Journal of Gerontology: BIOLOGICAL SCIENCES
2003, Vol. 58A, No. 7, 592–597
Copyright 2003 by The Gerontological Society of America
Passive Stretches Protect Skeletal Muscle
of Adult and Old Mice From Lengthening
Contraction-Induced Injury
Timothy J. Koh,1 Jennifer M. Peterson,2 Francis X. Pizza,2 and Susan V. Brooks3
1
School of Kinesiology, University of Illinois at Chicago.
Department of Kinesiology, University of Toledo, Ohio.
3
Institute of Gerontology, University of Michigan, Ann Arbor.
2
We tested the hypothesis that a single bout of training with passive stretches or isometric
contractions protects the extensor digitorum longus muscle in old mice from contraction-induced
injury. Lengthening contractions produced similar decreases in force (~70%–80%) and numbers of
overtly injured fibers (~15%–20%) in adult and old mice, but twofold greater inflammatory cell
accumulation above untreated control values in old versus adult mice. For both age groups, prior
training with passive stretches improved postinjury force almost twofold compared with untrained
muscles and reduced injured fibers by one half. Training with passive stretches or isometric
contractions reduced inflammatory cell accumulation following lengthening contractions by as
much as two thirds in old mice, but not in adult mice. The data indicate that passive stretches
provide some protection against contraction-induced injury in old mice, and that accumulation of
inflammatory cells does not correlate strongly with force deficit and number of injured fibers.
R
EPEATED stretches of active skeletal muscle (lengthening contractions) can produce muscle damage, pain,
loss of force production, and loss of mobility (1–4). Postulated mechanisms of damage include disruption of sarcomeres and other cytoskeletal elements, membrane injury,
loss of calcium homeostasis, inflammation, and oxidative
stress (5–8). Aging may increase susceptibility to lengthening contraction-induced injury as identical protocols of
lengthening contractions produced larger force deficits in
muscle of old versus adult mice (9,10), and greater muscle
fiber damage in muscles of old versus young men (11).
Aging also appears to impair recovery from muscle injury as
muscles of old animals recover more slowly than those of
young animals following protocols of lengthening contractions that produced similar force deficits (11–13).
Exercise training can protect skeletal muscle from
lengthening contraction-induced injury (14–17). In humans,
a single prior bout of lengthening contractions has been
shown to reduce subsequent lengthening contraction-induced force deficits, muscle soreness, and serum levels of
creatine kinase (14,16,18). In animals, a single bout of training with lengthening contractions decreased the force deficit
and the number of fibers demonstrating gross morphological
abnormalities at the light microscope level (overtly injured
fibers) in response to a second bout of lengthening
contractions (13,19,20). We showed recently that lengthening contractions are not required for inducing protection, as
training with passive stretches or isometric contractions
reduced the force deficit, the number of overtly injured
fibers, and the accumulation of inflammatory cells following
engthening contractions in extensor digitorum longus (EDL)
muscles of adult mice (21,22).
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The protective adaptation associated with exercise
training appears to be impaired in old animals. In adult rats
(6 months old), a protocol of 24 lengthening contractions of
the tibialis anterior provided complete protection against the
force deficit induced by an identical subsequent protocol
2 weeks later (13). Subjecting muscles of old rats (age:
32 months) to an identical protocol of 24 lengthening
contractions produced only partial protection. In addition,
dorsiflexor muscles of adult mice (age: 7 months) exposed
to weekly bouts of lengthening contractions showed progressive reductions in the force deficit until week 4 when
the force deficit was eliminated, whereas in old mice (age:
22 months) a similar level of protection was not demonstrated until week 5 (19). Whether training with passive
stretches or isometric contractions can induce protection
in muscles of old mice remains to be determined. Also,
a review of the literature revealed no data on inflammatory
cell accumulation in muscle from old animals following
lengthening contractions with or without prior training.
Thus, the purpose of the present study was to test the
hypothesis that training with passive stretches or isometric
contractions can protect skeletal muscle in old as well as
adult mice from lengthening contraction-induced injury as
assessed by force deficits, numbers of overtly injured fibers,
and accumulation of inflammatory cells.
METHODS
Animals
Three-month-old (adult; n ¼ 25) and 24-month-old (old;
n ¼ 23) specific pathogen-free male C57BL/6 mice were
obtained from the National Institute on Aging colony at
PROTECTING SKELETAL MUSCLE FROM INJURY
Harlan Sprague-Dawley (Indianapolis, IN). Breaches detected in Harlan’s breeding facilities after the completion
of this study placed some of the mice at risk for genetic
contamination. All results were compared for ‘‘at risk’’ mice
and mice with no known risk, and no differences were
observed for any of the measures associated with the present
study. All mice were housed at the University of Michigan
in a pathogen-free barrier facility until experimentation, and
then returned to a separate barrier facility between experimental procedures. All experimental procedures were approved by the University Committee for the Use and Care of
Animals at the University of Michigan.
