Extracellular matrix disruption and pain

after eccentric muscle action

WILLIAM T. STAUBER, PRISCILLA M. CLARKSON,

VALERIE 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 ions into the extracellular space, 2) from calcium influx
K. FRITZ, ANDWILLIAM J. [Link] matrix disrup- into a damaged myofiber with water accumulation within
tion and pain after eccentric muscle action. J. Appl. Physiol. the myofiber, and 3) from disruption of the extracellular
69(3): 868~874,1990.-Pain, stiffness, and indicators of muscle matrix and/or increase in its hydration state. In all cases
damage occur at different times after eccentric muscle action.
After a single bout of maximal resisted lengthening of the elbow there would be an apparent decrease in the resting length
flexors, elbow position, pain perception, and indicators of cel- of the muscle as the swollen tissue pushes against the
lular damage were measured. Immediately postexercise, a sig- fascia and shortens the muscles passively.
nificant decrease in resting muscle length was observed that The purpose of this study was to investigate the rela-
continued to 48 h. At this time, an increase in perceived muscle tionship, if any, among tenderness (pain perception),
soreness was noted (P c 0.05), and a biopsy of the biceps indicators of inflammation, and integrity of the extra-
brachii revealed mast cell degranulation, separations of the cellular matrix in human volunteers after a bout of
extracellular matrix from myofibers, and increased plasma eccentric muscle actions. Previous research with this
constituents in the extracellular space. It is proposed that mode of exercise-induced injury resulted in muscular
myofiber disruption allows intracellular proteins to escape and swelling and limitations in range of motion of the exer-
extracellular proteins and ions to enter, causing swelling,
whereas the disrupted extracellular matrix initiates the inflam- cised muscles before the appearance of indicators of
matory response, which includes the release of mast cell gran- muscle damage in the blood (11). Thus the nature of
ules seen at 48 h postexercise. Thus the delayed sensation of inflammation, pain, and muscle damage from repeated
pain (soreness) after repeated eccentric muscle actions probably eccentric muscle action is in question.
results from inflammation in response to extracellular matrix
disruption. MATERIALS AND METHODS

skeletal muscle injury; human; proteoglycans; inflammation; Five healthy nontrained volunteers (male and female)
exercise 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
MUSCLE INJURY after eccentric muscle action (10) is a the study was minimized by matching participants for
common experience in sports or occupational tasks es- age, height, and weight (Table 1). None of the partici-
pecially of a novel nature. The injury results in pain and pants was “weight-trained”; nor had they exercised for
muscular swelling after the exercise bout (19), but the at least 2 mo before the study. The volunteers were asked
symptoms disappear within a few days (1). As with other to refrain from any new or strenuous physical activity 7
tissue injuries, the muscle is repaired (6) without any days before and any time during the study.
residual dysfunction or scarring, and often the repair Using the passive mode of a computer-interfaced dy-
process results in a muscle that is able to resist even namometer (Biodex, Shirley, NY), each participant per-
greater applied forces (24). formed 70 maximal isokinetic resisted (eccentric muscle
The mechanical injury after eccentric muscle action action) movements of the elbow flexors using the non-
results in myofiber damage as well as alterations to the dominant arm at l2O”/s through a range of 120’. Re-
extracellular matrix (16), both of which may lead to straining straps were placed around the waist, across the
inflammation and pain. However, pain and inflammation chest, and loosely over the upper arm of the involved
follow a different time course than the indicators of limb. Each movement was a maximal effort to resist the
myofiber damage (23). Thus the specific processes that ability of the dynamometer to extend the elbow. Thus
follow muscle injury such as tissue swelling and extra- the elbow flexors were performing eccentric muscle ac-
cellular matrix disruption may be more important in the tions or resisted muscle lengthening. A 10-s rest between
production of pain and inflammation than the mechan- each exercise movement allowed the dynamometer to
ical damage to the myofiber itself (30). Swelling could return to the starting position.
result 1) from cell damage and release of proteins and By use of a subjective assessment scale of 1 (normal)-
868 0161-7567/90 $1.50 Copyright 0 1990 the American Physiological Society

