutrophils were elevated in both the Pln (interaction; Fig. 1A) and Sol (interaction; Fig. 2A) muscles at 6 and 24 h relative to contralateral control muscles. ED1⁺ macrophages in the Pln were elevated at 6 and 24 h (interaction; Fig. 1B). In the Sol, ED1⁺ macrophages were higher after HFES (main effect), but there was no significant interaction (Fig. 2B). ED2⁺ macrophages were elevated in the Pln (main effect; Fig. 1C) and were higher in the Sol at 24 h after HFES relative to controls (interaction; Fig. 2C).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Inflammatory cell concentrations after electrically stimulated concentric contractions of the plantaris muscles: neutrophils (HIS48+; A), ED1+ macrophages (B), and ED2+ macrophages (C). HFES, high-frequency electrical stimulation; CT, contralateral controls. Values are means ± SE; n, no. of rats. * Significantly higher for HFES relative to CT at specified time points, P  0.05. # Significantly elevated after HFES relative to CT (main effect), P  0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Inflammatory cell concentrations after electrically stimulated concentric contractions of the soleus muscles: neutrophils (HIS48+; A), ED1+ macrophages (B), and ED2+ macrophages (C). Values are means ± SE; n, no. of rats. * Significantly higher for HFES relative to CT at specified time points, P  0.05. # Significantly elevated after HFES relative to CT (main effect), P  0.05.

Eccentric contractions of the TA muscles resulted in higher neutrophil concentrations at 6 and 24 h after HFES relative to control muscles (interaction; Fig. 3A). ED1⁺ macrophages were higher in the TA muscles after HFES (main effect), but there was no significant interaction (Fig. 3B). ED2⁺ macrophages in the TA muscles were higher 72 h after HFES relative to control muscles (interaction; Fig. 3C).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Inflammatory cell concentrations after electrically stimulated eccentric contractions of the tibialis anterior muscles: neutrophils (HIS48+; A), ED1+ macrophages (B), and ED2+ macrophages (C). Values are means ± SE; n, no. of rats. * Significantly higher for HFES relative to CT at specified time points, P  0.05. # Significantly elevated after HFES relative to CT (main effect), P  0.05.

Histological evaluation of muscle cross sections by hematoxylin and eosin staining revealed that myofibers in the Pln (Fig. 4A) and Sol (Fig. 4B) muscles showed no gross histological signs of injury. In contrast to the Pln and Sol, the eccentrically contracted TA muscles exhibited gross histological abnormalities, evidenced by altered cytoplasmic staining and profound invasion by cells (Fig. 4C). None of the control muscles exhibited histological characteristics of overt muscle injury.

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Muscle cross sections obtained 72 h after HFES and stained with hematoxylin and eosin (magnification ×400). Concentric contractions of the plantaris (A) and soleus (B) muscles showed no gross histological signs of overt injury. In contrast to the plantaris and soleus, eccentric contractions of the tibialis anterior (C) muscles resulted in overt injury as indicated by a substantial number of myofibers that had a pale cytoplasm (arrowhead) and/or were invaded by cells (arrow).

	DISCUSSION

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The novel observations in the present study were the elevations in neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages in the Pln and Sol after concentric contractions (Figs. 1 and 2, respectively). As expected, inflammatory cells were also increased after eccentric contractions of the TA (Fig. 3). Because both the Pln and Sol showed no gross histological abnormalities (Fig. 4, A and B, respectively), the observed changes in these muscles may indicate that overt injury is not the sole determinant for the accumulation of neutrophils and macrophages in skeletal muscle. These observations may also indicate that inflammatory cells have biological functions in skeletal muscle that extend beyond their proposed role in the phagocytosis of tissue debris after overt muscle injury. Because of the vast array of inflammatory cell-derived products (23, 35), inflammatory cells in skeletal muscle (injured or noninjured) may also function as a source of cellular signals that contribute to skeletal muscle adaptations after mechanical loading.

Previous investigators have examined muscle neutrophils and/or macrophages after eccentric contractions (15, 33, 41, 43) and in the hindlimb-suspension reloading model of muscle injury (7, 42). In the present study, the elevations in neutrophils in the TA are consistent with our observations of rat Sol after downhill running (43) and with our findings in mice after in situ eccentric contractions of the EDL (33). Previous investigators using the hindlimb-suspension model of muscle injury have also reported elevations in neutrophils in rat Sol at 6 and 24 h after muscle reloading (7, 42). These studies, however, are in contrast to a recent study by Lapointe et al. (15), who reported that neutrophils are not elevated in the EDL muscles of young rats after in situ eccentric contractions. Conflicting neutrophil results are not easily reconciled but may be attributable to the age of the female Wistar rats (50-70 g) in the study by Lapointe et al. relative to ours (200-250 g). In addition, Lapointe et al. used an antibody against leukosialin (CD43; clone W3/13; Serotec) to identify neutrophils in cross sections. This antibody may not be an appropriate marker of neutrophils in skeletal muscle, because CD43 is expressed by other leukocytes and is shed from neutrophils when activated and when they adhere to endothelial cells (3, 35). The time course of elevations in ED1⁺ and ED2⁺ macrophages after eccentric contractions in the present study is consistent with the observations of Lapointe et al. (15) and with previous hindlimb-suspension studies (7, 42).

