Full text of DOI:10.1093/gerona/56.4.B163

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 4, B163–B171
Conditioning of Skeletal Muscles in Adult and Old
Mice for Protection From Contraction-Induced Injury
Susan V. Brooks, Julie A. Opiteck, and John A. Faulkner
Institute of Gerontology and Department of Physiology, University of Michigan, Ann Arbor.
The purpose of this study was to design a conditioning program that protected muscles in both
adult and old mice from a protocol of contractions that previously caused a significant number
of damaged fibers and a deficit in force. Hind-limb dorsiflexor muscles of adult (7 months) and
old (22 months) female B6D2F1 mice were exposed once a week to a protocol of repeated
forced stretches while maximally activated in vivo. By week 4, muscles of adult, but not old,
mice showed no force deficit. Conditioning was continued for 6 weeks, when both age groups
showed no force deficit for two consecutive weeks. Three days after the sixth contraction proto-
col, when morphological damage and force deficits are most severe, the numbers of damaged fi-
bers in muscles of adult and old mice were not different from those in uninjured control muscles
and the force deficits were reduced dramatically compared with unconditioned muscles. We
conclude that muscles of both adult and old mice conditioned successfully, but muscles of old
mice conditioned more slowly than those of adult mice.
OST activities involve complex body movements
that require muscles to perform combinations of con-
The demonstration of a muscle “conditioned” for pro-
tection from injury requires both the demonstration of
morphological damage and a significant force deficit in un-
conditioned muscles (9,10) and the attainment after a condi-
tioning program of no evidence of morphological damage to
muscle fibers or a force deficit. Previous investigations of
single and repeated exposure to a wide variety of exercise
protocols administered to both humans (11–16) and rodents
(17) concluded that conditioned muscles were produced.
Despite the conclusions of these studies that muscles were
conditioned, clear demonstrations of damage to muscles fol-
lowing protocols described as “injury-producing protocols”
were not presented. As a consequence, the degree of protection
could not be assessed in terms of the extent and magnitude
of damage to muscle fibers following the injury-producing
protocols in conditioned compared with unconditioned mus-
cles. The purpose of this study was to design and implement
a conditioning program that would protect skeletal muscles
of adult and old mice from contraction-induced injury. The
hypotheses were tested that (i) skeletal muscles of both
adult and old mice condition to repeated weekly protocols
of lengthening contractions such that they are not injured by
a contraction protocol that previously caused injury, but (ii)
conditioning occurs more slowly for muscles of old than
adult mice.
M
tractions wherein the muscles remain at fixed length,
shorten, or are stretched. Of the three types of contractions,
those in which activated muscles are stretched (lengthening
contractions) are most likely to cause significant injury to
skeletal muscle fibers (1). Initially, the injury is mechanical
and highly focal within single sarcomeres or small groups
of sarcomeres (2,3). The mechanical injury initiates a cas-
cade of events leading to a more severe secondary injury
throughout a segment of the injured fiber that peaks approx-
imately 3 days after the initial mechanical injury (1). Ath-
letes condition their muscles gradually to withstand the high
forces and strains of lengthening contractions for the pre-
vention of contraction-induced injury in specific sporting
activities. In contrast, much of the general population is not
conditioned for even minimally demanding stretches of ac-
tivated muscles that may occur unexpectedly in the activi-
ties of daily living.
The greater susceptibility of muscles in old animals, in-
cluding human beings, to injury (4–6) suggests that in older
individuals, conditioning may be required to withstand even
the mechanical requirements of lengthening contractions
encountered in daily life (7). Despite this potential require-
ment for conditioning in the elderly population, the im-
paired ability of muscles in old animals to recover from in-
jury (8) may make the achievement of a conditioned state
difficult. Furthermore, following severe injury, muscles in
old animals can sustain permanent deficits in force develop-
ment, mass, and number of fibers present in the cross sec-
tion (8). Consequently, a conditioning program that is too
demanding for muscles in elderly individuals could produce
prolonged and potentially irreversible structural and func-
tional deficits.
