Unaccustomed high-force eccentric exercise and extreme forms of exercise performed by athletes, such as marathon running, have been reported to cause muscle damage and accumulation of inflammatory cells (15,36). An acute, local inflammation is initiated by a rapid extravasation of fluid and blood-borne neutrophilic granulocytes into the damaged tissue (21). Peak accumulation of neutrophils occurs after 1-2 d, overlapped in time by accumulation of monocytes/macrophages, which peaks after several days (21). However, because of the methodological limitations of repeated biopsies and radionuclide imaging (radiolabeling of leukocytes), a detailed time course of the local inflammatory response to exercise in humans has so far not been established (23,29,37). In particular, the first 24 h after exercise has scarcely been investigated, and no study has hitherto aimed to explore development of the inflammatory reaction after exercise-induced muscle damage, using repeated biopsies during this time interval. Labeling and tracking of leukocytes (primarily neutrophils) with radionuclide imaging techniques have indicated that blood-borne leukocytes begin to accumulate in the exercised muscles immediately after exercise (20,33). However, such techniques give no information about the diapedesis of leukocytes. Thus, it is not possible to distinguish between the radiolabeled leukocytes that accumulate in the blood vessels and attach to the endothelial wall and those that actually have migrated into the tissue. Furthermore, it has recently been pointed out that published claims concerning animal and human studies are inconsistent in respect to the accumulation of neutrophilic granulocytes in exercise-injured muscles: neutrophils, in contrast to macrophages, have in some experiments not been observed at all (37).
From animal studies, it appears that inflammatory events can explain certain changes in muscle function (9). Interruption of recovery and a secondary loss of force, seen 1-3 d after exercise, have repeatedly been observed together with a marked tissue damage and an inflammatory reaction (9). Furthermore, pharmacological, antibody-mediated, and genetic (knockout) inhibition of the inflammatory reaction have reduced the secondary loss of muscle force (5,19,30). Altogether, these findings suggest a cause-effect relationship between the inflammatory response and the secondary loss of force. Because the exercise protocols used in animal studies in some extent are meant to model hard, human exercise, it is not unreasonable to assume that the inflammatory process may affect the recovery of muscle function in humans as well (9,21). In fact, biphasic or bimodal recovery of muscle function has been observed in human subjects (20,31,33), and accumulation of radiolabeled leukocytes in the exercised muscles have been found to forerun the secondary loss of force-generating capacity (20,33). Nevertheless, this has not been convincingly supported by biopsy methods documenting and enumerating leukocytes in the muscle tissue (23).
The time course of leukocyte accumulation after eccentric exercise seems to mirror not only changes in muscle function but also that of delayed onset muscle soreness (DOMS) (9,21,38). The mechanistic link might be a leukocyte-mediated release of cytotoxic and algetic substances (6,9). In particular, reactive oxygen species from neutrophils and macrophages seem to increase the muscle damage (41), whereas eicosanoids may be mediators of DOMS (1,6,38). However, the relationship between these events has not been explored thoroughly in humans. Noticeably, some investigators have even questioned the view that eccentric exercise can initiate an inflammatory response in the exercised muscles and/or that DOMS can be a consequence of this inflammation (see, e.g., [23,26,44]).
The purpose of this study was to explore the time course and the location of leukocyte accumulation after unaccustomed eccentric exercise, with emphasis on the first 24 h. This is the first study in which analyses of multiple biopsies from exercised and a nonexercised control muscles have been combined with scintigraphic imaging of transfused, autologous, radiolabeled leukocytes. These combined procedures make it possible to determine both exact tissue localization and overall distribution of leukocytes in whole muscle. Our main hypothesis has been that leukocytes begin infiltrating the exercised muscle tissue shortly after a bout of hard exercise, accumulating rapidly during the first 24 h, so that the accumulation of leukocytes would be closely related in time-and possibly also causally-to both the recovery of force-generating capacity and the development of muscle soreness.
MATERIALS AND METHODS
Subjects.
Eleven healthy male students (mean ± SD, age = 28 ± 4 yr, height = 1.80 ± 0.1 m, weight = 83 ± 6 kg) gave written, informed consent to participate in the study. The subjects' level of fitness varied due to a variable level of daily physical activity; three subjects were sedentary (subjects 4, 6, and 8), whereas the other eight ranged from "physically active" to "very active" (exercising 4-7 d·wk−1), but none of the subjects were engaged in heavy strength training. The study complied with the standards set by the Declaration of Helsinki and was approved by the Regional Ethics Committee of Southern Norway.
