Extracellular matrix disruption after eccentric muscle action WILLIAM VALERIE
T. STAUBER,
PRISCILLA
and pain
M. CLARKSON,
K. FRITZ, AND WILLIAM J. EVANS Departments of Physiology and Neurology, West Virginia University Health Science Center, Morgantown, West Virginia 26506; Department of Exercise Science, University of Massachusetts, Amherst 01003; and US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111 STAUBER,~ILLIAM T., PRISCILLAM. CLARKSON,~ALERIE K. FRITZ, ANDWILLIAM J. EVANS.Extracellular matrix disruption and pain after eccentric muscle action. J. Appl. Physiol. 69(3): 868~874,1990.-Pain, stiffness, and indicators of muscle damage occur at different times after eccentric muscle action. After a single bout of maximal resisted lengthening of the elbow flexors, elbow position, pain perception, and indicators of cellular damage were measured. Immediately postexercise, a significant decrease in resting muscle length was observed that continued to 48 h. At this time, an increase in perceived muscle soreness was noted (P c 0.05), and a biopsy of the biceps brachii revealed mast cell degranulation, separations of the extracellular matrix from myofibers, and increased plasma constituents in the extracellular space. It is proposed that myofiber disruption allows intracellular proteins to escape and extracellular proteins and ions to enter, causing swelling, whereas the disrupted extracellular matrix initiates the inflammatory response, which includes the release of mast cell granules seen at 48 h postexercise. Thus the delayed sensation of pain (soreness) after repeated eccentric muscle actions probably results from inflammation in response to extracellular matrix disruption.
ions into the extracellular space, 2) from calcium influx into a damaged myofiber with water accumulation within the myofiber, and 3) from disruption of the extracellular matrix and/or increase in its hydration state. In all cases there would be an apparent decrease in the resting length of the muscle as the swollen tissue pushes against the fascia and shortens the muscles passively. The purpose of this study was to investigate the relationship, if any, among tenderness (pain perception), indicators of inflammation, and integrity of the extracellular matrix in human volunteers after a bout of eccentric muscle actions. Previous research with this mode of exercise-induced injury resulted in muscular swelling and limitations in range of motion of the exercised muscles before the appearance of indicators of muscle damage in the blood (11). Thus the nature of inflammation, pain, and muscle damage from repeated eccentric muscle action is in question.
skeletal exercise
Five healthy nontrained volunteers (male and female) of college age participated in the study. A signed consent form was required of each individual before participation in the study. Variability among individuals chosen for the study was minimized by matching participants for age, height, and weight (Table 1). None of the participants was “weight-trained”; nor had they exercised for at least 2 mo before the study. The volunteers were asked to refrain from any new or strenuous physical activity 7 days before and any time during the study. Using the passive mode of a computer-interfaced dynamometer (Biodex, Shirley, NY), each participant performed 70 maximal isokinetic resisted (eccentric muscle action) movements of the elbow flexors using the nondominant arm at l2O”/s through a range of 120’. Restraining straps were placed around the waist, across the chest, and loosely over the upper arm of the involved limb. Each movement was a maximal effort to resist the ability of the dynamometer to extend the elbow. Thus the elbow flexors were performing eccentric muscle actions or resisted muscle lengthening. A 10-s rest between each exercise movement allowed the dynamometer to return to the starting position. By use of a subjective assessment scale of 1 (normal)-
muscle
injury;
human;
proteoglycans;
inflammation;
MUSCLE INJURY after eccentric
muscle action (10) is a common experience in sports or occupational tasks especially of a novel nature. The injury results in pain and muscular swelling after the exercise bout (19), but the symptoms disappear within a few days (1). As with other tissue injuries, the muscle is repaired (6) without any residual dysfunction or scarring, and often the repair process results in a muscle that is able to resist even greater applied forces (24). The mechanical injury after eccentric muscle action results in myofiber damage as well as alterations to the extracellular matrix (16), both of which may lead to inflammation and pain. However, pain and inflammation follow a different time course than the indicators of myofiber damage (23). Thus the specific processes that follow muscle injury such as tissue swelling and extracellular matrix disruption may be more important in the production of pain and inflammation than the mechanical damage to the myofiber itself (30). Swelling could result 1) from cell damage and release of proteins and 868
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MATERIALS AND METHODS
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ECM DISRUPTION
AFTER ECCENTRIC
10 (very, very sore), soreness of the biceps muscle was assessed before exercise and 48 h after exercise by questionnaire (33). Changes in muscle shortening of the elbow flexors were assessed before, immediately after, and 48 h after the exercise bout by use of a goniometer to measure the change in the relaxed elbow joint angle while the subject was standing with head facing forward and arm relaxed down at the side. Anatomic reference points for goniometer placement were the deltoid insertion, the lateral humeral epicondyle, and the ulnar styloid process with the hand in the neutral position. Each reference point was marked with a semipermanent ink. The data were analyzed using Friedman’s test for two-way classification to examine the changes in joint angle resulting from the exercise insult. At 48 h postexercise, needle biopsies were taken from the exercised and nonexercised biceps brachii muscles of each volunteer. This postexercise time was chosen because perceived muscle soreness would peak at 48 h postexercise (11), and only one biopsy sample was to be removed. The tissue samples were quickly frozen in 2 methylbutane cooled in liquid nitrogen. The tissue samples were mounted for sectioning in optimal cutting temperature compound. Then 12-pm sections were cut at -25°C in an American Optical cryostat and placed on alcohol-cleaned glass slides. Tissue samples were maintained at -2OC in airtight boxes until use. Sectioned material was examined using a histological stain for mast cells and immunohistochemical techniques for various proteoglycans, proteinases, and lymphocyte cell surface antigens (Table 2). Antisera to human fibrinogen and albumin were obtained commercially (Organon Teknika-Cappel, Malvern, PA). Monoclonal antibodies to the immune response antigen (13), B lymphocytes (Bl), and natural killer cells (NKH-1) were acquired from Coulter Immunology (Hialeah, FL). Fluoresceinconjugated concanavalin A (Con A, Vector Laboratories, Burlingame, CA) was used to localize glycoproteins rich in mannose residues such as found in the pericellular basement membrane of the basal lamina (17). Monoclonal antibodies directed against proteoglycans digested with chondroitinase ABC were provided by Dr. Bruce Caterson (University of North Carolina at Chapel Hill, Chapel Hill, NC). Antigen isolation, monoclonal antibody production, and characterization have been completely described previously (7, 9). Digestion of the tissue with chondroitinase ABC resulted in a proteoglycan fragment of small oligosaccharide stubs with at least one characteristic unsaturated disaccharide (0, 4, or 6 sulfated) of the chondroitin sulfate isomer and the linkage region attached to the proteoglycan core protein (8). TABLE 1. Physical characteristics of volunteers Subj NO.