METHODS
Animals
Experiments were performed on specific pathogen-free
(SPF), female B6D2F1 mice. The conditioning program
was initiated on 7-month-old and 22-month-old mice, with
final data collection completed at 9 and 24 months of age,
respectively. Female 7- to 9-month-old B6D2F1 mice have
B163

B164
BROOKS ET AL.
achieved ف90% of their final body mass (18), decreasing
the contribution of significant growth during the condition-
ing program that would occur in younger mice. The median
life span of female B6D2F1 mice housed under SPF condi-
tions is 28 months, and the 10th percentile survival point is
34 months (18). An age range of 22–24 months, which cor-
responds to the 70th to 80th percentile survival point, was
chosen in the present study to represent an age range prior
to that at which significant deficits in skeletal muscle mass
and force are observed in mice (19,20). We reasoned further
that the likelihood for mice to survive the repeated exposure
to anesthesia required for completion to the conditioning
program would decrease for mice beyond the median life
span. The 7- to 9-month-old and 22- to 24-month-old age
groups will be termed “adult” and “old,” respectively.
Prior to experimentation, all mice were housed in an SPF
facility in the Core Facility for Aged Rodents at the Univer-
sity of Michigan. Following the first day of conditioning,
mice were housed in microisolator cages in an SPF return
room located adjacent to the experimentation site and super-
vised by the University of Michigan Unit for Laboratory
Animal Medicine. All experimental procedures and housing
conditions were conducted in accordance with the National
Institutes of Health Guide for the Care and Use of Labora-
tory Animals.
the measured torque by known forces at different ankle an-
gles throughout the range of motion (22). The resultant mo-
ment arms were used for the determination from ankle
torque measurements of the isometric forces and the peak
forces developed during stretches of the dorsiflexor muscle
group.
Initial evaluations of isometric contractions were made
with the ankle positioned and maintained at 10Њ of plantar-
flexion, demonstrated in preliminary experiments to be the
optimum angle for force production. Fine needle electrodes
penetrated the skin and were placed parallel and adjacent to
the peroneal nerve for stimulation of the dorsiflexor muscle
group. The maximum isometric twitch force was deter-
mined by increasing the voltage of individual 0.2-millisec-
ond pulses until force no longer increased. A frequency–
force curve was generated for each muscle group from
records of the force exerted during periods of stimulation
with trains of pulses at increasing frequencies. The plateau
in the frequency–force curve was defined as the maximum
isometric tetanic force (Po); Po was typically elicited by a
stimulation frequency of 150 Hz to 200 Hz, consistent with
previous reports (21).
Conditioning Program
The conditioning program consisted of a once-a-week
protocol of repeated forced stretches of maximally activated
muscles (lengthening contractions). Each week, the Po of
the dorsiflexor muscle group was determined in vivo prior
to initiation of the lengthening contraction protocol. Values
for Po measured each week along with control Po values, de-
fined as the maximum value for Po measured during the
conditioning program, are shown in Figure 1. The decision
to report isometric forces relative to the highest Po attained
during the conditioning program was based on the wide
variation for when the maximum value was achieved for in-
dividual animals. For individual adult mice, maximum val-
ues occurred during every week except for week 2, and for
old mice during every week except weeks 2 and 3. Stimulus
pulses of 0.2-millisecond duration and the frequency that re-
sulted in Po were used for all lengthening contractions. Dur-
ing each lengthening contraction, the ankle was held sta-
tionary for the first 200 milliseconds to allow full activation
of the dorsiflexor muscles prior to being stretched. During
the final 500 milliseconds of the contraction, the ankle was
rotated from the starting position of 10Њ plantarflexion to a
final position of 50Њ plantarflexion.
The dorsiflexor muscle group consists of the tibialis ante-
rior (TBA), extensor digitorum longus (EDL), and extensor
hallicus longus (EHL) muscles. Our analysis was limited to
the TBA and the EDL muscles, as these muscles comprise
over 98% of the mass of the muscle group (23). Based on
overall mean optimum fiber lengths (Lf) and the previously
determined lever arms (22), the 40Њ rotation of the ankle re-
sulted in approximate displacements for TBA muscles of
10% Lf at 0.20 Lf/s and for EDL muscles of 13% Lf at 0.25
Lf/s. The isometric force developed just prior to the stretch
and the peak force developed during the stretch were re-
corded for each 10th contraction (21). Each lengthening
contraction protocol consisted of three bouts of contrac-
tions, with each bout separated by a 5-minute rest period
Measurement of Contractile Properties in vivo
All contraction protocols were administered by using an
apparatus designed for the evaluation in vivo of the biome-
chanical behavior of the muscles of the mouse ankle (21).
The apparatus permits repeated administration of contrac-
tion protocols. For each contraction protocol, mice were
anesthetized with an intraperitoneal injection of pentobar-
bital sodium, 40 mg/kg, with supplemental injections given
as needed to achieve and maintain a state with no response
to tactile stimuli. The total dose of anesthesia fluctuated
greatly, depending on the individual mouse and the week of
conditioning.