In Situ Model and Functional Evaluation
Each mouse was anesthetized with an intraperitoneal
injection of 2% avertin (tribromoethanol; 400 mg/kg). Supplemental doses were administered if the mouse responded
to a toe pinch. A small incision was made at the right ankle,
and the distal tendons of the EDL muscle were exposed. The
mouse was then placed on a heated platform maintained
at 378C. The hind limb was stabilized by fixing the distal
femur between sharpened screws and securing the foot with
tape to the platform. The intact tendon was tied with 4-0 silk
suture to the lever arm of a servomotor (Aurora Scientific,
model 305, Richmond Hill, ON, Canada), which controlled
the length of the muscle and measured the force developed
by the muscle. The servomotor was controlled by a computer
to move the lever arm through a given distance at a constant
velocity. The computer was also used to collect and analyze
force data. The small region of exposed tendon was kept
moist with warmed isotonic saline.
The EDL muscle was activated via the peroneal nerve
using an isolated stimulator (Grass Instruments, model S88,
West Warwick, RI) and needle electrodes were placed
transcutaneously adjacent to the nerve. A pulse duration
of 0.2 ms was used for all contractions. Stimulation voltage
was set at 6 V, adjusted in 1 V increments until maximal
twitch tension was achieved, and a voltage 1.2 times this
value was used for the remainder of the experiment (;8–
12 V). Muscle length was initially set to the length of
the EDL with the knee fully extended and the foot fully
plantarflexed, and then adjusted in 0.125-mm increments for
maximal twitch tension. Stimulation frequency was set to
200 Hz, and then adjusted in 50 Hz increments for maximum isometric force (typically 250 Hz). Finally, optimal
muscle length (Lo) was determined for maximum isometric
force (Po); this was either the same as, or slightly shorter
than, the optimum length for twitch contractions. Lo was
measured using well-defined anatomical landmarks as previously established (12). Optimal muscle fiber length (Lf)
was calculated by multiplying Lo by the Lf/Lo ratio of 0.44
(23). This evaluation protocol minimized the number of
isometric contractions performed to minimize the possible confounding influence on subsequent training and injury
protocols.
Training Protocols
EDL muscles were either untreated, untrained and
exposed to the injury protocol, or trained with stretches
with the muscle relaxed (passive stretches), or near-maximal
593
activation of the muscle held at Lo (isometric contractions),
and then exposed to the injury protocol. Training protocols
involved 75 repetitions performed at 0.25 Hz for a total
exercise duration of 5 minutes. Passive stretches were
initiated at Lo and were of 20% strain relative to Lf, at
a velocity of 1 Lf/s. Near-maximal activation for isometric
contractions of a duration of 300 ms was achieved using
a stimulation frequency (150 Hz) that elicited ;90 Po. Following the protocol, the incision at the ankle was closed
with 7-0 nylon and bathed with povidone-iodine solution,
and the mice were allowed to recover from anesthesia.
Injury Protocol and Postinjury Functional Evaluation
Two weeks after the training protocol of passive stretches
or isometric contractions, EDL muscles were exposed in
situ to 225 lengthening contractions given in 3 bouts of
75 repetitions. Repetitions were performed at 0.25 Hz and
bouts were separated by 5 minutes. Lengthening contractions were initiated from the plateau of an isometric contraction elicited by 150 Hz stimulation at Lo and were of 20%
strain relative to Lf, at a velocity of 1 Lf/s. Three days
after lengthening contractions, mice were anesthetized and
isometric contractile properties were again measured in
situ using a protocol identical to that for the pretraining
evaluation. Force deficits were calculated as the decrease
in Po produced at 3 days compared with Po before injury
expressed as a percentage of preinjury values. The protocol
of 225 lengthening contractions was chosen because previous studies showed that a protocol of 75 lengthening contractions resulted in more severe injury to muscles of old
compared with adult mice, whereas the 225 contraction
protocol produced similar magnitudes of injury between age
groups (10,12).
Histology and Immunohistochemistry
Following the final in situ functional evaluations, anesthetized mice were killed by cervical dislocation. EDL
muscles were dissected, blotted dry, weighed, mounted in
tissue freezing media, and frozen in isopentane chilled with
dry ice. Cross-sections were cut at 10 lm with a cryostat,
and some sections were stained with hematoxylin and eosin.