ASS1 Ass2 ss3 ss4 ss5 Means
* SE
Gender
Age, Yr
Height, cm
Weight, kg
M M M F F
23 19 19 17 23
159.5 175.3 165.1 170.1 159.5
60.2 59.4 59.9 62.6 60.2
165.9k3.0
60.5t0.5
20.2~1.2
MUSCLE ACTION
869
TABLE 2. Immunohistochemical reagents Antibody
Primary Goat Goat
anti-human anti-human
fibrinogen albumin
McAb
4/8/2B6
(C4SPG)
McAb
5/6/3B3
(CGSPG)
McAb McAb McAb
13 Bl NKH-1
C4SPG, chondroitin sulfate proteoglycan;
antisera
Delta unsaturated disaccharides of chondroitin 4-sulfate generated by digestion with chondroitinase ABC (3, 5) Delta unsaturated disaccharides of chondroitin and chondroitin 6-sulfate generated by digestion with chondroitinase ABC (3,5) Immune response antigen B lymphocytes Natural killer cells Labeled
Con A
antisera
Fibrinogen Albumin
Monoclonal
FITC-labeled
Specificity
lectins
Extracellular containing
matrix a-linked
glycoproteins mannose
4-sulfate proteoglycan; C6SPG, FITC, fluorescein isothiocyanate.
chondroitin
6-
The presence of mast cells was determined using a toluidine blue histological stain (22). Based on metachromatic properties of the mast cell granules, mast cells could be distinguished from other cell types by the redto-purple color of the granules. Tissue sections were stained for 10 min with 0.5% toluidine blue in 20% ethyl alcohol. After a rinse in phosphate-buffered saline (PBS, pH 7.4), coverslips were applied, and the sections were observed and photographed using an Olympus microscope with camera attachment. Briefly, the immunohistochemical staining procedure for the lymphocyte cell surface antigens, fibrinogen, and albumin included washing the slides three times for 5 min with filter sterile 0.001 M sodium phosphate in 0.15 M NaCl, pH 7.4 (PBS). Excess PBS was removed before specific antiserum was added, and 50 ~1 of the appropriate dilution of antiserum were applied to each slide. The slides were incubated at room temperature for 30 min in a moist chamber to prevent evaporation. The antisera were diluted to -1 mg/ml protein as determined by protein assay (21) using albumin as a standard. After incubation, all slides were washed for 5 min in PBS. Finally, 50 ~1 of fluorescein-labeled goat F(ab’)z antirabbit immunoglobulin (Ig) G, rabbit anti-goat IgG, or goat F(ab’)a anti-mouse IgG at a 1:20 dilution were added to all experimental and control slides. After another 30min incubation and a final wash for 5 min in PBS, glass coverslips were applied over the tissue sections with 50% glycerine-50% PBS containing 1 mg/ml p-phenylenediamine (Sigma Chemical, St. Louis, MO) and viewed on a Leitz Orthoplan fluorescence microscope (31). Localization of specific proteoglycans was performed using a modification of the previously described technique (3). The slides were washed for 5 min with 0.05 M tris(hydroxymethyl)aminomethane HCl and 0.05 M NaCl (pH 8.0) and predigested with 0.5 U/ml chondroitinase ABC (Sigma Chemical) diluted in the same buffer for 30 min at 37°C in a moist chamber. After a 5-min l
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870
ECM
DISRUPTION
AFTER
wash in PBS, 50 ~1 of diluted specific monoclonal antiserum were added to each slide and incubated as before. After incubation, all slides were washed again in PBS followed by the application of fluorescein-labeled goat F(ab’)2 anti-mouse IgG or IgM at a 1:20 dilution. After another 30-min incubation, the slides were washed with PBS, and glass coverslips were applied as described above . The slides were viewed on the fluorescence microscope as previously described. Fluorescein-labeled Con A at a 1:200 dilution in PBS was applied directly to slides of normal and injured muscle after an initial wash for 5 min in PBS. After a second wash in PBS, coverslips were applied as previously described. Method specificity and antibody specificity for the immunohistochemical reactions were determined according to the criteria of the reliability established by Petrusz et al. (26). The controls for the fluorescent immunohistochemical experiments included normal and injured tissue sections either incubated with normal rabbit serum instead of specific antibody or culture media instead of monoclonal antibody followed by fluorescein-labeled antiserum. Controls for the monoclonal antisera to proteoglycans included fresh frozen sections of rat ear as positive controls treated in the same manner as the normal and injured muscle tissue. The specificity of the monoclonal antibodies was tested by excluding the chondroitinase ABC digestion before monoclona-l antibody application followed by fluorescein-labeled second antibody. Another set of slides was incubated with chondroitinase ABC, diluted mouse serum, and fluorescein-conjugated second antibody. RESULTS
Evaluation of five subjects after a single bout of 70 maximal isokinetic resisted (eccentric muscle action) movements of the elbow flexors revealed significant increases in perceived muscle soreness of the exercised biceps and a significant decrease in the measured relaxed elbow joint angle. Before exercise, all subjects reported no perceived muscle soreness of the biceps muscle (average rating of 1; normal). At 48 h postexercise, however, the average score for perceived muscle soreness had increased to 8 (a0.7) with the range of responses between 7 and 9 on a scale of 10 (very, very sore). A significant decrease in the relaxed elbow joint angle was recorded immediately after exercise and 48 h later compared with baseline measurements taken before the exercise bout (Fig. 1). Immediately postexercise the relaxed elbow joint angle had significantly decreased an average of 7.2 t 1.3O (P 5 0.05) and by 48 h the decrease in angle averaged 10.4 t 5.9’ (P 5 0.05). The large variance among the joint angle measurements that occurred by 48 h was the result of two of the subjects experiencing a much greater decrease in angle than the others. Alterations to muscle fibers were observed histologically at 48 h by use of toluidine blue-stained sections of the exercised biceps muscle, although the muscle remained unchanged in sections from the contralateral limb. Sections of exercised muscle from all participants
ECCENTRIC
MUSCLE
ACTION
160 158
146. 144. l42*
. 1
PRE EXERCISE
I
POSTEXERCISE
1
48 HRS POST-
EXERCISE
1. Relaxed angle (elbow angle when arm is resting at side) before, immediately after, and 48 h after exercise bout. Values are means k SE. A significant decrease in relaxed elbow joint angle was recorded immediately after exercise and 48 h later (P 5 0.05). FIG.