The anesthetized mouse was placed in the experimental
apparatus upon a Plexiglas platform that was maintained at
37ЊC. The foot was aligned and stabilized within a plastic
“shoe” fixture, using wires surrounded by Silastic tubing
(Dow Corning, Midland, MI). The knee was stabilized with
a blunt screw pin at the femoral condyle; care was used so
that blood flow to the hind limb was not compromised. The
shoe fixture was attached to a torque transducer (model
QWFK-8M, Sensotec, Columbus, OH) such that the center
of rotation of the ankle joint was collinear with the axis of
the torque transducer. As a way to impose rotation of the an-
kle joint, the torque transducer was mounted on the shaft of
a custom-built servomotor. Control of the motor and collec-
tion of data from the torque transducer were accomplished
with LabVIEW software (Version 2.2.1, National Instru-
ments, Austin, TX) on a Macintosh computer (model
Quadra 700, Apple Computer, Cupertino, CA).
The muscle moment arm is the perpendicular distance
from the line of action of a muscle to the joint center of rota-
tion and transforms the linear movement of muscle into ro-
tation about a joint. In a separate group of mice of the same
strain, moment arms were calculated directly by dividing

CONDITIONING OF SKELETAL MUSCLES IN MICE
B165
the course of the conditioning program. Three groups of
muscles were evaluated and compared. For all mice, the
dorsiflexor muscles of the left leg were the experimental
muscles. Consequently, muscles from the left leg of the
mice that completed the conditioning program were desig-
nated conditioned muscles. In an additional group of mice,
the muscles in the left leg were subjected to only a single
protocol of lengthening contractions in vivo, and these mus-
cles were designated unconditioned muscles. The muscles
of the right legs from both groups of mice served as uncon-
ditioned and unexercised control muscles.
Final Evaluation of Conditioned and
Unconditioned Muscles
Final evaluations for evidence of injury to conditioned
muscles were conducted 3 days after administration of the
lengthening contraction protocol in the sixth week, and
evaluations of unconditioned muscles were made 3 days af-
ter administration of the single lengthening contraction pro-
tocol. A time period of 3 days was chosen because at this
time the decrease in force development and the morphologi-
cal evidence of the damage are most severe in EDL and
TBA muscles of mice (1,24). As a way to assess the magni-
tude of injury at the final evaluation, the following actions
were taken: (i) Po of the dorsiflexor muscle group was mea-
sured in vivo; (ii) Po of EDL muscles was measured in vitro;
and (iii) the percentages of damaged muscle fibers in single
cross sections of TBA and EDL muscles were determined.
Figure 1. Maximum isometric force (Po) developed by the dorsi-
flexor muscle groups in adult and old mice measured prior to the ad-
ministration of the lengthening contraction protocol. Data are pre-
sented as the mean Ϯ 1 SEM for Po measured each week as well as
for control Po, defined as the highest value of Po measured during the
conditioning program. *Indicates significant (p Ͻ .05) differences from
the respective control values for muscles in each age group. **Indi-
cates a significant difference between the value for muscles in adult
and old mice.
(8). Ten minutes following completion of the three bouts of
contractions, maximum isometric force was again mea-
sured.
For each bout, a lengthening contraction occurred every
5.0 seconds and continued for 60 contractions, or until iso-
metric force stabilized to within 50 mN of the prior 10th
contraction. Our rationale for termination of a bout upon
stabilization of isometric force was that the stabilization
indicated that the maximum extent of injury had been
achieved, a level of muscle fatigue was present that would
prevent further injury, or some combination of the two. As a
result of this design, bout 3 typically consisted of ف20%
fewer contractions than bout 1. Furthermore, the average
number of contractions during week 6 was ف20% lower
than during week 1, and individual mice differed with re-
spect to the total number of contractions during the condi-
tioning program. Despite the individual differences and
changes in contraction numbers throughout the conditioning
program, the conditioning programs administered to the
adult and old groups were not different.
Each week, following the lengthening contraction proto-
col, the mouse was removed from the apparatus and re-
turned to its cage for recovery. After all mice had been ad-
ministered the lengthening contraction protocol each week,
the mean group Po was calculated for both adult and old
mice and the control Po was recalculated. All mice contin-
ued the conditioning program until muscles in mice from
both age groups met the criterion of conditioned muscles.