Experimental design.
Changes in muscle function were monitored for 1 wk after a bout of one-legged, maximal voluntary eccentric exercise (300 repetitions) with the knee extensors (musculus quadriceps femoris). The other leg (randomly chosen dominant/nondominant) functioned as control for all tests and measurements. The recovery of muscle function was assessed with repeated tests of voluntary maximal, isokinetic knee extensions. The first test was performed before the workout to establish baseline values, the second test started approximately 3 min after exercise, and tests were thereafter repeated 6, 23, and 28 h and 2, 3, 4, and 7 d after exercise. Blood sampling for creatine kinase (CK) measurements and evaluation of muscle soreness were scheduled just before assessment of muscle function.
Distribution of 99mTechnetium-(radio)labeled leukocytes was followed for 24 h-the duration was limited by the 6-h half-life of 99mTechnetium. Radionuclide images (scintigrams) of both thighs (anterior and lateral views) were taken at 3, 7, 21, and 24 h after exercise. Blood for radionuclide labeling of leukocytes was obtained by venipuncture at 7:30 a.m., and the labeled leukocytes were reintroduced intravenously 2 h later. This was followed by the exercise protocol, which started at 11:20 a.m. and lasted for 40 min. Six subjects underwent radiolabeling of leukocytes and scintigraphy.
Biopsies were collected from both exercised and control muscle 0.5, 4, 8, 24, 96, and 168 h after exercise. With the frequent biopsy sampling during the first day, we wanted to obtain a detailed time course for the initial events, including tissue accumulation of leukocytes. The later biopsy times (4 and 7 d after exercise) were chosen to study the relation between the prolonged reduction in force-generating capacity and muscle soreness and the prolonged presence of leukocytes. To reduce the stress on the subjects and to reduce risk of contamination of tissue damage from previous biopsies, biopsies were collected from each subject at four of the six scheduled time points (from both the exercised and the control muscle). This means that biopsies from a total of seven subjects could be analyzed at each time point.
One-legged eccentric exercise.
The subjects performed 300 unilateral, voluntary maximal, isokinetic, eccentric actions (30°·s−1) with the musculus quadriceps femoris on a Cybex6000 (Lumex, Ronkonkoma, NY). The subjects sat with ∼90° in the hip joints, fastened with seat belts, and arms held crossed over the chest. The range of motion was from 35° to 105° (0° equals full extension in the knee joint), and the workout consisted of 30 sets of 10 repetitions with 30 s rest in between sets. The subjects were instructed to resist the movement maximally through the full range of motion in every repetition and received real-time visual feedback on their performance on a computer screen. They were also verbally encouraged during the exercise to ensure maximal effort.
Muscle function.
Maximal force-generating capacity was measured as voluntary maximal, isokinetic, concentric knee-extension peak torque at 60°·s−1 (on the Cybex6000). All subjects participated in four familiarization tests on separate days, before they entered the study. A warm-up of 5 min cycling at 100-150 W always preceded the concentric knee-extension test in addition to four warm-up contractions on the dynamometer. The intraindividual coefficient of variation of this test was <5%.
Radionuclide imaging: scintigraphic monitoring of leukocyte accumulation.
The method has been described in by Raastad et al. (33). In brief, 50 mL blood was drawn and leukocytes (mainly neutrophilic granulocytes) were isolated and labeled with 99mTechnetium before being reinfused. Accumulation of radiolabeled leukocytes in the subjects' thighs was quantified scintigraphically (with a gamma camera) on anterior and lateral view images (Fig. 1).
;)
FIGURE 1: Analysis of accumulation of 99mTechnetium-(radio)labeled leukocytes. A, Photo of the thighs of a subject with marks for regions of interest. B, C, Scintigrams obtained from anterior and lateral views, respectively. Note that dark areas reflect high radioactivity. The large gray square was used to analyze the radioactivity of the whole thigh. The smaller squares (1-5) were used to analyze the radioactivity in the musculus rectus femoris, the musculus vastus lateralis, the musculus vastus medialis, the muscle-tendon junction, and the hamstrings. Recordings were always performed on exactly corresponding locations on both sides-the exercised and the control thigh. The regions on panel A were also assessed (palpated) for muscle soreness.