The number of fibers exhibiting clear evidence of injury and
the total number of fibers in a single cross-section from the
midbelly of each muscle were counted with the aid of
a microscope imaging system (Bioquant, Nashville, TN).
Injured fibers were defined as those exhibiting cellular
infiltration, pale and/or discontinuous staining of the
cytoplasm, and/or a substantially swollen appearance. Fiber
counts were performed by an observer blinded to the
treatments, and another observer not blinded; no systematic
differences were found between sets of measurements, and
values were averaged. Injured fibers were reported as
a percentage of the total number of fibers in the crosssection.
Other sections were prepared for immunohistochemistry
to characterize differences in inflammatory cell accumulation following lengthening contractions between training
groups and between age groups. Sections were fixed in
cold acetone, quenched with hydrogen peroxide, and then
incubated for 3 hours at room temperature with primary
594
KOH ET AL.
antibody; neutrophils were identified using an anti-Ly6G
antibody [1:100 in phosphate buffered saline (PBS);
PharMingen, Franklin Lake, NJ] and macrophages were
identified using an anti-F4/80 antibody (1:100 in PBS;
Serotec, Raleigh, NC). Negative control slides had primary
antibody omitted. Sections were then washed in PBS and
incubated with biotinylated mouse adsorbed anti-rat IgG
(1:200 in PBS; Vector Laboratories, Burlingame, CA),
followed by avidin D horseradish peroxidase (1:1000 in
PBS). Finally, sections were developed with 3-amino-9ethylcarbazole (Vector). The number of inflammatory cells
were counted in 2 sections for each muscle by an observer
blinded to treatments, the area of the sections were measured
using a calibrated grid, and inflammatory cell concentrations
expressed per mm3 muscle (taking into account 10-lm
section thickness).
Data Analysis
Muscle physiological cross-sectional area (PCSA) was
calculated by dividing muscle mass by the product of Lf
and muscle density, 1.06 mg/mm3. Specific Po was calculated by dividing Po by PCSA. Mean values and standard
errors were determined for each variable for each group.
Variables were compared between groups using two-way
analysis of variance. The Student-Newman-Keuls post hoc
test was used to locate differences between means when the
F ratio was statistically significant. Pearson moment-product
correlations were also calculated to determine relationships
between postinjury force production and inflammatory cell
accumulation. The 0.05 level was taken to indicate statistical
significance.
RESULTS
Mean body mass was significantly greater for old (30.3 6
0.6 g) compared with adult (26.4 6 0.6 g) mice, whereas
EDL muscle mass (10.6 6 0.1 for old versus 10.3 6 0.2 mg
for adult), Po (396 6 11 versus 397 6 11 mN), and specific
Po (22.8 6 0.6 N/cm2 versus 23.4 6 0.6 N/cm2 ) were not
different between age groups.
As anticipated, for untrained muscles, the protocol of
225 lengthening contractions resulted in large decreases
in isometric force production of ;70%–80% at 3 days
compared with preinjury values and were not different
between age groups (Figure 1). Compared with untrained
muscles, muscles trained with passive stretches showed
a nearly twofold improvement in force production 3 days
following lengthening contractions in both adult and old
mice (Figure 1). Decreases in force induced by lengthening
contractions were not different for muscles trained with isometric contractions compared with untrained muscles, and
were also not different for muscles of old compared with
adult mice for any training protocol.
Three days following exposure of untrained muscles to
225 lengthening contractions, 15%–20% of the fibers in
cross-sections were classified as overtly injured in muscles
of both adult and old animals (Figure 2). The number of
injured fibers observed in cross-sections of muscles exposed
to lengthening contractions was smaller by one half for
muscles trained using passive stretches than for untrained
muscles or for muscles trained with isometric contractions
Figure 1. Postinjury force production 3 days after 225 lengthening
contractions in groups of adult or old mice that were not trained (n ¼ 10 and
n ¼ 8, respectively), trained with passive stretches (n ¼ 8 for both), or trained
with isometric contractions (n ¼ 7 for both). *Force production for the group
trained with passive stretches was significantly larger than for the untrained
group ( p , .05).
(Figure 2). The percentage of injured fibers was not different
between adult and old mice for any training condition.
In untrained muscle, lengthening contractions induced
large increases in inflammatory cells at 3 days in both adult
and old mice (Figure 3). Neutrophil accumulation was
twofold greater in old compared with adult mice and there
was a trend of increased macrophage accumulation in old
versus adult mice ( p ¼ .09). For macrophages, the power for
detecting an interaction between age and treatment was only
0.12, suggesting that the sample size was too small to detect
statistically significant interactions in the macrophage data.