Our working definition for conditioned dorsiflexor muscles
in adult and old mice was the point at which the mean group
Po was not different from the control Po for 2 weeks in suc-
cession. A working definition for conditioned muscles was
necessary because a strict definition in terms of the elimina-
tion of damage to muscle fibers could not be applied during
Measurements of force as an assessment of injury.—
Final evaluations of Po of the dorsiflexor muscles were
made as described above and were used to determine the
force deficit induced in conditioned and unconditioned
muscles by the lengthening contraction protocol. The force
deficit, which provided a measure of the magnitude of in-
jury, was calculated as the difference in Po measured prior
to and 3 days following the lengthening contraction proto-
col expressed as a percentage of Po prior to the lengthening
contractions. Clearly, the presence of multiple muscles in
the dorsiflexor group complicates the interpretation of mea-
surements made in vivo. Because of the dominance of the
TBA muscles in the dorsiflexor group, immediately follow-
ing the collection of final data in vivo, conditioned, uncon-
ditioned, and contralateral control EDL muscles were ana-
lyzed for force production in vitro.
EDL muscles were isolated and 5-0 silk suture was tied
securely to the proximal and distal tendons. The muscles
were removed from the limb and immersed in a bath con-
taining buffered physiological salt solution (composition:
NaCl, 137 mM; NaHCO3, 24 mM; glucose, 11 mM; KCL, 5
mM; CaCl2, 2 mM; MgSO4, 1 mM; NaH2PO4, 1 mM; and
tubocurarine chloride, 0.025 mM) maintained at 25ЊC (1).
The pH was maintained at approximately 7.4 by bubbling
with a gas mixture of 95% O2 and 5% CO2. The distal ten-
don was attached to a fixed post and the proximal tendon
was tied to the lever arm of a servomotor (Model 300H, Au-
rora Scientific Inc., Richmond Hill, Ontario, Canada). Mus-
cles were stimulated directly by an electrical field generated
between two platinum plate electrodes with 0.2 millisecond
pulses of supramaximal intensity. Stimulation voltage and

B166
BROOKS ET AL.
muscle length were adjusted until a single stimulus pulse
elicited maximum twitch force. Force was measured during
periods of stimulation at increasing frequencies until a pla-
teau was reached at Po. For EDL muscles, force deficits re-
sulting from the lengthening contraction protocol adminis-
tered in vivo were calculated as the difference in Po of
conditioned or unconditioned EDL muscles and Po of their
respective contralateral control EDL muscles expressed as a
percentage of the Po developed by the control muscle.
Optimal length (Lo) was measured with the EDL muscle
attached and in the bath. For the Lo of TBA muscles to be
obtained, the ankle of each leg was positioned with 10Њ of
plantarflexion, and muscle lengths were measured in situ.
Subsequently, TBA muscles were removed and the mice
were euthanized with an overdose of anesthetic. The Lf for
each muscle was calculated as the product of Lo and Lf/Lo
ratios (23).
periment, nor were differences observed in body mass be-
tween the conditioned and unconditioned animals.
Conditioning Program
At the beginning of the experiment, the Po of dorsiflexor
muscles in adult and old mice were not different (Figure 1).
During weeks 2 and 3, the Po of muscles in adult mice de-
creased to 75–80% of the control value, whereas that of the
old mice remained decreased to a similar level from week 2
through week 4. As a result, the Po for muscles of adult
mice recovered and stabilized at control values by week 4
and that of old mice by week 5. Consequently, by the sixth
week, dorsiflexor muscles in both age groups met our work-
ing criterion for conditioned muscles of developing Po not
different from the control Po for 2 weeks in succession, and
the final lengthening contraction protocol was administered
during week 6.
Morphology as an assessment of injury.—For all EDL
and TBA muscles, tendons were trimmed; muscles were
blotted, weighed, and cut transversely in half. The proximal
end of each muscle was weighed, dehydrated at 80ЊC for a
minimum of 1 week, and then weighed again for calculation
of dry mass/wet mass ratios. The distal end of each muscle
was mounted in tissue-freezing medium (Triangle Biomedi-
cal Sciences, Durham, NC) and frozen in isopentane cooled
with dry ice. Muscles were sectioned, stained with hema-
toxylin and eosin (H&E), and analyzed for mean single fi-
ber cross-sectional area (CSA) and percentage of the fibers
in a cross section that were damaged. Criterion for a dam-
aged fiber was either that the fiber was infiltrated with mac-
rophages or was in the process of degeneration.