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 ECM DISRUPTION AFTER ECCENTRIC MUSCLE ACTION 869

10 (very, very sore), soreness of the biceps muscle was TABLE 2. Immunohistochemical reagents
assessed before exercise and 48 h after exercise by ques-
tionnaire (33). Changes in muscle shortening of the elbow Antibody Specificity
flexors were assessed before, immediately after, and 48 h
Primary antisera
after the exercise bout by use of a goniometer to measure
Goat anti-human fibrinogen Fibrinogen
the change in the relaxed elbow joint angle while the Goat anti-human albumin Albumin
subject was standing with head facing forward and arm
relaxed down at the side. Anatomic reference points for Monoclonal antisera
goniometer placement were the deltoid insertion, the McAb 4/8/2B6 (C4SPG) Delta unsaturated disaccharides
lateral humeral epicondyle, and the ulnar styloid process of chondroitin 4-sulfate generated
by digestion with chondroitinase
with the hand in the neutral position. Each reference ABC (3, 5)
point was marked with a semipermanent ink. The data McAb 5/6/3B3 (CGSPG) Delta unsaturated disaccharides
were analyzed using Friedman’s test for two-way classi- of chondroitin and chondroitin
fication to examine the changes in joint angle resulting 6-sulfate generated by digestion
with chondroitinase ABC (3,5)
from the exercise insult. McAb 13 Immune response antigen
At 48 h postexercise, needle biopsies were taken from McAb Bl B lymphocytes
the exercised and nonexercised biceps brachii muscles of McAb NKH-1 Natural killer cells
each volunteer. This postexercise time was chosen be- Labeled lectins
cause perceived muscle soreness would peak at 48 h
FITC-labeled Con A Extracellular matrix glycoproteins
postexercise (11), and only one biopsy sample was to be containing a-linked mannose
removed. The tissue samples were quickly frozen in 2
C4SPG, chondroitin 4-sulfate proteoglycan; C6SPG, chondroitin 6-
methylbutane cooled in liquid nitrogen. The tissue sam- sulfate proteoglycan; FITC, fluorescein isothiocyanate.
ples 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 The presence of mast cells was determined using a
alcohol-cleaned glass slides. Tissue samples were main- toluidine blue histological stain (22). Based on meta-
tained at -2OC in airtight boxes until use. chromatic properties of the mast cell granules, mast cells
Sectioned material was examined using a histological could be distinguished from other cell types by the red-
stain for mast cells and immunohistochemical techniques to-purple color of the granules. Tissue sections were
for various proteoglycans, proteinases, and lymphocyte stained for 10 min with 0.5% toluidine blue in 20% ethyl
cell surface antigens (Table 2). Antisera to human fibrin- alcohol. After a rinse in phosphate-buffered saline (PBS,
ogen and albumin were obtained commercially (Organon pH 7.4), coverslips were applied, and the sections were
Teknika-Cappel, Malvern, PA). Monoclonal antibodies observed and photographed using an Olympus micro-
to the immune response antigen (13), B lymphocytes scope with camera attachment.
(Bl), and natural killer cells (NKH-1) were acquired Briefly, the immunohistochemical staining procedure
from Coulter Immunology (Hialeah, FL). Fluorescein- for the lymphocyte cell surface antigens, fibrinogen, and
conjugated concanavalin A (Con A, Vector Laboratories, albumin included washing the slides three times for 5
Burlingame, CA) was used to localize glycoproteins rich min with filter sterile 0.001 M sodium phosphate in 0.15
in mannose residues such as found in the pericellular M NaCl, pH 7.4 (PBS). Excess PBS was removed before
basement membrane of the basal lamina (17). specific antiserum was added, and 50 ~1 of the appropri-
Monoclonal antibodies directed against proteoglycans ate dilution of antiserum were applied to each slide. The
digested with chondroitinase ABC were provided by Dr. slides were incubated at room temperature for 30 min in
Bruce Caterson (University of North Carolina at Chapel a moist chamber to prevent evaporation. The antisera
Hill, Chapel Hill, NC). Antigen isolation, monoclonal were diluted to -1 mg/ml protein as determined by
antibody production, and characterization have been protein assay (21) using albumin as a standard. After
completely described previously (7, 9). Digestion of the incubation, all slides were washed for 5 min in PBS.
tissue with chondroitinase ABC resulted in a proteogly- Finally, 50 ~1 of fluorescein-labeled goat F(ab’)z anti-
can fragment of small oligosaccharide stubs with at least rabbit immunoglobulin (Ig) G, rabbit anti-goat IgG, or
one characteristic unsaturated disaccharide (0, 4, or 6 goat F(ab’)a anti-mouse IgG at a 1:20 dilution were added
sulfated) of the chondroitin sulfate isomer and the link- to all experimental and control slides. After another 30-
age region attached to the proteoglycan core protein (8). min incubation and a final wash for 5 min in PBS, glass
coverslips were applied over the tissue sections with 50%
TABLE 1. Physical characteristics of volunteers glycerine-50% PBS containing 1 mg/ml p-phenylenedi-
amine (Sigma Chemical, St. Louis, MO) and viewed on
Subj Age, Height, Weight,
NO.
Gender
Yr cm
a Leitz Orthoplan fluorescence microscope (31).
kg
Localization of specific proteoglycans was performed
ASS1 M 23 159.5 60.2 using a modification of the previously described tech-
Ass2 M 19 175.3 59.4
nique (3). The slides were washed for 5 min with 0.05 M
ss3 M 19 165.1 59.9
ss4 F 17 170.1 62.6 tris(hydroxymethyl)aminomethane HCl and 0.05 M l