revealed widened perimysial areas between fascicles and separation of the myofibers from one another within fascicles (endomysial region) (Fig. 2A ). Some mononuclear cells were observed in the perimysial and endomysial regions; however, they did not fill the intracellular spaces. Mast cell degranulation was evident in sections of exercised muscle from all participants. Degranulating mast cells were localized primarily in the perimysial area near blood vessels (Fig. 2B). However, a few were also present in the endomysial area near damaged myofibers (Fig. 2C). Occasionally, a few necrotic fibers were observed because they stained darker with toluidine blue (loss of viability) and/or contained infiltrating mononuclear cells and flocculent degeneration of the sarcoplasm. The frequency of necrotic fibers per section of between 300 and 500 fibers was -2%, with the remainder of the fibers appearing unaltered. Further evidence of muscle alterations from eccentric muscle action was found using immunohistochemical techniques to observe changes in localization of specific serum components, mononuclear cells, and extracellular matrix (ECM) components. In normal muscle sections, the serum components albumin and fibrinogen were localized in capillaries with some diffuse staining around myofibers for albumin. In exercise-injured muscle, both of these serum components filled areas of the widened perimysial and endomysial spaces around fibers (Fig. 20). Some damaged myofibers were easily identified because both albumin and fibrinogen were localized within these compromised fibers. Major alterations to the ECM were obvious in sections of exercised biceps muscle. Cross sections of normal biceps muscle showed chondroitin 6-sulfate proteoglycan localization in the endomysial area around muscle fibers,
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ECM
DISRUPTION
AFTER
ECCENTRIC
MUSCLE
ACTION
871
FIG. 2. Cross sections of human biceps muscle 48 h after exercise. A: toluidine blue stain (arrows indicate gaps; see Fig. 30). B: toluidine blue stain. deeranulating mast cell (arrow). C: toluydine blue stain, degranulating mast cell (arrow). D: immunohistochemical localization of albumin. x300.
blood vessels, and nerve fibers (Fig. 3A). Likewise, Con A-stained material was observed in a similar pattern in the perimysial region in addition to the endomysial area of normal muscle sections (Fig. 3B). Both chondroitin 6sulfate proteoglycan localization and Con A staining accentuated the close association existing between myofibers and the ECM. With the use of both chondroitin 6-sulfate proteoglycan and Con A, muscle sections from all exercised volunteers revealed that a portion of the ECM was actually pulled away from the surface of myofibers into the widened interstitial spaces, leaving the perimeter of the fiber with no fluorescent material present (Fig. 3, C and D). This was usually observed along at least one edge of the myofibers, but occasionally separation was observed in two or three regions of the
perimeter. Occasionally these gaps could also be found filled in with Con A- and chondroitin B-sulfate proteoglycan-positive material. No mononuclear cell infiltration was observed in these fibers with ECM gaps at this time. Localization of chondroitin 4-sulfate proteoglycan had also changed after the exercise bout compared with the contralateral limb. In normal muscle, chondroitin 4sulfate proteoglycan was localized predominantly around blood vessels (Fig. 4A). A narrow band of stained material was also observed in the perimysium and endomysium and around capillaries. In exercised muscle, chondroitin 4-sulfate proteoglycan staining was observed filling widened perimysial areas (Fig. 4B). However, it was absent from the endomysial region along the perimeter
FIG. 3. Chondroitin 6-sulfate proteoglycan (CGSPG) localization and Con A staining of ECM in cross sections of human biceps muscle. A: CGSPG localization in normal biceps muscle. B: Con A staining of ECM in normal biceps muscle. C: CGSPG localization in exercise-injured biceps muscle; ECM is pulled away from surface of myofibers (arrows). D: Con A stainining of exercise-injured biceps muscle. ECM is pulled away from surface of myofibers (arrow). X300.
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872
ECM DISRUPTION
AFTER
ECCENTRIC
MUSCLE
ACTION
FIG. 4. Localization of chondroitin 4sulfate proteoglycan (C4SPG), immune response antigen (13), and B lymphocyte cell surface antigen (Bl) in cross sections of human biceps muscle. A: C4SPG localization in normal biceps muscle. B: C4SPG localization in exercise-iniured biceps muscle (arrows indicate areas without staining). C: 13 localization in exercise-injured biceps muscle; macrophages were observed around disrupted myofibers (arrow). D: Bl localization in exercise-injured biceps muscle; no localization of B lymphocytes was observed. x300.
of myofibers that were no longer in contact (Fig. 4B). Experimental controls for the chondroitin proteoglycans were negative without any fluorescent localization when either chondroitinase ABC or the specific monoclonal antibody was excluded from incubation with the tissue sections. The identity of mononuclear cells observed in the exercised muscle samples was investigated using monoclonal antibodies specific for cells of lymphoid origin. 13 (Ia antigen)-positive cells were observed in small numbers in the interstitial spaces near blood vessels and occasionally in or near the few necrotic fibers found in the exercised muscle sections (Fig. 4C). Because the Ia antigen is expressed by other monocytes besides macrophages, monoclonal antibody specific for B cells (Bl) and natural killer cells (NKH-1) was used to investigate the presence of these cell types. No Bl- or NKH-lpositive cells were localized in either normal or exercised muscle sections from any of the participants (Fig. 40). DISCUSSION
It is generally accepted that skeletal muscle pain caused by exercise involving eccentric muscle action results from mechanical rather than metabolic factors (23). Presumably the mechanical strain overcomes the ability of the active muscle to resist and results in disruption of some myofibers (2). Although appreciable damage to myofibers can result (23), no established relationship exists between myofiber damage and pain. In a previous study using eccentric muscle action to produce soreness, it was noted that pain sensation was maximal 2 days after the exercise bout, but the resting muscle length was decreased immediately postexercise (11). In other studies, the clinical indicator of myofiber damage, creatine kinase, only appeared in the blood after the exercise bout was finished and continued to increase without further muscular activity (14). The nature of the
divergence in responses has not yet been adequately explained (23). It is possible that pain and myofiber damage are not related but coexist after eccentric muscle action. For example, muscle swelling can be seen immediately after the exercise (15) along with limitations in range of motion (11). Yet only in muscles with rigid compartments does this lead to an increase in interstitial pressure sufficient to impair local microcirculation necessary to produce a state of partial ischemia (23)-the biceps brachii are not such a muscle. So it must be concluded that pain sensation in the biceps muscle was not the result solely of muscle swelling or edema. Myofiber damage also occurs during the exercise bout (2, 12) and is demonstrable immediately postexercise (23). However, the intracellular proteins that escape, such as creatine kinase, are not seen in the blood immediately after exercise but appear later and continue to rise without additional injury. This delayed response may reflect the time necessary for I) the proteins to enter the vascular system by way of the lymphatics or 2) a change to occur in vascular permeability that would allow their entry into the microcirculation, In either case, the proteins would act as osmotically active molecules while in the interstitial space before their appearance in the blood. Little is known about how intracellular proteins such as lactate dehydrogenase, creatine kinase, and myoglobin released from muscle enter the general circulation. From the observations in this study it would appear that the vasculature has become permeable to some macromolecules (e.g., fibrinogen) at 48 h after the exercise bout. This occurred at the same time that mast cell degranulation with its known effect on vascular permeability was observed (32). Although these observations might help establish a link between mast cell degranulation and transcystosis of creatine kinase, no definitive informa-