The radioactivity in different parts of the musculus quadriceps and hamstrings was calculated with custom-made software (GE healthcare, Oslo, Norway). Regions of interest were divided into thirteen 16-cm2 squares (Fig. 1). For each square, the number of counts corrected for background radioactivity was calculated. One large square was positioned over the whole thigh to quantify total radioactivity in the musculus quadriceps, and the smaller squares were used to quantify the radioactivity in different parts of the muscle. The biopsied areas in the musculus vastus lateralis were omitted. The accumulation of radiolabeled leukocytes in the exercised leg was expressed as the percentage difference in radioactivity from the control leg.
Before exercise, marks with a waterproof pen were placed on the musculus vastus lateralis, the musculus vastus medialis, the musculus rectus femoris, and the muscle-tendon junction of each subject. The subjects' thighs were then photographed (Fig. 1). The pictures were adjusted in size, using Adobe PhotoDeluxe (Home Edition 3.1), to fit the scintigrams. The scintigrams, which were printed on transparent article, could be superpositioned on the photos to visualize quantification of accumulated radiolabeled leukocytes in the different parts of the musculus quadriceps.
Muscle biopsies.
A 5-mm Pelomi-needle (Albertslund, Denmark) with manual suction was used to obtain tissue samples (usually 3 × 50-100 mg) from the midsection of the musculus vastus lateralis. Subjects were in a supine position, and the procedure was performed under local anesthesia (Xylocain® adrenaline, 10 mg·mL−1 + 5 μg·mL−1, AstraZeneca, Sweden). Each needle insertion was placed approximately 3 cm proximal to the last insertion to avoid affected tissue from previous biopsies. The muscle samples were rinsed in saline before visible fat and connective tissue were removed and subsequently frozen in isopentane on dry ice and stored at −80°C until analysis.
Immunohistochemistry.
Five-micrometer-thick serial transverse sections were cut with a cryostat microtome (Microm, Walldorf, Germany) at −22°C and mounted on glass slides, air-dried, and stored at −80°C until further analysis. Serial sections were immunohistochemically stained for CD16 (M7006; DAKO, Copenhagen, Denmark) and CD68 (M0718; DAKO). Dilution used for both primary antibodies was 1:200. Biotin-conjugated goat anti-mouse (E0433; DAKO) was used as the secondary antibody, followed by complexes of avidin and biotinylated enzymes (ABComplexes; DAKO). Specific antibodies were visualized with the Fuchsin Substrate-Chromogen System (K0624; DAKO). The sections were finally counterstained with hematoxylin and mounted with coverslips. Negative and positive controls for both antibodies were always included. During quantification of cells positive for CD16 and CD68, sample identity was concealed. Four microscopic fields (20×) were randomly chosen, and the number of positive cells per square millimeter was manually counted. The area of muscle section assessed was measured by imaging software TEMA (CheckVision, Hadsund, Denmark).
The anti-CD16 antibody (against the Fc gamma receptor III found on granulocytes, monocytes/macrophages, NK cells, and reactive T cells) was chosen because we wanted to be able to detect most blood-borne leukocytes that might infiltrate the muscle due to exercise-induced muscle damage. The anti-CD68 antibody (associated with lysosomal granules) was chosen to more specifically detect monocytes and macrophages.
Blood sampling.
Blood was drawn from an antecubital vein into a 10-mL serum vacutainer tube. After coagulating for 30-45 min at room temperature (∼20°C), the blood was centrifuged at 2700g for 10 min at 4°C. Serum was then immediately pipetted into Eppendorf tubes and stored at −80°C until analysis. CK was analyzed with the Hitachi 917 Automated Biochemistry Analyzer (Roche®, Basel, Switzerland); analytic coefficient of variation being <2.8%.
Muscle soreness in the musculus quadriceps femoris.
Muscle soreness was rated on a visual analog scale where 0 mm represented "not sore at all" and 100 mm represented "extremely sore." The subjects stretched, contracted (isolated contractions and squats), and palpated the quadriceps muscle to assess general soreness in the whole musculus quadriceps and local soreness in the musculus vastus medialis and musculus vastus lateralis and centrally (including musculus rectus femoris and musculus vastus intermedius) as well as the muscle-tendon junction just above patella (Fig. 1). During palpation, the subjects pressed two fingers against each region of interest, applying enough force to cause light discomfort in the control muscle. The subjects were instructed to evaluate the soreness in the exercised muscle in comparison with the corresponding site in the control muscle. Two evaluations of soreness were thus obtained: during stretching and active muscle use and during palpation of relaxed muscles.
Statistics.