ss5 F 23 159.5 60.2 NaCl (pH 8.0) and predigested with 0.5 U/ml chondroi-
tinase ABC (Sigma Chemical) diluted in the same buffer
Means * SE 20.2~1.2 165.9k3.0 60.5t0.5 for 30 min at 37°C in a moist chamber. After a 5-min

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 870 ECM DISRUPTION AFTER ECCENTRIC MUSCLE ACTION

wash in PBS, 50 ~1 of diluted specific monoclonal anti- 160

serum were added to each slide and incubated as before.
After incubation, all slides were washed again in PBS 158
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 micro-
scope 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 previ-
ously described.
Method specificity and antibody specificity for the 146.
immunohistochemical reactions were determined accord-
ing to the criteria of the reliability established by Petrusz 144.
et al. (26). The controls for the fluorescent immunohis-
tochemical experiments included normal and injured tis- l42*
.
sue sections either incubated with normal rabbit serum
instead of specific antibody or culture media instead of 1 I 1

monoclonal antibody followed by fluorescein-labeled PRE - POST- 48 HRS

antiserum. Controls for the monoclonal antisera to pro- EXERCISE EXERCISE POST- EXERCISE
teoglycans included fresh frozen sections of rat ear as FIG. 1. Relaxed angle (elbow angle when arm is resting at side)
positive controls treated in the same manner as the before, immediately after, and 48 h after exercise bout. Values are
normal and injured muscle tissue. The specificity of the means k SE. A significant decrease in relaxed elbow joint angle was
monoclonal antibodies was tested by excluding the chon- recorded immediately after exercise and 48 h later (P 5 0.05).
droitinase ABC digestion before monoclona-l antibody
application followed by fluorescein-labeled second anti- revealed widened perimysial areas between fascicles and
body. Another set of slides was incubated with chondroi- separation of the myofibers from one another within
tinase ABC, diluted mouse serum, and fluorescein-con- fascicles (endomysial region) (Fig. 2A ). Some mononu-
jugated second antibody. clear cells were observed in the perimysial and endomy-
sial regions; however, they did not fill the intracellular
RESULTS spaces. Mast cell degranulation was evident in sections
of exercised muscle from all participants. Degranulating
Evaluation of five subjects after a single bout of 70 mast cells were localized primarily in the perimysial area
maximal isokinetic resisted (eccentric muscle action) near blood vessels (Fig. 2B). However, a few were also
movements of the elbow flexors revealed significant in- present in the endomysial area near damaged myofibers
creases in perceived muscle soreness of the exercised (Fig. 2C). Occasionally, a few necrotic fibers were ob-
biceps and a significant decrease in the measured relaxed served because they stained darker with toluidine blue
elbow joint angle. Before exercise, all subjects reported (loss of viability) and/or contained infiltrating mononu-
no perceived muscle soreness of the biceps muscle (av- clear cells and flocculent degeneration of the sarcoplasm.
erage rating of 1; normal). At 48 h postexercise, however, The frequency of necrotic fibers per section of between
the average score for perceived muscle soreness had 300 and 500 fibers was -2%, with the remainder of the
increased to 8 (a0.7) with the range of responses between fibers appearing unaltered.
7 and 9 on a scale of 10 (very, very sore). Further evidence of muscle alterations from eccentric