First Exposure to Lengthening Contraction Protocol
In addition to the similarity of both initial and control Po
between dorsiflexor muscles of adult and old mice, a high
degree of consistency was observed between the age groups
for the forces developed throughout the entire lengthening
contraction protocol in week 1. Essentially no differences
between the age groups were observed for either the isomet-
ric force or the peak force during the stretch during adminis-
tration of the first 180 lengthening contraction protocol
(Figures 2 and 3). For muscles in both adult and old mice,
force decreased continuously in each bout such that by the
end of the protocol in week 1, the isometric force and peak
force during the stretch were 35% and 55%, respectively, of
the initial values (Figures 2 and 3). In addition, the recovery
in force during the rest periods between bouts was not dif-
ferent for muscles in adult and old mice. Furthermore, 10
minutes after completion of the protocol, the recovery of
isometric force to 81 Ϯ 6% and 76 Ϯ 5% of the initial val-
ues for muscles in adult and old mice, respectively, was not
different between the two age groups.
Statistics
All data were reported as the mean Ϯ 1 standard error of
the mean (SEM). A two-way analysis of variance (ANOVA)
was used to analyze data for effects of muscle group (condi-
tioned, unconditioned, control) and age (adult, old), and for
interactions between these two groups. A two-way repeated
measures (one factor repetition) ANOVA was used to test
for effects of the conditioning program and of age on iso-
metric forces and peak forces developed during the stretch
during the initial and final lengthening contraction proto-
cols. In all cases, Tukey post hoc analyses were used to de-
termine individual differences between groups. For all sta-
tistical tests, the accepted level of significance was set a
priori at p Յ .05.
Changes With Conditioning in Isometric and
Peak Forces
For muscles in both adult and old mice, the 6-week con-
ditioning program had no effect on Po (Figures 1 and 2). Al-
though the conditioning program did not affect Po directly,
the ability of the dorsiflexor muscles to maintain isometric
force during the lengthening contraction protocol was in-
creased (Figure 2). Compared with week 1 when the isomet-
ric force decreased during the lengthening contraction pro-
tocol to 35% of the initial value for both adult and old mice,
during week 6, isometric force was maintained at levels of
50% of the initial value for old mice and almost 60% for
adult mice. The greater decrease in isometric force for con-
ditioned muscles in old compared with adult mice suggests
that the effect of conditioning to increase the ability of mus-
cles to maintain isometric force was less for muscles in old
compared with adult mice. The smaller conditioning effect
in old mice was also apparent from the higher isometric
forces developed in week 6 compared with week 1 by mus-
RESULTS
Nine adult and eight old mice that completed the 6-week
conditioning program had body masses of 27.0 Ϯ 1.2 g and
31.6 Ϯ 1.3 g, respectively, at the end of the 6 weeks. The
additional five adult and six old unconditioned mice that
were exposed to only a single lengthening contraction pro-
tocol had mean body masses of 25.7 Ϯ 0.5 g and 34.9 Ϯ 2.2
g, respectively. Although in all cases the old mice had larger
body masses than the adult mice, no changes in body mass
occurred within an age group during the course of the ex-

CONDITIONING OF SKELETAL MUSCLES IN MICE
B167
force was not due to changes in the passive force character-
istics.
Final Evaluations of Conditioned and
Unconditioned Muscles
The force deficits measured for conditioned and uncondi-
tioned muscles 3 days following the lengthening contraction
protocol are shown in Figure 4. No effect of age was ob-
served on the force deficits of either the whole dorsiflexor
muscle groups evaluated in vivo or the EDL muscles evalu-
ated in vitro. Consequently, the data from both age groups
were pooled. For dorsiflexor muscles, the force deficit of
11.2 Ϯ 4.6% for conditioned muscles was less than half of
that for unconditioned muscles, 27.4 Ϯ 5.4%. Compared
with measurements made on the whole dorsiflexor muscle
groups, the effectiveness of conditioning to decrease the
force deficit was even greater based on evaluations of EDL
muscles. The pooled force deficit for conditioned EDL mus-
cles from adult and old mice of 7.8 Ϯ 2.9% was less than
one third of the force deficit of 25.4 Ϯ 2.9% measured for
unconditioned EDL muscles (Figure 4).
Consistent with the smaller force deficits measured for
conditioned compared with unconditioned muscles, the 2%
damaged fibers in single cross sections of conditioned EDL
and TBA muscles was lower than the 10% damaged fibers
in unconditioned EDL and TBA muscles (Tables 1 and 2).