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ECM
DISRUPTION
AFTER
tion about the role of lymphatic removal of intracellular proteins or matrix components has been reported. In contrast, in the skeletal muscles of uninjured rats, the lymphatics have been reported to be responsible for removal of about three-fourths of the transcytosed albumin (28). So the lymphatic circulation may indeed be important in the removal of muscle proteins released by injury or disease. To summarize at this point, pain sensation does not seem to be related to events associated with myofiber damage but seems more closely aligned with delayed responses such as the release of mast cell products that include histamine, a known algesic. It is not surprising then that common medications for the relief of pain have been unsuccessful in treating delayed-onset muscle soreness (20) because they do not block histamine receptors (4). However, calcium-blocking drugs, which could also block the calcium-mediated degranulation of mast cells, have been of some benefit (18, 34). Perhaps it is not the myofiber injury that mediates the pain at all but the disruption of the connective tissue (1) or extracellular matrix surrounding the myofibers. When the extracellular matrix is -damaged, it may release cytokines or peptide degradation products (27) that alert the immune system and subsequently produce a delayedonset inflammation. Also inflammation is a common feature in diseases of connective tissues including polymyositis (l3), which are characterized by inflammatory responses, activation of the immune system, and often pain. Thus the matrix alteration unique to this type of injury may be paramount in the evolution of pain. Another factor in delayed-onset muscle pain and elevated indicators of muscle damage might result from a cytotoxic response -an overreaction of the immune system to injury. By alerting the immune system, the strain injury might produce further damage if specific cellular cytotoxicity such as that seen in inflammatory muscle diseases (13) were operative. Cytotoxic cell involvement has not received much attention, but the few studies appear contradictory. In exercise-induced muscle damage in rats, lymphoid cells were observed within the muscles (32), but no such inflammatory cell response was reported after eccentric muscle action in humans (29). Even though we too were unable to find any cytotoxic cells other than macrophages, it can be argued that the human biopsy sample was too small to make any conclusive statements. However, in another study the cytokine interleukin-lp increased in human muscle biopsies and remained elevated for 5 days after repeated eccentric muscle action (5). Thus a very complex reaction of extracellular matrix, cells, and inflammation mediators exists after muscle damage, some of which may be unique to eccentric muscle actions. This work was supported in part by funds from Comptex, Inc., to W. T. Stauber. This work was presented in part at the American College of Sports Medicine Meeting in Baltimore, MD, 31 May-3 June 1989. Address for reprint requests: W. T. Stauber, Dept. of Physiology, West Virginia University Health Science Center, Morgantown, WV 26506. Received
6 Sentember 1
1989: I accented I
in final
form
3 Mav 4 1990. ~~~-
ECCENTRIC
MUSCLE
ACTION
873
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24. NEWHAM, D. J., AND P. M. CLARKSON. Repeated high-force eccentric exercise: effects on muscle pain and damage. J. Appl. Physiol. 63: 1381-1386,1987. 25. NEWHAM, D. J., K. R. MILLS, B. M. QUIGLEY, AND R. H. T. EDWARDS. Pain and fatigue after concentric and eccentric muscle contractions. CZin. Sci. Lond. 64: 55-62, 1983. 26. PETRUSZ, P., P. ORDRONNEAU, AND J. C. W. FINELY. Criteria of reliability for light microscopic immunocytochemical staining. Histochem. J. 12: 333-348,198O. 27. POSTLETHWAITE, A. E., AND A. H. KANG. Collagenand collagen peptide-induced chemotaxis of human blood monocytes. J. Exp. A4ed. 143: 1299-1307,1976. 28. REED, R. F., S. JOHANSEN, AND H. NODDELAND. Turnover rate of interstitial albumin in rat skin and skeletal muscle. Effects of limb movements and motor activity. Acta Physiol. Stand. 125: 711-718, 1985. 29. ROUND, J. M., D. A. JONES, AND G. CAMBRIDGE. Cellular infil-
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30.
31.
32.
33.
34.
MUSCLE
ACTION
trates in human skeletal muscle: exercise induced damage as a model for inflammatory disease? J. Neural. Sci. 82: l-11, 1987. STAUBER, W. T. Eccentric action of muscles: physiology, injury and adaptation. In: Exercise and Sport Sciences Review, edited by K. B. Pandolf. Baltimore, MD: Williams & Wilkins, 1989, vol. 17, p. 157-185. STAUBER, W. T., V. K. FRITZ, B. DAHLMANN, AND H. REINAUER. Immunofluorescent localization of an alkaline proteinase in skeletal muscle from diabetic rats. Basic Appl. Histochem. 30: 147-152, 1986. STAUBER, W. T., V. K. FRITZ, D. W. VOGELBACH, AND B. DAHLMANN. Characterization of muscles injured by forced lengthening. I. Cellular infiltrates. Med. Sci. Sports Exercise 20: 345-353, 1988. TIIDUS. P. M.. AND C. D. IANUZZO. Effects of intensity and duration of muscular exercise on delayed soreness and serum enzvme activities. A4ed. Sci. SDorts Exercise 15: 461-465, 1983. WA~TON, J. Diffuse exercise-induced muscle pain of undetermined cause relieved by verapamil. Lancet 1: 993, 1981.
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T. STAUBER,
PRISCILLA
and pain
M. CLARKSON,
K. FRITZ, AND WILLIAM J. EVANS Departments of Physiology and Neurology, West Virginia University Health Science Center, Morgantown, West Virginia 26506; Department of Exercise Science, University of Massachusetts, Amherst 01003; and US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111 STAUBER,~ILLIAM T., PRISCILLAM. CLARKSON,~ALERIE K. FRITZ, ANDWILLIAM J. EVANS.Extracellular matrix disruption and pain after eccentric muscle action. J. Appl. Physiol. 69(3): 868~874,1990.-Pain, stiffness, and indicators of muscle damage occur at different times after eccentric muscle action. After a single bout of maximal resisted lengthening of the elbow flexors, elbow position, pain perception, and indicators of cellular damage were measured. Immediately postexercise, a significant decrease in resting muscle length was observed that continued to 48 h. At this time, an increase in perceived muscle soreness was noted (P c 0.05), and a biopsy of the biceps brachii revealed mast cell degranulation, separations of the extracellular matrix from myofibers, and increased plasma constituents in the extracellular space. It is proposed that myofiber disruption allows intracellular proteins to escape and extracellular proteins and ions to enter, causing swelling, whereas the disrupted extracellular matrix initiates the inflammatory response, which includes the release of mast cell granules seen at 48 h postexercise. Thus the delayed sensation of pain (soreness) after repeated eccentric muscle actions probably results from inflammation in response to extracellular matrix disruption.