A significant decrease in the relaxed elbow joint angle muscle action was found using immunohistochemical
was recorded immediately after exercise and 48 h later techniques to observe changes in localization of specific
compared with baseline measurements taken before the serum components, mononuclear cells, and extracellular
exercise bout (Fig. 1). Immediately postexercise the re- matrix (ECM) components. In normal muscle sections,
laxed elbow joint angle had significantly decreased an the serum components albumin and fibrinogen were lo-
average of 7.2 t 1.3O (P 5 0.05) and by 48 h the decrease calized in capillaries with some diffuse staining around
in angle averaged 10.4 t 5.9’ (P 5 0.05). The large myofibers for albumin. In exercise-injured muscle, both
variance among the joint angle measurements that oc- of these serum components filled areas of the widened
curred by 48 h was the result of two of the subjects perimysial and endomysial spaces around fibers (Fig.
experiencing a much greater decrease in angle than the 20). Some damaged myofibers were easily identified
others. because both albumin and fibrinogen were localized
Alterations to muscle fibers were observed histologi- within these compromised fibers.
cally at 48 h by use of toluidine blue-stained sections of Major alterations to the ECM were obvious in sections
the exercised biceps muscle, although the muscle re- of exercised biceps muscle. Cross sections of normal
mained unchanged in sections from the contralateral biceps muscle showed chondroitin 6-sulfate proteoglycan
limb. Sections of exercised muscle from all participants 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 bi-

ceps muscle 48 h after exercise. A: tolui-
dine blue stain (arrows indicate gaps; see
Fig. 30). B: toluidine blue stain. deeran-
ulating mast cell (arrow). C: toluydine
blue stain, degranulating mast cell (ar-
row). D: immunohistochemical localiza-
tion of albumin. x300.

blood vessels, and nerve fibers (Fig. 3A). Likewise, Con perimeter. Occasionally these gaps could also be found
A-stained material was observed in a similar pattern in filled in with Con A- and chondroitin B-sulfate proteo-
the perimysial region in addition to the endomysial area glycan-positive material. No mononuclear cell infiltra-
of normal muscle sections (Fig. 3B). Both chondroitin 6- tion was observed in these fibers with ECM gaps at this
sulfate proteoglycan localization and Con A staining time.
accentuated the close association existing between my- Localization of chondroitin 4-sulfate proteoglycan had
ofibers and the ECM. With the use of both chondroitin also changed after the exercise bout compared with the
6-sulfate proteoglycan and Con A, muscle sections from contralateral limb. In normal muscle, chondroitin 4-
all exercised volunteers revealed that a portion of the sulfate proteoglycan was localized predominantly around
ECM was actually pulled away from the surface of my- blood vessels (Fig. 4A). A narrow band of stained mate-
ofibers into the widened interstitial spaces, leaving the rial was also observed in the perimysium and endomy-
perimeter of the fiber with no fluorescent material pres- sium and around capillaries. In exercised muscle, chon-
ent (Fig. 3, C and D). This was usually observed along droitin 4-sulfate proteoglycan staining was observed fill-
at least one edge of the myofibers, but occasionally ing widened perimysial areas (Fig. 4B). However, it was
separation was observed in two or three regions of the absent from the endomysial region along the perimeter