For variables that were normally distributed, a one-way repeated-measures ANOVA with Dunnett's and Tukey post hoc tests was performed to identify statistically significant changes from baseline. Exceptions were CK and DOMS, where Friedman's test with Dunn's post hoc test was applied. We assessed the differences from baseline for both exercised and control legs as well as differences between the legs. Because different subjects were biopsied at each time point, a Student's paired t-test or a Wilcoxon signed rank test (choice dependent on a normality test for Gaussian distribution) was used to analyze differences detected histologically (exercised vs control). The Mann-Whitney test was applied for testing differences between subgroups of subjects. Selected bivariate relationships were examined with the Pearson product-moment correlation coefficient test or the Spearman rank correlation test (choice dependent on a normality test for Gaussian distribution). Because scintigrams were obtained from six subjects and biopsies from 7 of the 11 subjects at each biopsy time point, the number of subjects will vary from 6 to 11 in the different analyses. P ≤ 0.05 was used for establishing statistical significance. Data are presented as means with SEM, if not otherwise stated in the text. The statistics were performed with Microsoft® Excel 2003 and InStat® 3.06 (GraphPad Software Inc., San Diego, CA).
RESULTS
Muscle function.
Three hundred maximal voluntary, isokinetic, eccentric actions (a total work of −50 ± 4 kJ) resulted in a 47 ± 5% reduction in maximal voluntary, concentric torque (P < 0.01; n = 11). The recovery of the force-generating capacity was biphasic: during the first 6 h, there was a fast recovery, whereas between 6 and 95 h (4 d) after exercise, there was no significant recovery. Thereafter, the force-generating capacity recovered slowly, and after 1 wk (167 h) the subjects were 13 ± 4% below baseline values (P < 0.01). The force-generating capacity of the control leg was statistically unaffected during the week of experiment.
Three subjects (subjects 4, 6, and 8) had an extraordinarily large acute loss of force-generating capacity (66%, 64%, and 73%, respectively) and suffered a secondary loss of force (Fig. 2). In addition, these three "high responders" had the greatest accumulation of radiolabeled leukocytes in the exercised muscles (see below). They recovered to 49 ± 2% under baseline values during the first 6 h after exercise, but then their force-generating capacity declined again to a 60 ± 6% reduction 47 h after exercise. One week after exercise, they were still 32 ± 6% weaker than before exercise. We continued to test these subjects every week, and they did not fully recover before 2 wk to 2 months after exercise. It is, however, important to state that the halted recovery 1-3 d after exercise was a general trend for all subjects, not just the high responders (Fig. 2).
FIGURE 2: Changes in maximal voluntary knee-extension torque (60°·s−1) in high (n = 3) and moderate (n = 8) responders. Values are means + SEM. The force-generating capacity of the control leg was not affected. #Significantly different from baseline (P < 0.05). *Significant difference between high and moderate responders (P < 0.05).
Muscle soreness in the musculus quadriceps femoris.
Perceived muscle soreness during stretching, unloaded contractions, and squats with body weight increased after exercise and peaked with a value of 53 ± 9 mm in the musculus vastus medialis 47 h after exercise (P < 0.01; n = 11; Fig. 3). Muscle soreness evaluated by palpation followed the same time course as the soreness during stretching and contractions and peaked at 47 h after exercise: 31 ± 5, 25 ± 5, and 24 ± 4 mm for musculus vastus medialis, musculus vastus lateralis, and musculus rectus femoris, respectively. Individual peak soreness (over time) during palpation was significantly higher in the musculus vastus medialis than in the musculus vastus lateralis and musculus rectus femoris (P < 0.05). Only minor (not significant) soreness was reported for the muscle-tendon junction (just above patella).
FIGURE 3: Time course of muscle soreness (visual analog scale) in different parts of the musculus quadriceps femoris. Values are means + SEM, n = 11. #Significantly different from control muscle (P < 0.05).
Accumulation of 99mTechnetium-(radio)labeled leukocytes.