ions into the extracellular space, 2) from calcium influx into a damaged myofiber with water accumulation within the myofiber, and 3) from disruption of the extracellular matrix and/or increase in its hydration state. In all cases there would be an apparent decrease in the resting length of the muscle as the swollen tissue pushes against the fascia and shortens the muscles passively. The purpose of this study was to investigate the relationship, if any, among tenderness (pain perception), indicators of inflammation, and integrity of the extracellular matrix in human volunteers after a bout of eccentric muscle actions. Previous research with this mode of exercise-induced injury resulted in muscular swelling and limitations in range of motion of the exercised muscles before the appearance of indicators of muscle damage in the blood (11). Thus the nature of inflammation, pain, and muscle damage from repeated eccentric muscle action is in question.
skeletal exercise
Five healthy nontrained volunteers (male and female) of college age participated in the study. A signed consent form was required of each individual before participation in the study. Variability among individuals chosen for the study was minimized by matching participants for age, height, and weight (Table 1). None of the participants was “weight-trained”; nor had they exercised for at least 2 mo before the study. The volunteers were asked to refrain from any new or strenuous physical activity 7 days before and any time during the study. Using the passive mode of a computer-interfaced dynamometer (Biodex, Shirley, NY), each participant performed 70 maximal isokinetic resisted (eccentric muscle action) movements of the elbow flexors using the nondominant arm at l2O”/s through a range of 120’. Restraining straps were placed around the waist, across the chest, and loosely over the upper arm of the involved limb. Each movement was a maximal effort to resist the ability of the dynamometer to extend the elbow. Thus the elbow flexors were performing eccentric muscle actions or resisted muscle lengthening. A 10-s rest between each exercise movement allowed the dynamometer to return to the starting position. By use of a subjective assessment scale of 1 (normal)-
muscle
injury;
human;
proteoglycans;
inflammation;
MUSCLE INJURY after eccentric
muscle action (10) is a common experience in sports or occupational tasks especially of a novel nature. The injury results in pain and muscular swelling after the exercise bout (19), but the symptoms disappear within a few days (1). As with other tissue injuries, the muscle is repaired (6) without any residual dysfunction or scarring, and often the repair process results in a muscle that is able to resist even greater applied forces (24). The mechanical injury after eccentric muscle action results in myofiber damage as well as alterations to the extracellular matrix (16), both of which may lead to inflammation and pain. However, pain and inflammation follow a different time course than the indicators of myofiber damage (23). Thus the specific processes that follow muscle injury such as tissue swelling and extracellular matrix disruption may be more important in the production of pain and inflammation than the mechanical damage to the myofiber itself (30). Swelling could result 1) from cell damage and release of proteins and 868
0161-7567/90
$1.50
Copyright
MATERIALS AND METHODS
0 1990 the American
Physiological
Society
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ECM DISRUPTION
AFTER ECCENTRIC
10 (very, very sore), soreness of the biceps muscle was assessed before exercise and 48 h after exercise by questionnaire (33). Changes in muscle shortening of the elbow flexors were assessed before, immediately after, and 48 h after the exercise bout by use of a goniometer to measure the change in the relaxed elbow joint angle while the subject was standing with head facing forward and arm relaxed down at the side. Anatomic reference points for goniometer placement were the deltoid insertion, the lateral humeral epicondyle, and the ulnar styloid process with the hand in the neutral position. Each reference point was marked with a semipermanent ink. The data were analyzed using Friedman’s test for two-way classification to examine the changes in joint angle resulting from the exercise insult. At 48 h postexercise, needle biopsies were taken from the exercised and nonexercised biceps brachii muscles of each volunteer. This postexercise time was chosen because perceived muscle soreness would peak at 48 h postexercise (11), and only one biopsy sample was to be removed. The tissue samples were quickly frozen in 2 methylbutane cooled in liquid nitrogen. The tissue samples were mounted for sectioning in optimal cutting temperature compound. Then 12-pm sections were cut at -25°C in an American Optical cryostat and placed on alcohol-cleaned glass slides. Tissue samples were maintained at -2OC in airtight boxes until use. Sectioned material was examined using a histological stain for mast cells and immunohistochemical techniques for various proteoglycans, proteinases, and lymphocyte cell surface antigens (Table 2). Antisera to human fibrinogen and albumin were obtained commercially (Organon Teknika-Cappel, Malvern, PA). Monoclonal antibodies to the immune response antigen (13), B lymphocytes (Bl), and natural killer cells (NKH-1) were acquired from Coulter Immunology (Hialeah, FL). Fluoresceinconjugated concanavalin A (Con A, Vector Laboratories, Burlingame, CA) was used to localize glycoproteins rich in mannose residues such as found in the pericellular basement membrane of the basal lamina (17). Monoclonal antibodies directed against proteoglycans digested with chondroitinase ABC were provided by Dr. Bruce Caterson (University of North Carolina at Chapel Hill, Chapel Hill, NC). Antigen isolation, monoclonal antibody production, and characterization have been completely described previously (7, 9). Digestion of the tissue with chondroitinase ABC resulted in a proteoglycan fragment of small oligosaccharide stubs with at least one characteristic unsaturated disaccharide (0, 4, or 6 sulfated) of the chondroitin sulfate isomer and the linkage region attached to the proteoglycan core protein (8). TABLE 1. Physical characteristics of volunteers Subj NO.
ASS1 Ass2 ss3 ss4 ss5 Means
* SE
Gender
Age, Yr
Height, cm
Weight, kg
M M M F F
23 19 19 17 23
159.5 175.3 165.1 170.1 159.5
60.2 59.4 59.9 62.6 60.2
165.9k3.0
60.5t0.5
20.2~1.2
MUSCLE ACTION
869
TABLE 2. Immunohistochemical reagents Antibody
Primary Goat Goat
anti-human anti-human
fibrinogen albumin
McAb
4/8/2B6
(C4SPG)
McAb
5/6/3B3
(CGSPG)
McAb McAb McAb
13 Bl NKH-1
C4SPG, chondroitin sulfate proteoglycan;
antisera
Delta unsaturated disaccharides of chondroitin 4-sulfate generated by digestion with chondroitinase ABC (3, 5) Delta unsaturated disaccharides of chondroitin and chondroitin 6-sulfate generated by digestion with chondroitinase ABC (3,5) Immune response antigen B lymphocytes Natural killer cells Labeled
Con A
antisera
Fibrinogen Albumin
Monoclonal
FITC-labeled
Specificity
lectins
Extracellular containing
matrix a-linked
glycoproteins mannose
4-sulfate proteoglycan; C6SPG, FITC, fluorescein isothiocyanate.