FIG. 3. Chondroitin 6-sulfate proteo-

glycan (CGSPG) localization and Con A
staining of ECM in cross sections of
human biceps muscle. A: CGSPG local-
ization in normal biceps muscle. B: Con
A staining of ECM in normal biceps
muscle. C: CGSPG localization in exer-
cise-injured biceps muscle; ECM is
pulled away from surface of myofibers
(arrows). D: Con A stainining of exer-
cise-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 4-

sulfate proteoglycan (C4SPG), immune
response antigen (13), and B lymphocyte
cell surface antigen (Bl) in cross sections
of human biceps muscle. A: C4SPG lo-
calization 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; macro-
phages were observed around disrupted
myofibers (arrow). D: Bl localization in
exercise-injured biceps muscle; no local-
ization of B lymphocytes was observed.
x300.

of myofibers that were no longer in contact (Fig. 4B). divergence in responses has not yet been adequately
Experimental controls for the chondroitin proteoglycans explained (23).
were negative without any fluorescent localization when It is possible that pain and myofiber damage are not
either chondroitinase ABC or the specific monoclonal related but coexist after eccentric muscle action. For
antibody was excluded from incubation with the tissue example, muscle swelling can be seen immediately after
sections. the exercise (15) along with limitations in range of mo-
The identity of mononuclear cells observed in the tion (11). Yet only in muscles with rigid compartments
exercised muscle samples was investigated using mono- does this lead to an increase in interstitial pressure
clonal antibodies specific for cells of lymphoid origin. 13 sufficient to impair local microcirculation necessary to
(Ia antigen)-positive cells were observed in small num- produce a state of partial ischemia (23)-the biceps
bers in the interstitial spaces near blood vessels and brachii are not such a muscle. So it must be concluded
occasionally in or near the few necrotic fibers found in that pain sensation in the biceps muscle was not the
the exercised muscle sections (Fig. 4C). Because the Ia result solely of muscle swelling or edema.
antigen is expressed by other monocytes besides macro- Myofiber damage also occurs during the exercise bout
phages, monoclonal antibody specific for B cells (Bl) (2, 12) and is demonstrable immediately postexercise
and natural killer cells (NKH-1) was used to investigate (23). However, the intracellular proteins that escape,
the presence of these cell types. No Bl- or NKH-l- such as creatine kinase, are not seen in the blood im-
positive cells were localized in either normal or exercised mediately after exercise but appear later and continue to
muscle sections from any of the participants (Fig. 40). rise without additional injury. This delayed response may
reflect the time necessary for I) the proteins to enter the
DISCUSSION vascular system by way of the lymphatics or 2) a change
It is generally accepted that skeletal muscle pain to occur in vascular permeability that would allow their
caused by exercise involving eccentric muscle action entry into the microcirculation, In either case, the pro-
results from mechanical rather than metabolic factors teins would act as osmotically active molecules while in
(23). Presumably the mechanical strain overcomes the the interstitial space before their appearance in the
ability of the active muscle to resist and results in dis- blood.
ruption of some myofibers (2). Although appreciable Little is known about how intracellular proteins such
damage to myofibers can result (23), no established re- as lactate dehydrogenase, creatine kinase, and myoglobin
lationship exists between myofiber damage and pain. In released from muscle enter the general circulation. From
a previous study using eccentric muscle action to produce the observations in this study it would appear that the
soreness, it was noted that pain sensation was maximal vasculature has become permeable to some macromole-
2 days after the exercise bout, but the resting muscle cules (e.g., fibrinogen) at 48 h after the exercise bout.
length was decreased immediately postexercise (11). In This occurred at the same time that mast cell degranu-
other studies, the clinical indicator of myofiber damage, lation with its known effect on vascular permeability was
creatine kinase, only appeared in the blood after the observed (32). Although these observations might help
exercise bout was finished and continued to increase establish a link between mast cell degranulation and
without further muscular activity (14). The nature of the transcystosis of creatine kinase, no definitive informa-