The number of 99mTechnetium leukocytes (measured as radioactivity) was higher in the exercised muscles than in the control muscles at all time points, but large individual differences were observed (Fig. 4). The radioactivity increased in the musculus vastus medialis and the musculus vastus lateralis between 3 and 8 h after exercise (P < 0.05; n = 6; Fig. 5). In the three high responders (subjects 4, 6, and 8), there was a steady increase of accumulated leukocytes until 24 h after exercise, and massive accumulation was observed in the musculus rectus femoris (Figs. 4 and 5). The muscle-tendon junction was the other location where the accumulation of leukocytes was large-four of the six subjects had the largest leukocyte accumulation in this region. The peak difference between the muscle-tendon junction in the exercised and control muscle ranged from 27% to 764% (mean value = 223%). We found positive correlations between peak accumulation of radiolabeled leukocytes (over time) in the whole thigh and muscle weakness at all time points from 23 to 71 h after exercise (r = 0.81-0.85; P < 0.05). A negative correlation was observed between peak accumulation of radiolabeled leukocytes in the musculus quadriceps and muscle soreness (r = −0.96, P < 0.01); the correlation between accumulation of radiolabeled leukocytes and muscle soreness at 24 h after exercise was r = −0.88. There was no consistent relationship between soreness and accumulation of leukocytes in different regions of the exercised muscle at the intraindividual level (e.g., the degree of DOMS was only moderate in the musculus rectus femoris in the high-responder subjects).
FIGURE 4: Whole thigh scintigrams of six subjects obtained 7 h after exercise. The graphs show changes in the accumulation of radiolabeled, autologous leukocytes (radioactivity) in the exercised muscles (musculus quadriceps; % difference from control) at 3, 7, 21, and 24 h after exercise. Note the different y-axis scale for subject 4 and the massive accumulation of radiolabeled leukocytes in the musculus rectus femoris of this subject. Subjects 1, 2, and 10 exercised with their right leg, whereas subjects 4, 6, and 8 exercised with their left leg. HR, high responders.
FIGURE 5: Radioactivity in the musculus vastus lateralis and medialis (left panel) and musculus rectus femoris (right panel). The values are percentage difference between the exercised muscle and the corresponding control muscle. In the graph to the right, the high and moderate responders are separated to illustrate the large difference between subgroups in the musculus rectus femoris; note the logarithmic scale on the y-axis. Values are means ± SEM, n = 6. #Significantly different from control muscle (P < 0.05). *Significantly different from the previous time point (P < 0.05).
There was no detectable accumulation of radiolabeled leukocytes in the hamstrings muscle of the exercised leg. The difference between exercised and control leg was −4 ± 4% at the time point when individual leukocyte accumulation peaked in the musculus quadriceps.
Immunohistochemistry.
We observed an increased number of CD16+ and/or CD68+ cells in the exercised muscle compared with control muscle at all time points (0.5, 4, 8, 24, 96, and 168 h after exercise; P < 0.05; n = 7; Fig. 6). The highest individual numbers of CD16+ and CD68+ cells were observed 4 and 7 d (96 or 168 h) after exercise. The peak values in the exercised leg were 30 ± 5 and 34 ± 6 cells per square millimeter for CD16 and CD68, respectively, whereas the corresponding values for the control muscle were 8 ± 1 and 13 ± 3 cells per square millimeter. CD16+ and CD68+ cells were mainly observed in the endomysium and perimysium (Fig. 7). Intracellular infiltration was seen only occasionally: in 4 of the 11 subjects, 0.2%-1.35% of the myofibers of the exercised muscle was heavily infiltrated by CD16+ and/or CD68+ cells (Fig. 7, inserted picture). There was a positive correlation between the number of CD16+ cells and the reduced force-generating capacity 4 d (96 h) after exercise (r = 0.69; n = 7; P < 0.05).
FIGURE 6: Changes in the number of CD16+ cells (left panel) and CD68+ cells (right panel). Values are means ± SEM, n = 7 at each time point. #Significantly different from control muscle (P < 0.05). *Significant difference between the numbers of CD16+ and CD68+ cells found in samples from the exercised muscles obtained 0.5-24 h after exercise and those obtained at 96 and 168 h after exercise (P < 0.01).
FIGURE 7: A, CD16+ cells (red stain) were observed in the interstitial spaces of the exercising leg (musculus vastus lateralis) shown here at 96h (4d) after exercise; the inserted picture shows CD68+ cells (red) inside a muscle cell (scale bar = 50 μm). B, A small number of CD16+ cells were noted in the control leg. Blue stain (hematoxylin) shows nuclei. Scale bar = 100 μm.
The muscle sections were also stained with an antibody against CD56 (data not presented), which showed no clear signs of NK cells. Because the number of activated T cells in response to sterile muscle damage in healthy subjects was expected to be rather low (22,24,36) and because the number of basophilic and eosinophilic granulocytes in the blood stream is very and relatively low, respectively, we assumed that the great majority of CD16+ cells (especially at early time points) should be either neutrophilic granulocytes or monocytes/macrophages. Consequently, any difference between the CD16 and the CD68 staining presumably gave us a fair estimate of the presence of neutrophilic granulocytes.