chondroitin
6-
The presence of mast cells was determined using a toluidine blue histological stain (22). Based on metachromatic properties of the mast cell granules, mast cells could be distinguished from other cell types by the redto-purple color of the granules. Tissue sections were stained for 10 min with 0.5% toluidine blue in 20% ethyl alcohol. After a rinse in phosphate-buffered saline (PBS, pH 7.4), coverslips were applied, and the sections were observed and photographed using an Olympus microscope with camera attachment. Briefly, the immunohistochemical staining procedure for the lymphocyte cell surface antigens, fibrinogen, and albumin included washing the slides three times for 5 min with filter sterile 0.001 M sodium phosphate in 0.15 M NaCl, pH 7.4 (PBS). Excess PBS was removed before specific antiserum was added, and 50 ~1 of the appropriate dilution of antiserum were applied to each slide. The slides were incubated at room temperature for 30 min in a moist chamber to prevent evaporation. The antisera were diluted to -1 mg/ml protein as determined by protein assay (21) using albumin as a standard. After incubation, all slides were washed for 5 min in PBS. Finally, 50 ~1 of fluorescein-labeled goat F(ab’)z antirabbit immunoglobulin (Ig) G, rabbit anti-goat IgG, or goat F(ab’)a anti-mouse IgG at a 1:20 dilution were added to all experimental and control slides. After another 30min incubation and a final wash for 5 min in PBS, glass coverslips were applied over the tissue sections with 50% glycerine-50% PBS containing 1 mg/ml p-phenylenediamine (Sigma Chemical, St. Louis, MO) and viewed on a Leitz Orthoplan fluorescence microscope (31). Localization of specific proteoglycans was performed using a modification of the previously described technique (3). The slides were washed for 5 min with 0.05 M tris(hydroxymethyl)aminomethane HCl and 0.05 M NaCl (pH 8.0) and predigested with 0.5 U/ml chondroitinase ABC (Sigma Chemical) diluted in the same buffer for 30 min at 37°C in a moist chamber. After a 5-min l
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870
ECM
DISRUPTION
AFTER
wash in PBS, 50 ~1 of diluted specific monoclonal antiserum were added to each slide and incubated as before. After incubation, all slides were washed again in PBS followed by the application of fluorescein-labeled goat F(ab’)2 anti-mouse IgG or IgM at a 1:20 dilution. After another 30-min incubation, the slides were washed with PBS, and glass coverslips were applied as described above . The slides were viewed on the fluorescence microscope as previously described. Fluorescein-labeled Con A at a 1:200 dilution in PBS was applied directly to slides of normal and injured muscle after an initial wash for 5 min in PBS. After a second wash in PBS, coverslips were applied as previously described. Method specificity and antibody specificity for the immunohistochemical reactions were determined according to the criteria of the reliability established by Petrusz et al. (26). The controls for the fluorescent immunohistochemical experiments included normal and injured tissue sections either incubated with normal rabbit serum instead of specific antibody or culture media instead of monoclonal antibody followed by fluorescein-labeled antiserum. Controls for the monoclonal antisera to proteoglycans included fresh frozen sections of rat ear as positive controls treated in the same manner as the normal and injured muscle tissue. The specificity of the monoclonal antibodies was tested by excluding the chondroitinase ABC digestion before monoclona-l antibody application followed by fluorescein-labeled second antibody. Another set of slides was incubated with chondroitinase ABC, diluted mouse serum, and fluorescein-conjugated second antibody. RESULTS
Evaluation of five subjects after a single bout of 70 maximal isokinetic resisted (eccentric muscle action) movements of the elbow flexors revealed significant increases in perceived muscle soreness of the exercised biceps and a significant decrease in the measured relaxed elbow joint angle. Before exercise, all subjects reported no perceived muscle soreness of the biceps muscle (average rating of 1; normal). At 48 h postexercise, however, the average score for perceived muscle soreness had increased to 8 (a0.7) with the range of responses between 7 and 9 on a scale of 10 (very, very sore). A significant decrease in the relaxed elbow joint angle was recorded immediately after exercise and 48 h later compared with baseline measurements taken before the exercise bout (Fig. 1). Immediately postexercise the relaxed elbow joint angle had significantly decreased an average of 7.2 t 1.3O (P 5 0.05) and by 48 h the decrease in angle averaged 10.4 t 5.9’ (P 5 0.05). The large variance among the joint angle measurements that occurred by 48 h was the result of two of the subjects experiencing a much greater decrease in angle than the others. Alterations to muscle fibers were observed histologically at 48 h by use of toluidine blue-stained sections of the exercised biceps muscle, although the muscle remained unchanged in sections from the contralateral limb. Sections of exercised muscle from all participants
ECCENTRIC
MUSCLE
ACTION
160 158
146. 144. l42*
. 1
PRE EXERCISE
I
POSTEXERCISE
1
48 HRS POST-
EXERCISE
1. Relaxed angle (elbow angle when arm is resting at side) before, immediately after, and 48 h after exercise bout. Values are means k SE. A significant decrease in relaxed elbow joint angle was recorded immediately after exercise and 48 h later (P 5 0.05). FIG.
revealed widened perimysial areas between fascicles and separation of the myofibers from one another within fascicles (endomysial region) (Fig. 2A ). Some mononuclear cells were observed in the perimysial and endomysial regions; however, they did not fill the intracellular spaces. Mast cell degranulation was evident in sections of exercised muscle from all participants. Degranulating mast cells were localized primarily in the perimysial area near blood vessels (Fig. 2B). However, a few were also present in the endomysial area near damaged myofibers (Fig. 2C). Occasionally, a few necrotic fibers were observed because they stained darker with toluidine blue (loss of viability) and/or contained infiltrating mononuclear cells and flocculent degeneration of the sarcoplasm. The frequency of necrotic fibers per section of between 300 and 500 fibers was -2%, with the remainder of the fibers appearing unaltered. Further evidence of muscle alterations from eccentric muscle action was found using immunohistochemical techniques to observe changes in localization of specific serum components, mononuclear cells, and extracellular matrix (ECM) components. In normal muscle sections, the serum components albumin and fibrinogen were localized in capillaries with some diffuse staining around myofibers for albumin. In exercise-injured muscle, both of these serum components filled areas of the widened perimysial and endomysial spaces around fibers (Fig. 20). Some damaged myofibers were easily identified because both albumin and fibrinogen were localized within these compromised fibers. Major alterations to the ECM were obvious in sections of exercised biceps muscle. Cross sections of normal biceps muscle showed chondroitin 6-sulfate proteoglycan localization in the endomysial area around muscle fibers,
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ECM
DISRUPTION
AFTER
ECCENTRIC
MUSCLE
ACTION
871
FIG. 2. Cross sections of human biceps muscle 48 h after exercise. A: toluidine blue stain (arrows indicate gaps; see Fig. 30). B: toluidine blue stain. deeranulating mast cell (arrow). C: toluydine blue stain, degranulating mast cell (arrow). D: immunohistochemical localization of albumin. x300.