Furthermore, the number of damaged fibers in conditioned
EDL or TBA muscles 3 days following the lengthening con-
traction protocol was not different from the fewer than 1%
damaged fibers in control muscles. A more severe injury to
unconditioned compared with conditioned muscles was also
indicated by the 11% lower muscle mass of unconditioned
compared with conditioned and control EDL muscles 3
days following the lengthening contraction protocol (1, Ta-
ble 1).
Figure 2. Isometric forces just prior to the stretch developed dur-
ing each 10th contraction for dorsiflexor muscles during the initial,
week 1, and final, week 6, lengthening contraction protocols for A,
adult, and B, old mice. Data are presented as the mean Ϯ 1 SEM.
Contractions are numbered 1 to 60 during each of three bouts of
lengthening contractions. Bouts are separated by 5-min rest periods
(R). Following completion of the contraction protocol, maximum iso-
metric force (Po) was measured following 10 min of rest (R R). Data
are plotted only in cases in which the point represents a mean of over
half of the total sample of muscles. Actual sample sizes for each point
are given above or below the symbols. *Indicates significant (p Ͻ .05)
differences between values for weeks 1 and 6.
cles in adult mice during most of bout 2, all of bout 3, and
10 minutes following the lengthening contraction protocol
but only during part of bout 3, for old mice (Figure 2).
In contrast to the lack of an effect of the 6-week condi-
tioning program on Po, for muscles in both adult and old
mice, the peak force developed during the stretch was sig-
nificantly increased in week 6 compared with week 1 (Fig-
ure 3). The effect of conditioning to increase peak force was
not different for muscles in adult and old mice. The contri-
bution of passive force to the total force measured during
stretches was considerable and variable, with passive exten-
sion properties of the muscles, tendons, and ankle joint be-
ing negligible at 15Њ of plantarflexion but contributing
ف15% of the force at 25Њ and ف45% at 50Њ. Despite the
magnitude of the contribution, no consistent effect of the
conditioning program on the passive tension was observed.
Consequently, we conclude that the increase in peak stretch
Figure 3. Peak forces developed during each 10th lengthening con-
traction for dorsiflexor muscles during the initial, week 1, and final,
week 6, lengthening contraction protocols. Pooled data for muscles in
adult and old mice are presented as the mean Ϯ 1 SEM. Contractions
are numbered 1 to 60 during each of three bouts of lengthening con-
tractions. Bouts are separated by 5-min rest periods (R). Data are
plotted only in cases in which the point represents a mean of over half
of the total sample of muscles. Sample sizes for each point are the
same as in Figure 2. *Indicates significant (p Ͻ .05) differences be-
tween values for weeks 1 and 6.

B168
BROOKS ET AL.
adult mice (Tables 1 and 2). The single age-associated dif-
ference was in the number of damaged fibers in cross sec-
tions 3 days after the lengthening contraction protocol,
when unconditioned TBA muscles in old mice had two
times the number of damaged fibers observed in TBA mus-
cles of adult mice (Table 2).
Conditioned muscles.—In both adult and old mice, the
protection from contraction-induced injury provided by the
conditioning program was not accompanied by changes in
muscle mass, Lf, single fiber CSA, or dry mass/wet mass
(Tables 1 and 2). Muscle mass, Lf, and single fiber CSA
were not different for conditioned compared with control
EDL muscles (Table 1). In addition, no differences were ob-
served for dry mass/wet mass among any of the groups of
EDL muscles in adult mice (Table 1). In old mice, dry mass/
wet mass of conditioned EDL muscles was 7% lower than
that of control EDL muscles, but was not different from that
of unconditioned muscles. Similarly, no differences were
observed between conditioned and control TBA muscles for
mass, single fiber CSA, or dry mass/wet mass in either adult
or old mice (Table 2). In old mice, the mean Lf of condi-
tioned TBA muscles was 4% lower than that of control
TBA muscles but not different from the Lf of unconditioned
TBA muscles. Finally, the similarity of the effects of the
conditioning program on the properties of muscles in adult
and old mice was supported by values for Po of conditioned
EDL muscles, 243 Ϯ 10 kN/m2 and 244 Ϯ 12 kN/m2, that
were not different.
Figure 4. Force deficits measured 3 days after administration of in
vivo lengthening contraction protocol to unconditioned and condi-
tioned dorsiflexor muscles. Force deficits are shown for dorsiflexor
muscles evaluated in vivo and for extensor digitorum longus (EDL)
muscles evaluated in vitro. Pooled data for muscles in adult and old
mice are presented as the mean Ϯ 1 SEM. *Indicates significant (p Ͻ
.05) differences between values for conditioned and unconditioned
muscles.