In the nonexercised control muscle, there was a trend to moderately increased numbers of CD68+ cells over time. The mean values ranged from 2 to 5 cells per square millimeter at 0.5-24 h after exercise compared with 11 and 10 cells per square millimeter at 96 and 168 h after exercise, respectively (0.5 vs 168 h gave P = 0.1; unpaired t-test).
Creatine kinase.
Serum CK levels were increased 6 h after exercise and remained high throughout the experimental week; the median value was 457 U·L−1 (range = 210-25000 U·L−1) 95 h after exercise (P < 0.01; n = 11; Fig. 8). The three subjects (subjects 4, 6, and 8) with the largest loss of force and leukocyte accumulation had the highest peak values, ranging between 13,000 and 25,000 U·L−1 95 h after exercise, and their peak values were significantly higher than in the other eight subjects (P < 0.01; Fig. 8). The values of the high responders followed a biphasic pattern; that is, the CK levels increased during the first 23 h after exercise, declined slightly (not significantly) at 47 h, then increased again and peaked 95 h after exercise.
FIGURE 8: Changes in serum CK. The figure shows values for the high (n = 3) and moderate responders (n = 8); note the logarithmic scale on the y-axis. Values are means ± SEM. #Significantly different from baseline (P < 0.05). *Significant difference between high and moderate responders (P < 0.05).
Individual responses and high versus moderate responders.
In general, large individual differences were seen in the different variables, and this is outlined in Table 1. Subjects 4, 6, and 8 diverged markedly from the other eight subjects with greater acute loss of force-generating capacity and slower recovery as well as larger accumulation of radiolabeled leukocytes (of the whole thigh) and higher serum CK levels. In addition, they did less work during the exercise protocol because of greater reduction in work capacity during exercise. However, the soreness and the accumulation of leukocytes in the musculus vastus lateralis did not differ detectably between high and moderate responders; still, the soreness tended to be lower, whereas the number of CD16+ cells and radiolabeled leukocytes tended to be higher in the high responders. Intracellular infiltration of CD16+ and/or CD68+ cells was seen in the three responders, but in only one of the other eight subjects. Moreover, the three high responders acquired more ultrastructural (myofibrillar) disruptions than the others but exhibited low/moderate expression of N-terminal propeptide of procollagen type III (PIIINP)-compared with some of the other subjects that experienced intense DOMS. These latter data are presented in Raastad et al. (32). In terms of body mass, height, and age, subjects 4, 6, and 8 did not differ significantly from the other eight subjects, but the high responders were the least physically trained based on the self-report of regular physical activity.
TABLE 1: Individual data for main variables.
DISCUSSION
The main finding of this study was that the fast accumulation of radiolabeled leukocytes (assessed by scintigraphy) in the exercised muscles was observed concomitantly with histologically detection of CD16- and CD68-positive cells. Together, these findings demonstrated a local inflammatory cell response to muscle-damaging eccentric exercise in humans. Furthermore, a positive correlation was observed between accumulation of radiolabeled leukocytes and muscle weakness the first 3 d after exercise and between accumulation of CD16-positive cells and reduced force-generating capacity 4 d after exercise. During this period (1-4 d after exercise), there was no detectable recovery of force-generating capacity. Surprisingly, there was a strong, negative correlation between accumulation of radiolabeled leukocytes and sensation of DOMS. Hence, large accumulation of radiolabeled leukocytes was correlated with low degree of DOMS (Table 2).
TABLE 2: Overview of the changes of measured variables during the experimental week (hours after exercise).
Time course and types of leukocytes accumulated locally.
This study focused on the early local inflammatory response to exercised-induced muscle damage. As anticipated (20,33), the radionuclide imaging technique (scintigraphy) showed an early accumulation of radiolabeled leukocytes in the exercised muscles (3-24 h after exercise). The histology data verified increased numbers of both CD16- and CD68-positive cells in the endomysium and perimysium at the early time points (0.5-24 h). The radiolabeled leukocytes are primarily neutrophils (8), but significant accumulation of neutrophils in the exercised muscle tissue was, however, not supported by the histological data. Although we could not distinguish explicitly between neutrophils and monocytes/macrophages with our immunohistochemical approach, the dominating leukocyte subtype detected appeared to be monocytes/macrophages. This assumption is because the mean numeric values of CD16- and CD68-positive cells were very similar at all time points and that CD16 is found on both neutrophilic granulocytes and monocytes/macrophages whereas CD68 is primarily found on monocytes/macrophages.