blood vessels, and nerve fibers (Fig. 3A). Likewise, Con A-stained material was observed in a similar pattern in the perimysial region in addition to the endomysial area of normal muscle sections (Fig. 3B). Both chondroitin 6sulfate proteoglycan localization and Con A staining accentuated the close association existing between myofibers and the ECM. With the use of both chondroitin 6-sulfate proteoglycan and Con A, muscle sections from all exercised volunteers revealed that a portion of the ECM was actually pulled away from the surface of myofibers into the widened interstitial spaces, leaving the perimeter of the fiber with no fluorescent material present (Fig. 3, C and D). This was usually observed along at least one edge of the myofibers, but occasionally separation was observed in two or three regions of the
perimeter. Occasionally these gaps could also be found filled in with Con A- and chondroitin B-sulfate proteoglycan-positive material. No mononuclear cell infiltration was observed in these fibers with ECM gaps at this time. Localization of chondroitin 4-sulfate proteoglycan had also changed after the exercise bout compared with the contralateral limb. In normal muscle, chondroitin 4sulfate proteoglycan was localized predominantly around blood vessels (Fig. 4A). A narrow band of stained material was also observed in the perimysium and endomysium and around capillaries. In exercised muscle, chondroitin 4-sulfate proteoglycan staining was observed filling widened perimysial areas (Fig. 4B). However, it was absent from the endomysial region along the perimeter
FIG. 3. Chondroitin 6-sulfate proteoglycan (CGSPG) localization and Con A staining of ECM in cross sections of human biceps muscle. A: CGSPG localization in normal biceps muscle. B: Con A staining of ECM in normal biceps muscle. C: CGSPG localization in exercise-injured biceps muscle; ECM is pulled away from surface of myofibers (arrows). D: Con A stainining of exercise-injured biceps muscle. ECM is pulled away from surface of myofibers (arrow). X300.
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872
ECM DISRUPTION
AFTER
ECCENTRIC
MUSCLE
ACTION
FIG. 4. Localization of chondroitin 4sulfate proteoglycan (C4SPG), immune response antigen (13), and B lymphocyte cell surface antigen (Bl) in cross sections of human biceps muscle. A: C4SPG localization in normal biceps muscle. B: C4SPG localization in exercise-iniured biceps muscle (arrows indicate areas without staining). C: 13 localization in exercise-injured biceps muscle; macrophages were observed around disrupted myofibers (arrow). D: Bl localization in exercise-injured biceps muscle; no localization of B lymphocytes was observed. x300.
of myofibers that were no longer in contact (Fig. 4B). Experimental controls for the chondroitin proteoglycans were negative without any fluorescent localization when either chondroitinase ABC or the specific monoclonal antibody was excluded from incubation with the tissue sections. The identity of mononuclear cells observed in the exercised muscle samples was investigated using monoclonal antibodies specific for cells of lymphoid origin. 13 (Ia antigen)-positive cells were observed in small numbers in the interstitial spaces near blood vessels and occasionally in or near the few necrotic fibers found in the exercised muscle sections (Fig. 4C). Because the Ia antigen is expressed by other monocytes besides macrophages, monoclonal antibody specific for B cells (Bl) and natural killer cells (NKH-1) was used to investigate the presence of these cell types. No Bl- or NKH-lpositive cells were localized in either normal or exercised muscle sections from any of the participants (Fig. 40). DISCUSSION
It is generally accepted that skeletal muscle pain caused by exercise involving eccentric muscle action results from mechanical rather than metabolic factors (23). Presumably the mechanical strain overcomes the ability of the active muscle to resist and results in disruption of some myofibers (2). Although appreciable damage to myofibers can result (23), no established relationship exists between myofiber damage and pain. In a previous study using eccentric muscle action to produce soreness, it was noted that pain sensation was maximal 2 days after the exercise bout, but the resting muscle length was decreased immediately postexercise (11). In other studies, the clinical indicator of myofiber damage, creatine kinase, only appeared in the blood after the exercise bout was finished and continued to increase without further muscular activity (14). The nature of the
divergence in responses has not yet been adequately explained (23). It is possible that pain and myofiber damage are not related but coexist after eccentric muscle action. For example, muscle swelling can be seen immediately after the exercise (15) along with limitations in range of motion (11). Yet only in muscles with rigid compartments does this lead to an increase in interstitial pressure sufficient to impair local microcirculation necessary to produce a state of partial ischemia (23)-the biceps brachii are not such a muscle. So it must be concluded that pain sensation in the biceps muscle was not the result solely of muscle swelling or edema. Myofiber damage also occurs during the exercise bout (2, 12) and is demonstrable immediately postexercise (23). However, the intracellular proteins that escape, such as creatine kinase, are not seen in the blood immediately after exercise but appear later and continue to rise without additional injury. This delayed response may reflect the time necessary for I) the proteins to enter the vascular system by way of the lymphatics or 2) a change to occur in vascular permeability that would allow their entry into the microcirculation, In either case, the proteins would act as osmotically active molecules while in the interstitial space before their appearance in the blood. Little is known about how intracellular proteins such as lactate dehydrogenase, creatine kinase, and myoglobin released from muscle enter the general circulation. From the observations in this study it would appear that the vasculature has become permeable to some macromolecules (e.g., fibrinogen) at 48 h after the exercise bout. This occurred at the same time that mast cell degranulation with its known effect on vascular permeability was observed (32). Although these observations might help establish a link between mast cell degranulation and transcystosis of creatine kinase, no definitive informa-
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ECM
DISRUPTION
AFTER