For muscles of old mice, training with passive stretches
reduced neutrophil accumulation following subsequent
lengthening contractions by one third, and training with
isometric contractions reduced neutrophil accumulation by
two thirds (Figure 3). For muscles of adult mice, training
with either passive stretches or isometric contractions
had no effect on neutrophil concentrations. Similar to the
neutrophil data, there was a trend of a reduction in macrophage accumulation following lengthening contractions for
training with passive stretches ( p ¼ .052) or isometric contractions ( p ¼ .061), and visually, this reduction in macrophage accumulation appeared to occur in muscles of old but
not adult mice. Again, the power for detecting an interaction between age and treatment was only 0.12, suggesting
that the sample size was too small to detect such interactions. Supporting this idea, one-way analysis of variance
performed on each age group separately indicated significant reductions in macrophage accumulation with training
in muscles of old but not adult mice.
DISCUSSION
The primary finding of this study was that training with
passive stretches provides some protection to skeletal
muscle in old as well as adult mice from lengthening contraction-induced decreases in force production and overt
muscle fiber injury. These data are consistent with a previous
study (21) demonstrating that training with passive stretches
PROTECTING SKELETAL MUSCLE FROM INJURY
595
Figure 2. Percentages of overtly injured fibers in extensor digitorum longus
muscles 3 days after 225 lengthening contractions in groups of adult and old
mice that were not trained (n ¼ 10 and n ¼ 8, respectively), trained with passive
stretches (n ¼ 8 for both), or trained with isometric contractions (n ¼ 7 for both).
Percentage of injured fibers calculated as the percentage of the total number of
fibers in a cross-section exhibiting clear signs of degeneration. Values are means
with standard error bars. *Percentage of injured fibers for the group trained with
passive stretches was significantly smaller than for the untrained group ( p , .05).
reduced lengthening contraction-induced injury in adult
mice. In the previous study, injury was induced with a
protocol of 75 lengthening contractions that decreased
isometric force production by ;55% at 3 days in untrained
muscle. The injury protocol in the present study consisted
of 225 lengthening contractions that induced decreases in
force of ;70%–80% in untrained muscle. Despite the more
severe injury induced by the 225 contraction protocol in the
present study, training with passive stretches still induced
some protection, increasing postinjury force production
twofold and reducing the number of overtly injured fibers
by one half, indicating the robust nature of the protective
response to training with passive stretches. Consistent with
previous findings (12), the 225-contraction protocol resulted
in similar amounts of injury to untrained muscles of adult
and old mice, and thus allowed comparisons of the 2 age
groups for the amount of protection provided by the training
protocols. The finding of larger decreases in force compared
with percentages of injured fibers for all treatment groups
is consistent with previous studies (10,23) and suggests
that factors other than overt muscle fiber injury at the
light microscope level determine the force impairment [e.g.,
disruption of small groups of sarcomeres (24), excitationcontraction uncoupling (25)]. The observation of no differences between age groups for force production or numbers
of overtly damaged fibers following lengthening contractions in muscles trained with passive stretches does not support the hypothesis suggested by previous studies (13,19)
that the protective adaptation associated with exercise
training is impaired in old animals.
The findings in the present study that training with
isometric contractions did not improve postinjury force
production or reduce the number of overtly injured fibers
resulting from 225 lengthening contractions contrasts with
our previous observation in adult mice that the same isometric training protocol improved force production 3 days
Figure 3. A, Neutrophil concentrations (Ly6Gþ cells) in extensor digitorum
longus muscles in untreated control muscles from adult or old mice (n ¼ 10 and
n ¼ 8, respectively) or after 225 lengthening contractions in groups of adult or
old mice that were not trained (n ¼ 10 and n ¼ 8, respectively), trained with
passive stretches (n ¼ 8 for both), or trained with isometric contractions (n ¼ 7
for both). Values are means with standard error bars. *Lengthening contractions
induced significant accumulation of neutrophils in untrained muscle of both age
groups with significantly greater accumulation in old versus adult mice;
**Training with passive stretches significantly reduced lengthening contractioninduced neutrophil accumulation in old mice; ***Training with isometric
contractions produced significantly larger reductions in neutrophil accumulation
than training with passive stretches in old mice ( p , .05). Training did not
influence neutrophil accumulation in adult mice. B, Macrophage concentrations
(F4/80þ cells) in extensor digitorum longus muscles in control muscles from
adult or old mice (n ¼ 10 and n ¼ 8, respectively) or after 225 lengtheningcontractions groups of adult or old mice that were not trained (n ¼10 and n ¼ 8,
respectively), trained with passive stretches (n ¼ 8 for both), or trained with
isometric contractions (n ¼ 7 for both). *Lengthening contractions induced
significant accumulation of macrophages in untrained muscle of both age groups
( p , .05).
after 75 lengthening contractions (21). The severity of the
injury induced by the 225 lengthening contractions may
have overwhelmed any protective response induced by
training with isometric contractions. Whether optimization
of training protocols or repeated training bouts demonstrate
greater protection than the single bout used in the present
study remains to be determined.