Properties of Conditioned, Unconditioned, and Control
EDL and TBA Muscles
Unconditioned and control muscles.—Consistent with
the similar functional properties of the dorsiflexor muscles
of adult and old mice, the properties of EDL and TBA mus-
cles of old compared with adult mice were essentially not
different. The Po of control EDL muscles evaluated in vitro
of 271 Ϯ 6 kN/m2 for adult mice and 265 Ϯ 6 kN/m2 for old
mice were not different. The Po of unconditioned EDL mus-
cles from adult and old mice were also not different, but the
values of 219 Ϯ 14 kN/m2 and 198 Ϯ 12 kN/m2 for adult
and old mice, respectively, were both lower than the respec-
tive control values reflecting the injured state of the uncon-
ditioned muscles. In addition, no differences were observed
for muscle mass, Lf, single fiber CSA, or dry mass/wet mass
of unconditioned or control muscles of old compared with
DISCUSSION
Both the functional and morphological data of the present
study provide strong evidence to support our hypothesis that
6 weeks of once-a-week exposure to a protocol of lengthen-
ing contractions conditioned dorsiflexor muscles of both
adult and old mice. The evidence for the conditioning of
muscles in old mice was in every way as impressive as the
evidence for the conditioning of muscles in adult mice.
Three days after a protocol of lengthening contractions, the
threefold greater force deficit and fivefold greater number
of damaged fibers observed for unconditioned compared
with conditioned muscles strongly supported the condi-
tioned state of EDL and TBA muscles. Although the num-
Table 1. Morphological Data for EDL Muscles of Adult and Old Mice
Adult Mice
Old Mice
EDL Musc.
Control Musc.
Uncond. Musc.
Cond. Musc.
Control Musc.
Uncond. Musc.
Cond. Musc.
Muscle Mass (mg)
Lf (mm)
Single Fiber CSA (␮m2)
Dry Mass/Wet Mass
Damaged Fibers (%)
9.6 Ϯ 0.2 (14)
6.0 Ϯ 0.1 (14)
1629 Ϯ 35 (10)
0.27 Ϯ 0.004 (12)
0.5 Ϯ 0.3 (10)
8.6 Ϯ 0.3 (5)*
5.9 Ϯ 0.1 (5)
1479 Ϯ 11 (4)
0.27 Ϯ 0.01 (4)
10.5 Ϯ 0.5 (4)*
9.7 Ϯ 0.3 (9)†
6.0 Ϯ 0.1 (9)
1560 Ϯ 96 (6)
0.25 Ϯ 0.01 (7)
1.7 Ϯ 0.8 (6)†
8.8 Ϯ 0.2 (14)
6.0 Ϯ 0.1 (14)
1497 Ϯ 75 (8)
0.27 Ϯ 0.01 (13)
0.3 Ϯ 0.2 (8)
8.7 Ϯ 0.2 (5)
6.2 Ϯ 0.1 (5)
1572 Ϯ 124 (3)
0.26 Ϯ 0.01 (4)
11.0 Ϯ 1.0 (3)*
9.1 Ϯ 0.3 (8)
6.0 Ϯ 0.04 (8)
1382 Ϯ 126 (5)
0.25 Ϯ 0.01 (6)*
0.2 Ϯ 0.2 (5)†
Notes: EDL ϭ extensor digitorum longus. Values are given for control, unconditioned, and conditioned muscles and are expressed as means Ϯ SEM (sample size)
for muscle mass, optimum fiber length (Lf), single fiber cross-sectional area (CSA), dry mass/wet mass ratio, and the number of damaged fibers in single cross sections
(damaged fibers, %). Significant differences were determined by a two-factor analysis of variance and are indicated by symbols as described below. Significance was
set at p Ͻ .05.
*Significantly different from value for control muscles of the same age group.
†Significantly different from value for unconditioned muscles of the same age group.

CONDITIONING OF SKELETAL MUSCLES IN MICE
B169
Table 2. Morphological Data for TBA Muscles of Adult and Old Mice
Adult Mice
Old Mice
TBA Musc.
Control Musc.
Uncond. Musc.
Cond. Musc.
Control Musc.
Uncond. Musc.
Cond. Musc.