tion about the role of lymphatic removal of intracellular proteins or matrix components has been reported. In contrast, in the skeletal muscles of uninjured rats, the lymphatics have been reported to be responsible for removal of about three-fourths of the transcytosed albumin (28). So the lymphatic circulation may indeed be important in the removal of muscle proteins released by injury or disease. To summarize at this point, pain sensation does not seem to be related to events associated with myofiber damage but seems more closely aligned with delayed responses such as the release of mast cell products that include histamine, a known algesic. It is not surprising then that common medications for the relief of pain have been unsuccessful in treating delayed-onset muscle soreness (20) because they do not block histamine receptors (4). However, calcium-blocking drugs, which could also block the calcium-mediated degranulation of mast cells, have been of some benefit (18, 34). Perhaps it is not the myofiber injury that mediates the pain at all but the disruption of the connective tissue (1) or extracellular matrix surrounding the myofibers. When the extracellular matrix is -damaged, it may release cytokines or peptide degradation products (27) that alert the immune system and subsequently produce a delayedonset inflammation. Also inflammation is a common feature in diseases of connective tissues including polymyositis (l3), which are characterized by inflammatory responses, activation of the immune system, and often pain. Thus the matrix alteration unique to this type of injury may be paramount in the evolution of pain. Another factor in delayed-onset muscle pain and elevated indicators of muscle damage might result from a cytotoxic response -an overreaction of the immune system to injury. By alerting the immune system, the strain injury might produce further damage if specific cellular cytotoxicity such as that seen in inflammatory muscle diseases (13) were operative. Cytotoxic cell involvement has not received much attention, but the few studies appear contradictory. In exercise-induced muscle damage in rats, lymphoid cells were observed within the muscles (32), but no such inflammatory cell response was reported after eccentric muscle action in humans (29). Even though we too were unable to find any cytotoxic cells other than macrophages, it can be argued that the human biopsy sample was too small to make any conclusive statements. However, in another study the cytokine interleukin-lp increased in human muscle biopsies and remained elevated for 5 days after repeated eccentric muscle action (5). Thus a very complex reaction of extracellular matrix, cells, and inflammation mediators exists after muscle damage, some of which may be unique to eccentric muscle actions. This work was supported in part by funds from Comptex, Inc., to W. T. Stauber. This work was presented in part at the American College of Sports Medicine Meeting in Baltimore, MD, 31 May-3 June 1989. Address for reprint requests: W. T. Stauber, Dept. of Physiology, West Virginia University Health Science Center, Morgantown, WV 26506. Received
6 Sentember 1
1989: I accented I
in final
form
3 Mav 4 1990. ~~~-
ECCENTRIC
MUSCLE
ACTION
873
REFERENCES 1. ABRAHAM, W. M. Factors in delayed muscle soreness. 1Med. Sci. Sports Exercise 9: 11-20, 1977. 2. ARMSTRONG, R. B. Mechanisms of exercise-induced delayed onset muscular soreness: a brief review. Med. Sci. Sports Exercise 16: 529-538,1984. 3. BERTOLOTTO, A., L. PALMUCCI, A. GAGLIANO, T. MONGINI, AND G. TARONE. Immunohistochemical localization of chondroitin sulfate in normal and pathological human muscle. J. Neurol. Sci. 73: 233-244, 1986. 4. BLACK, J. W., W. A. M. DUNCAN, C. J. DURANT, C. R. GANELLIN, AND E. M. PARSONS. Definition and antagonism of histamine H2receptors. Nature Lond. 236: 385-390, 1972. 5. CANNON, J. G., R. A. FIELDING, M. A. FIATARONE, S. F. ORENCOLE, C. A. DINARELLO, AND W. J. EVANS. Increased interleukin lp in human skeletal muscle after exercise. Am. J. Physiol. 257 (Regulatory Integrative Comp. Physiol. 26): R451-R455, 1989. 6. CARLSON, B. M., AND J. A. FAULKNER. The regeneration of skeletal muscle fibers following injury: a review. Med. Sci. Sports Exercise 15: 187-198,1983. 7. CATERSON, B., J. R. BAKER, J. E. CHRISTNER, J. F. KEARNEY, AND R. L. STROHRER. The characterization of clonal antibodies directed against bovine nasal cartilage proteoglycan and like protein. In: Monoclonal Antibodies and T-Cell Hybridomas, edited by G. J. Hammerling, B. Hammerling, and J. F. Kearney. New York: Elsevier/North-Holland, 1981, p. 259-268. 8. CATERSON, B., T. CALABRO, AND A. HAMPTON. Monoclonal antibodies as probes for elucidating proteoglycan structure and function. In: Biology of Proteoglycans, edited by T. N. Wight and R. P. Mecham. New York: Academic, 1987, p. l-26. 9. CATERSON, B., J. E. CHRISTNER, J. R. BAKER, AND J. R. COUCHMAN. Production of monoclonal antibodies directed against connective tissue proteoglycans. Federation Proc. 44: 386-393, 1985. 10. CAVANAGH, P. R. On “muscle action” vs. “muscle contraction.” J. Biomech. 21: 69, 1988. 11. CLARKSON, P. M., AND I. TREMBLAY. Exercise-induced muscle damage, repair, and adaptation in humans. J. Appl. Physiol. 65: I6, 1988. 12. DICK, R. W., AND P. CAVANAGH. An explanation of the upward drift in oxygen uptake during prolonged sub-maximal downhill running. Med. Sci. Sports Exercise 19: 310-317, 1987. 13. ENGEL, A. G., AND K. ARAHATA. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigen-specific cell mediated cytotoxicity in some diseases and implication for the pathogenesis of the different inflammatory myopathies. Hum. Pathol. 17: 704-721, 1986. 14. EVANS, W. J., C. N. MEREDITH, J. G. CANNON, C. A. DINARELLO, W. R. FRONTERA, V. A. HUGHES, B. H. JONES, AND H. G. KNUTTGEN. Metabolic changes following eccentric exercise in trained and untrained men. J. Appl. Physiol. 61: 1864-1868, 1986. 15. FRIDEN, J., P. N. STAKIANOS, A. R. HARGENS, AND W. H. AKESON. Residual muscular swelling after repetitive eccentric contractions. J. Orthop. Res. 6: 493-498, 1988. 16. FRITZ, V. K., AND W. T. STAUBER. Characterization of muscles injured by forced lengthening. II. Proteoglycans. Med. Sci. Sports Exercise 20: 354-361, 1988. 17. GULATI, A. K., A.H. REDDI, AND A. A. ZALEWSKI. Changes in the basement membrane zone components during skeletal muscle fiber degeneration and regeneration. J. Cell Biol. 97: 957-962, 1983. 18. HAVERKORT-POELS, P. J. E., E. M. G. JOOSTEEN, AND W. RUITENBECK. Prevention of recurrent exertional rhabdomyolysis by dantrolene sodium. Muscle Nerve 10: 45-46, 1987. 19. HOUGH, T. Ergographic studies in muscle soreness. Am. J. Physiol. 7: 76-92,1902. 20. KUIPERS, H., H. A. KEIZER, F. T. J. VERSTAPPEN, AND D. L. COSTILL. Influence of prostaglandin-inhibiting drug on muscle soreness after ecentric work. Int. J. Sports Med. 6: 336-339, 1985. 21. LOWRY, 0. H., J. N. ROSENBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 22. MCMANUS, J. F. A., AND R. W. MOWRY. Staining Methods, Histologic and Histochemical. New York: Hoeber, 1960, p. 132 and 261. 23. NEWHAM, D. J. The consequences of eccentric contractions and their relationship to delayed-onset muscle pain. Eur. J. Appl. Physiol. Occup. Physiol. 57: 353-359, 1988.
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DISRUPTION
AFTER
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