When the relationship between inflammatory cells and overt signs of muscle injury is based on temporal analysis, the accumulation of inflammatory cells in skeletal muscle has traditionally been attributed to events associated with muscle injury and the subsequent regeneration (9, 36). This interpretation is a reasonable explanation for the elevated inflammatory cells in the TA (Fig. 3) because 1) all of the exercised TA muscles in the present study showed gross histological abnormalities at 72 h post-HFES (Fig. 4C), and 2) the same electrical muscle stimulation model used in the present study has been reported to cause overt injury to the TA, as indicated by a 35% force deficit 2 days after the stimulation protocol (20, 21). In contrast, concentric contractions of the Pln (Fig. 1) and Sol (Fig. 2) caused an elevation in neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages in the absence of gross histological abnormalities (Fig. 4, A and B, respectively). The lack of histological abnormalities in the Pln and Sol is consistent with previous studies that have established that concentric contractions do not cause overt muscle injury (6, 23, 30). Taken together, these observations may indicate that overt muscle injury is not the sole prerequisite for the accumulation of inflammatory cells in skeletal muscle after mechanical loading. This interpretation is consistent with our laboratory's recent report (33) of elevated neutrophils, but not macrophages, in the absence of overt injury in mice after isometric contractions and passive stretches. An alternative explanation of our findings is that some degree of minor injury (i.e., injury that does not result in a functional impairment or gross histological abnormalities) may have occurred after concentric contractions, and thus one or more chemoattractant(s) for inflammatory cells may have been produced and/or released.

Because the amount of force produced by the TA, Sol, and Pln in this model is unknown, the influence of force development on the accumulation of inflammatory cells in these muscles cannot be addressed. Our laboratory has previously demonstrated that passive stretching elevates neutrophils, but not macrophages, to the same level as maximal isometric contractions (33). These observations may indicate that the amount of force produced by a muscle per se is not a critical determinant for the accumulation of inflammatory cells in skeletal muscle.

Unfortunately, because very little is known about factors that orchestrate the accumulation of inflammatory cells in skeletal muscle, it is difficult to speculate as to which chemoattractants may have been produced and/or released after eccentric and concentric contractions. Potential candidates include fragments of complement proteins (e.g., C5a) and chemoattractants derived from endothelial cells, skeletal muscle cells, and/or resident cells in skeletal muscle (reviewed in Refs. 4, 9, 14, 18, 40). The vascular endothelium is a potential source of chemoattractants because it represents an initial barrier that neutrophils and monocytes must cross before entering skeletal muscle, and because the vascular endothelium is capable of producing numerous chemoattractants for neutrophils and macrophages, including chemokines (e.g., IL-8 and monocyte chemotactic protein-1) and lipid mediators (e.g., platelet-activating factor and leukotriene B₄) (14). Recent evidence indicates that skeletal muscle cells are also capable of producing chemoattractants for either neutrophils and/or macrophages under basal and/or proinflammatory conditions, after injury, and in inflammatory myopathies. Such factors include IL-8 (5), monocyte chemotactic protein-1 (5), complement proteins (8, 16, 17), transforming growth factor- (28), and lipopolysaccharide-inducible CXC chemokine (38). Finally, resident fibroblasts and/or inflammatory cells in skeletal muscle may also be a source of chemoattractants for inflammatory cells (4, 40), with the latter source possibly serving as a positive feedback mechanism whereby greater numbers of neutrophils and macrophages could be attracted to skeletal muscle. Which, if any, of the above chemoattractants and/or sources contributed to the accumulation of neutrophils and/or macrophages in the present study remains to be determined.

Interestingly, some chemoattractants for inflammatory cells have been shown to activate and/or enhance their function, whereas others do not. For example, IL-8, a chemoattractant for neutrophils but not monocytes, has been shown to be capable of causing and enhancing the release of reactive oxygen species and proteases from neutrophils (4). Theoretically, the release of reactive oxygen species from neutrophils after mechanical loading could cause minor or overt injury to skeletal muscle, because they have been demonstrated to damage skeletal muscle during the reperfusion of ischemic tissue (37) and have been reported to injure cultured myotubes (26). On the other hand, transforming growth factor-₁, a potent chemoattractant for both neutrophils and monocytes, does not appear to influence reactive oxygen species production from neutrophils (4, 34) but stimulates monocytes to produce cytokines (e.g., tumor necrosis factor-, platelet-derived growth factor BB, and fibroblast growth factor) (22) that could influence skeletal muscle growth (9, 10, 46). Thus, depending on the chemoattractants produced within skeletal muscle (injured or noninjured), inflammatory cells and their derived products may have dichotomous actions in skeletal muscle after mechanical loading.

Neutrophils and ED1⁺ macrophages are believed to be responsible for the removal of cellular debris after overt injury based on their ability to perform phagocytosis and on limited qualitative observations (25, 31). ED2⁺ macrophages, which have a limited phagocytic capacity (24), have been hypothesized to contribute to the early events of muscle regeneration after overt injury by causing satellite cell activation and myoblast proliferation (9, 10). Thus the function of inflammatory cells in skeletal muscle has traditionally been limited to events that follow overt injury. The accumulation of neutrophils, ED1⁺ macrophages, and ED2⁺ macrophages in the Sol and Pln after concentric contractions, however, may indicate that these cells influence skeletal muscle in the absence of overt injury. Because inflammatory cells are capable of producing reactive oxygen species, reactive nitrogen species, cytokines, and growth factors that individually are known to influence skeletal muscle, the presence of inflammatory cells in noninjured muscle may serve as an additional source of cellular signals for muscle adaptations to mechanical loading. Such adaptations may include protection from injury, muscle hypertrophy, and angiogenesis. Further work is needed before the potential implications of our results can be fully appreciated.