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 ECM DISRUPTION AFTER ECCENTRIC MUSCLE ACTION 873

tion about the role of lymphatic removal of intracellular REFERENCES

proteins or matrix components has been reported. In 1. ABRAHAM, W. M. Factors in delayed muscle soreness. 1Med. Sci.
contrast, in the skeletal muscles of uninjured rats, the Sports Exercise 9: 11-20, 1977.
lymphatics have been reported to be responsible for 2. ARMSTRONG, R. B. Mechanisms of exercise-induced delayed onset
muscular soreness: a brief review. Med. Sci. Sports Exercise 16:
removal of about three-fourths of the transcytosed al- 529-538,1984.
bumin (28). So the lymphatic circulation may indeed be 3. BERTOLOTTO, A., L. PALMUCCI, A. GAGLIANO, T. MONGINI, AND
important in the removal of muscle proteins released by G. TARONE. Immunohistochemical localization of chondroitin sul-
injury or disease. fate in normal and pathological human muscle. J. Neurol. Sci. 73:
233-244, 1986.
To summarize at this point, pain sensation does not 4. BLACK, J. W., W. A. M. DUNCAN, C. J. DURANT, C. R. GANELLIN,
seem to be related to events associated with myofiber AND E. M. PARSONS. Definition and antagonism of histamine H2-
damage but seems more closely aligned with delayed receptors. Nature Lond. 236: 385-390, 1972.
responses such as the release of mast cell products that 5. CANNON, J. G., R. A. FIELDING, M. A. FIATARONE, S. F. OREN-
COLE, C. A. DINARELLO, AND W. J. EVANS. Increased interleukin
include histamine, a known algesic. It is not surprising lp in human skeletal muscle after exercise. Am. J. Physiol. 257
then that common medications for the relief of pain have (Regulatory Integrative Comp. Physiol. 26): R451-R455, 1989.
been unsuccessful in treating delayed-onset muscle so- 6. CARLSON, B. M., AND J. A. FAULKNER. The regeneration of skeletal
reness (20) because they do not block histamine receptors muscle fibers following injury: a review. Med. Sci. Sports Exercise
15: 187-198,1983.
(4). However, calcium-blocking drugs, which could also
7. CATERSON, B., J. R. BAKER, J. E. CHRISTNER, J. F. KEARNEY,
block the calcium-mediated degranulation of mast cells, AND R. L. STROHRER. The characterization of clonal antibodies
have been of some benefit (18, 34). directed against bovine nasal cartilage proteoglycan and like pro-
Perhaps it is not the myofiber injury that mediates the tein. In: Monoclonal Antibodies and T-Cell Hybridomas, edited by
pain at all but the disruption of the connective tissue (1) G. J. Hammerling, B. Hammerling, and J. F. Kearney. New York:
Elsevier/North-Holland, 1981, p. 259-268.
or extracellular matrix surrounding the myofibers. When 8. CATERSON, B., T. CALABRO, AND A. HAMPTON. Monoclonal anti-
the extracellular matrix is -damaged, it may release cy- bodies as probes for elucidating proteoglycan structure and func-
tokines or peptide degradation products (27) that alert tion. In: Biology of Proteoglycans, edited by T. N. Wight and R. P.
the immune system and subsequently produce a delayed- Mecham. New York: Academic, 1987, p. l-26.
9. CATERSON, B., J. E. CHRISTNER, J. R. BAKER, AND J. R. COUCH-
onset inflammation. Also inflammation is a common MAN. Production of monoclonal antibodies directed against con-