Thus, our observations indicate that circulating neutrophilic granulocytes are captured in the microvessels in the exercised musculus vastus lateralis during or shortly after exercise, but apparently few cells cross the endothelial lining. However, based on the scintigraphy, it is plausible that higher numbers of neutrophils infiltrated the tissue in the musculus rectus femoris and the muscle-tendon junction. More evident than neutrophils, monocytes (i.e., CD68-positive cells) appeared to transmigrate and accumulate gradually in the musculus vastus lateralis during the days after exercise. Our findings are in line with those of previous human studies. Among 11 publications (4,7,10,12,14,16,22,24,25,28,44) reporting on biopsies obtained in the first hour after muscle-damaging exercise (0-6 h), a significant increase in the numbers of leukocytes was only observed in two (10,16). Of these, only Fielding et al. (10) reported on the presence of neutrophils. At later time points (from 48 h to ≥7 d), increased numbers of macrophages (or mononuclear cells) have been a more consistent histological finding (e.g., [17,22,39]).
Histological observations of macrophages and neutrophils might be biased because macrophages are presumably more easily detectable than neutrophils due to size differences and a longer life span in inflamed tissue. Furthermore, neutrophils could be difficult to detect histologically in human muscles if substantial tissue accumulation only occurs in some parts of the exercised muscle; for example, the musculus rectus femoris and the muscle-tendon junction, which are difficult to obtain tissue samples from. We do believe that the high levels of radioactivity registered in these regions reflected the magnitude of initial tissue damage. The anatomical structure of the musculus rectus femoris and the muscle-tendon junction seems to predispose these sites to both exercise-induced muscle damage and sport injuries (2,10,18).
Accumulation of leukocytes and recovery of muscle function.
In accordance with previous studies (20,33), accumulation of radiolabeled leukocytes preceded the halted recovery of muscle function-and a secondary force loss in the high responders. We now report for the first time a strong correlation between individual changes in muscle function and accumulation of radiolabeled leukocytes in humans. In addition, we observed increased numbers of CD16- and CD68-positive cells present in the muscle tissue during the whole period of halted recovery (1-4 d after exercise). This supports the hypothesis that local inflammation in the exercised muscles can affect the force-generating capacity of the myofibers during the days after unaccustomed eccentric exercise (9,21). Although a portion of the leukocytes are located on the luminal side of the endothelial blood vessel wall, it is still plausible that they may affect the myofibers through their interaction with endothelial cells (14). However, it could also be that the changes in force-generating capacity are primarily due to the initial muscle damage, the severity of which is reflected by the accumulation of radiolabeled leukocytes. Our experiment does not allow us to establish whether leukocyte accumulation is a cause or effect of exercise-induced muscle damage and loss of force-generating capacity. It has, however, been shown that leukocytes can damage cells in inflamed tissues (41). A cause-effect relationship has been indicated by Pizza et al. (30), who showed that the secondary damage after eccentric "exercise" (in situ lengthening contractions) was reduced in mice deficient in CD18, which hinders local accumulation of neutrophils. Similarly, Lapointe et al. (19) observed a reduced secondary loss of force-generating capacity after lengthening contractions in rats, when the accumulation of ED1+ (phagocytic) macrophages was reduced by nonsteroidal anti-inflammatory drug treatment. Observations from other animal studies have shown no association between changes in muscle function and changes in inflammatory cell accumulation (e.g., [11]). The discrepancy is hard to explain but could be due to differences between species (experimental animals), the number of infiltrated leukocytes, and their activation state in addition to the ability of the muscle cells to buffer leukocyte-derived damaging agents and oxidative stress.
Biphasic CK response.
The serum CK levels increased in a biphasic way in our three high responders. The first peak, observed 1 d after exercise, may reflect increased permeability of the sarcolemma, caused by the exercise per se and/or accumulation of leukocytes. However, the peak after 4 d was more likely due to myofiber necrosis (2). In support of this assumption, we observed areas with necrotic fibers (heavily infiltrated by leukocytes) in the exercised musculus vastus lateralis samples from the high responders. This makes it very likely that their musculus rectus femoris suffered even more necrosis, considering the large accumulation of radiolabeled leukocytes in this muscle. Moreover, necrosis has been observed in human studies, and it seems that the large increase in circulating CK levels (i.e., approximately above 10,000 U·L−1) precedes the end stage of this process, which is not histologically apparent until several days after exercise (17). Finally, it seems reasonable to propose that long-lasting depression in force-generating capacity (weeks) in the high responders was a result of such necrosis.