596
KOH ET AL.
Lengthening contractions were associated with greater
accumulation of inflammatory cells in untrained muscles
from old versus adult mice despite no difference in force
deficits and numbers of overtly injured fibers between age
groups. A number of factors may play a role in the increased
inflammatory cell response in old versus adult mice including differences in chemoattractant release, in chemotaxis to injured sites, and in the function and life span of
inflammatory cells. The influence of aging on inflammatory
cell chemotaxis to injured skeletal muscle has not been
previously investigated. Studies of the migratory response
of neutrophils to a bacterial infection show no impairment in
elderly humans, but the ability of neutrophils to perform
phagocytosis has been reported to be impaired [reviewed
in (26)]. Consequently, one might speculate that greater
neutrophil accumulation in muscles of old compared with
adult mice after lengthening contractions may be attributable to a compensatory mechanism that results in greater
recruitment to injured muscle to make up for diminished
phagocytic capacity.
Training with isometric contractions reduced lengthening contraction-induced inflammatory cell accumulation in
muscles of old mice but did not influence force production
or numbers of overtly injured fibers. Furthermore, training
with passive stretches had no effect on inflammatory cell
accumulation in adult mice but did reduce the post injury
force deficit and number of overtly injured fibers. These data
support previous findings showing a lack of association
between inflammatory cell accumulation and measures of
injury (22). The dissociation between inflammatory cell responses and measures of injury indicates that overt muscle
fiber injury is not the sole determinant for the accumulation
of inflammatory cells in skeletal muscle after exercise and
that inflammatory cells may play a role in muscle beyond
repair of damaged fibers (27–29). In total, the findings of
this study suggest that measures of force production,
numbers of overtly injured fibers, and inflammatory cell
accumulation each provide overlapping but not identical
information about the response of muscle to lengthening
contractions.
Although the phenomenon of exercise-induced protection
of skeletal muscle from lengthening contraction-induced
injury is well established, the underlying mechanisms
remain to be elucidated. One proposed mechanism of
exercise-induced protection from injury is an increase in
the number of sarcomeres in series in muscle fibers (30).
Contrary to this hypothesis, experiments using training with
controlled lengthening contractions have produced no
significant increase in sarcomere number in rabbit dorsiflexor muscles (31) or in mouse or rat EDL muscles (Koh
and Brooks, unpublished observations). Another potential
protective mechanism is increasing the strength of the
cytoskeletal protein network that surrounds sarcomeres
(32) and transmits tension through the membrane (33,34).
Training-induced up-regulation of proteins in this network
(e.g., desmin, talin, vinculin, dystrophin) could help to
stabilize sarcomeres during lengthening contractions and
thus protect muscle fibers from the initial mechanical injury. Inflammatory cell accumulation following lengthening
contractions has been associated with increased reactive
oxygen species generation (35) and increased oxidation of
glutathione (36). Administration of exogenous superoxide
dismutase reduced the force deficit and percentage of
injured fibers 3 days after lengthening contractions in the
EDL muscles of young, adult, and old mice (10), suggesting
that superoxide or its byproducts contribute to the delayed
injury process. Thus, training-induced up-regulation of freeradical scavenging pathways could protect muscle from
such secondary injury.
Conclusion
In conclusion, we found that passive stretches provide
some protection against a severe lengthening contractioninduced injury in old as well as adult mice. Because contraction-induced injury is associated with pain, weakness,
and loss of mobility in elderly people (6), our finding that
training with passive stretches protects muscle in old mice
from injury may have therapeutic implications. The modest
level of protection observed may have resulted from the
severe injury protocol used in this study. Whether optimization of training protocols or increasing the number of
training bouts increase their protective effect remains to be
determined. In addition, the mechanisms underlying the
protection provided by training await further investigation.
ACKNOWLEDGMENTS
Financial support was provided by National Institute on Aging grants
AG-15434 and AG-00114.
Address correspondence to Dr. Susan V. Brooks, Institute of Gerontology, University of Michigan, 300 N. Ingalls, Ann Arbor, MI 48109. E-mail:
[email protected]
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Received December 11, 2002
Accepted April 2, 2003
Decision Editor: James R. Smith, PhD