feature in diseases of connective tissues including poly- nective tissue proteoglycans. Federation Proc. 44: 386-393, 1985.
myositis (l3), which are characterized by inflammatory 10. CAVANAGH, P. R. On “muscle action” vs. “muscle contraction.” J.
responses, activation of the immune system, and often Biomech. 21: 69, 1988.
11. CLARKSON, P. M., AND I. TREMBLAY. Exercise-induced muscle
pain. Thus the matrix alteration unique to this type of
damage, repair, and adaptation in humans. J. Appl. Physiol. 65: I-
injury may be paramount in the evolution of pain. 6, 1988.
Another factor in delayed-onset muscle pain and ele- 12. DICK, R. W., AND P. CAVANAGH. An explanation of the upward
vated indicators of muscle damage might result from a drift in oxygen uptake during prolonged sub-maximal downhill
running. Med. Sci. Sports Exercise 19: 310-317, 1987.
cytotoxic response -an overreaction of the immune sys-
13. ENGEL, A. G., AND K. ARAHATA. Mononuclear cells in myopathies:
tem to injury. By alerting the immune system, the strain quantitation of functionally distinct subsets, recognition of anti-
injury might produce further damage if specific cellular gen-specific cell mediated cytotoxicity in some diseases and impli-
cytotoxicity such as that seen in inflammatory muscle cation for the pathogenesis of the different inflammatory myopa-
diseases (13) were operative. Cytotoxic cell involvement thies. Hum. Pathol. 17: 704-721, 1986.
14. EVANS, W. J., C. N. MEREDITH, J. G. CANNON, C. A. DINARELLO,
has not received much attention, but the few studies W. R. FRONTERA, V. A. HUGHES, B. H. JONES, AND H. G.
appear contradictory. In exercise-induced muscle damage KNUTTGEN. Metabolic changes following eccentric exercise in
in rats, lymphoid cells were observed within the muscles trained and untrained men. J. Appl. Physiol. 61: 1864-1868, 1986.
(32), but no such inflammatory cell response was re- 15. FRIDEN, J., P. N. STAKIANOS, A. R. HARGENS, AND W. H. AKESON.
Residual muscular swelling after repetitive eccentric contractions.
ported after eccentric muscle action in humans (29). J. Orthop. Res. 6: 493-498, 1988.
Even though we too were unable to find any cytotoxic 16. FRITZ, V. K., AND W. T. STAUBER. Characterization of muscles
cells other than macrophages, it can be argued that the injured by forced lengthening. II. Proteoglycans. Med. Sci. Sports
human biopsy sample was too small to make any conclu- Exercise 20: 354-361, 1988.
17. GULATI, A. K., A.H. REDDI, AND A. A. ZALEWSKI. Changes in the
sive statements. However, in another study the cytokine
basement membrane zone components during skeletal muscle fiber
interleukin-lp increased in human muscle biopsies and degeneration and regeneration. J. Cell Biol. 97: 957-962, 1983.
remained elevated for 5 days after repeated eccentric 18. HAVERKORT-POELS, P. J. E., E. M. G. JOOSTEEN, AND W. RUI-
muscle action (5). Thus a very complex reaction of ex- TENBECK. Prevention of recurrent exertional rhabdomyolysis by
tracellular matrix, cells, and inflammation mediators dantrolene sodium. Muscle Nerve 10: 45-46, 1987.
19. HOUGH, T. Ergographic studies in muscle soreness. Am. J. Physiol.
exists after muscle damage, some of which may be unique 7: 76-92,1902.