Accumulation of leukocytes and delayed onset muscle soreness.
Variations were seen in all variables measured, but there was a general association between the individual changes in force-generating capacity, ultrastructural damage, accumulation of radiolabeled leukocytes, and CK activity (moderate vs high responders). However, there was no such association between the DOMS and the other indicators of muscle damage (see Table 1). Interesting, the generally small intrasubject variability in DOMS between different parts of the musculus quadriceps was in sharp contrast to the large differences in the accumulation of radiolabeled leukocytes. This was especially evident for the musculus rectus femoris and the muscle-tendon junction where high numbers of radiolabeled leukocytes were found together with low degree of DOMS. In the musculus vastus lateralis, where the biopsies were obtained, there seems to be no support for the assumption that leukocyte accumulation and inflammation per se cause DOMS (6). Furthermore, the accumulation of CD16- and CD68-positive cells among the myofibers seemed to increase from day 2 (24 h after exercise) to day 5, whereas the degree of DOMS was higher on day 2 than on day 5. Others have also questioned the relationship between inflammation and DOMS because DOMS is present on time points when no accumulation of leukocytes could be detected among the myofibers (13,24-26,44). Malm et al. (25) did, however, suggest that activation of leukocytes present in the epimysium already before exercise could be involved in DOMS. The idea that DOMS is related to activity in the connective tissue was suggested many years ago (3), and it is still an attractive hypothesis. We did indeed find increased expression of tenacin C and PIIINP (see Table 1 and Raastad et al. [32]), which might suggest that damage and remodeling of the extracellular matrix are somehow related to DOMS (7). If leukocytes do not cause DOMS, the activity of fibroblasts and mast cells could potentially play a role in DOMS (39). Interestingly, the high-responder subjects demonstrated severe muscle weakness but reported quite low DOMS, and correspondingly they clearly exhibited most ultrastructural myofibrillar damage but only low/moderate tenacin C and PIIINP responses (see Table 1 and Raastad et al. [32]). In support of our findings, ultrastructural disruptions are related to changes in force-generating capacity (42), but changes in muscle function are not related to DOMS (27). Swelling and increased intramuscular pressure may also play a part in DOMS (13), but contradictory results have been reported (26). Finally, DOMS could also have mechanistic components in the central nervous system (43).
The strong negative correlation between overall accumulation of radiolabeled leukocytes and DOMS was unexpected. It suggests the unorthodox idea that neutrophilic granulocytes somehow reduce or counteract pain-provoking substances and/or reduce the sensitivity of the pain receptors. Corroborating our finding, Rittner et al. (35) observed that selective accumulation of neutrophilic granulocytes in rat paws did not cause pain (measured by paw withdrawal). Neutrophils have in fact the potential to reduce pain due to their content of opioid peptides, which act as antinociceptive mediators (34). Moreover, administration of a peripheral acting opioid (morphine-6-β-glucuronide) has been demonstrated to effectively reduce DOMS (40)-indicating that activation of opioid receptors in the exercised muscle reduces muscle soreness.
CONCLUSION
We have documented the inflammatory response in muscles exposed to maximal eccentric exercise in humans using repeated biopsies from both exercised and nonexercised control muscles combined with noninvasive radionuclide imaging. The local inflammatory response was observed immediately after exercise, but leukocyte accumulation appeared predominantly on the luminal side of the blood vessels, at least during the first hours. During the following days, mostly macrophages accumulated in the extravascular space, and they remained there for at least 1 wk. Three subjects were classified as high responders, with severe muscle weakness and heavy accumulation of radiolabeled leukocytes in the musculus rectus femoris. The magnitude of the inflammatory cell accumulation in the exercised muscles was correlated to the halted recovery of force-generating capacity that occurred 1-4 d after exercise. Accordingly, the accumulation of leukocytes can possibly have contributed to the muscle weakness during this period, or alternatively leukocyte accumulation may simply reflect the degree of muscle damage and the need for regeneration. On the basis of the serum CK levels, substantial necrosis of myofibers occurred, which certainly could have contributed to the long-term suppression of muscle function, especially in the high responders. We found no evidence for the notion that accumulation of blood-borne leukocytes causes DOMS. On the contrary, our data indicate that DOMS may be relieved by local accumulation of neutrophils.
No external funding was received for this work. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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