to eccentric muscle actions. 20. KUIPERS, H., H. A. KEIZER, F. T. J. VERSTAPPEN, AND D. L.
COSTILL. Influence of prostaglandin-inhibiting drug on muscle
This work was supported in part by funds from Comptex, Inc., to soreness after ecentric work. Int. J. Sports Med. 6: 336-339, 1985.
W. T. Stauber. 21. LOWRY, 0. H., J. N. ROSENBROUGH, A. L. FARR, AND R. J.
This work was presented in part at the American College of Sports RANDALL. Protein measurement with the folin phenol reagent. J.
Medicine Meeting in Baltimore, MD, 31 May-3 June 1989. Biol. Chem. 193: 265-275, 1951.
Address for reprint requests: W. T. Stauber, Dept. of Physiology, 22. MCMANUS, J. F. A., AND R. W. MOWRY. Staining Methods, His-
West Virginia University Health Science Center, Morgantown, WV tologic and Histochemical. New York: Hoeber, 1960, p. 132 and 261.
26506. 23. NEWHAM, D. J. The consequences of eccentric contractions and
their relationship to delayed-onset muscle pain. Eur. J. Appl.
Received 6 Sentember
1 1989: I accented
I in final form 3 Mav 4 1990.
~~~- Physiol. Occup. Physiol. 57: 353-359, 1988.

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874 ECM DISRUPTION AFTER ECCENTRIC MUSCLE ACTION

24. NEWHAM, D. J., AND P. M. CLARKSON. Repeated high-force eccen- trates in human skeletal muscle: exercise induced damage as a
tric exercise: effects on muscle pain and damage. J. Appl. Physiol. model for inflammatory disease? J. Neural. Sci. 82: l-11, 1987.
63: 1381-1386,1987. 30. STAUBER, W. T. Eccentric action of muscles: physiology, injury
25. NEWHAM, D. J., K. R. MILLS, B. M. QUIGLEY, AND R. H. T. and adaptation. In: Exercise and Sport Sciences Review, edited by
EDWARDS. Pain and fatigue after concentric and eccentric muscle K. B. Pandolf. Baltimore, MD: Williams & Wilkins, 1989, vol. 17,
contractions. CZin. Sci. Lond. 64: 55-62, 1983. p. 157-185.
26. PETRUSZ, P., P. ORDRONNEAU, AND J. C. W. FINELY. Criteria of 31. STAUBER, W. T., V. K. FRITZ, B. DAHLMANN, AND H. REINAUER.
Immunofluorescent localization of an alkaline proteinase in skel-
reliability for light microscopic immunocytochemical staining. His-
etal muscle from diabetic rats. Basic Appl. Histochem. 30: 147-152,
tochem. J. 12: 333-348,198O.
1986.
27. POSTLETHWAITE, A. E., AND A. H. KANG. Collagen- and collagen 32. STAUBER, W. T., V. K. FRITZ, D. W. VOGELBACH, AND B. DAHL-
peptide-induced chemotaxis of human blood monocytes. J. Exp. MANN. Characterization of muscles injured by forced lengthening.
A4ed. 143: 1299-1307,1976. I. Cellular infiltrates. Med. Sci. Sports Exercise 20: 345-353, 1988.
28. REED, R. F., S. JOHANSEN, AND H. NODDELAND. Turnover rate of 33. TIIDUS. P. M.. AND C. D. IANUZZO. Effects of intensity and
interstitial albumin in rat skin and skeletal muscle. Effects of limb duration of muscular exercise on delayed soreness and serum
movements and motor activity. Acta Physiol. Stand. 125: 711-718, enzvme activities. A4ed. Sci. SDorts Exercise 15: 461-465, 1983.
1985. 34. WA~TON, J. Diffuse exercise-induced muscle pain of undetermined
29. ROUND, J. M., D. A. JONES, AND G. CAMBRIDGE. Cellular infil- cause relieved by verapamil. Lancet 1: 993, 1981.

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