Muscle Mass (mg)
Lf (mm)
Single Fiber CSA (␮m2)
Dry Mass/Wet Mass
Damaged Fibers (%)
48.3 Ϯ 0.9 (14)
8.8 Ϯ 0.1 (14)
2409 Ϯ 73 (10)
0.23 Ϯ 0.003 (13)
0.5 Ϯ 0.2 (10)
44.5 Ϯ 1.5 (5)
8.3 Ϯ 0.2 (5)*
2279 Ϯ 228 (4)
0.22 Ϯ 0.004 (5)
6.3 Ϯ 1.7 (4)*
44.7 Ϯ 1.7 (9)
8.6 Ϯ 0.2 (8)
2197 Ϯ 111 (6)
0.23 Ϯ 0.004 (8)
2.8 Ϯ 1.1 (6)
45.3 Ϯ 1.2 (14)
9.0 Ϯ 0.1 (14)
2377 Ϯ 78 (9)
0.22 Ϯ 0.01 (13)
0.1 Ϯ 0.1 (9)
45.4 Ϯ 1.2 (6)
8.6 Ϯ 0.2 (6)*
2425 Ϯ198 (4)
0.23 Ϯ 0.003 (5)
12.5 Ϯ 1.8 (4)*,‡
43.4 Ϯ 1.3 (8)
8.6 Ϯ 0.1 (8)*
2303 Ϯ 159 (5)
0.23 Ϯ 0.01 (7)
2.8 Ϯ 1.3 (5)†
Notes: TBA ϭ tibialis anterior. Values are given for control, unconditioned, and conditioned muscles and are expressed as means Ϯ SEM (sample size) for muscle
mass, optimum fiber length (Lf), single fiber cross-sectional area (CSA), dry mass/wet mass ratio, and the number of damaged fibers in single cross sections (damaged fi-
bers, %). Significant differences were determined by a two-factor analysis of variance and are indicated by symbols as described below. Significance was set at p Ͻ .05.
*Significantly different from value for control muscles of the same age group.
†Significantly different from value for unconditioned muscles of the same age group.
‡Significantly different from value for muscles in adult mice of the same experimental group.
ber of damaged fibers in the conditioned muscles at 3 days
was not different from that in uninjured control muscles, the
presence of a small but significant force deficit of ف9% was
not consistent in the strictest sense with our hypothesis that
conditioned muscles would not be injured. Devor and
Faulkner (9) also concluded that, following degeneration in-
duced by treatment with the myotoxic agent, bupivacaine,
newly regenerated fibers in muscles of old rats were pro-
tected from contraction-induced injury although injury was
not completely eliminated in their bupivacaine-treated mus-
cles. Most certainly a lengthening contraction protocol
could be designed that would produce no evidence of con-
traction-induced injury to conditioned muscles, but these re-
sults emphasize the rigor that must be used when conclu-
sions are drawn regarding the degree of protection provided
by a conditioning program or treatment.
The secondary hypothesis, that the achievement of a con-
ditioned muscle requires a longer period of conditioning for
muscles in old compared with adult mice, was also sup-
ported. Although not tested rigorously, the hypothesis was
supported on the basis of our working definition of condi-
tioned muscles that dorsiflexor muscles in adult mice gener-
ated Po not different from control values by week 4,
whereas muscles in old mice did not achieve control values
for Po until week 5. Although 6 weeks of once-a-week
lengthening contraction protocols clearly conditioned mus-
cles in both adult and old mice to be more resistant to con-
traction-induced injury, the lack of precision in the weekly
evaluations with the shoe apparatus prevented determina-
tion of exactly when any given individual within a group
was conditioned. A distinct possibility is that within groups
of both adult and old mice, some muscles conditioned more
slowly and others more rapidly than the remainder of the
group.
nantly lengthening contractions is only 25% of that required
for comparable exercise composed largely of shortening
contractions (25). Under these circumstances, the metabolic
cost of the lengthening contraction protocol may have been
such that fatigability was not a factor for muscles in either
adult or old mice. In addition, muscles of old mice are more
susceptible to injury during protocols of stretches of maxi-
mally activated muscles (4,6). Despite greater susceptibility
to injury reported previously for muscles in old compared
with younger animals (4,6), Gosselin (26) recently reported
that a moderate protocol of 20 lengthening contractions re-
sulted in a force deficit of ف25% that was similar for mus-
cles from adult and old rats. In the present study, the nature
of the contraction protocol was apparently mild enough as
to produce equivalent force deficits of ف25% at 3 days for
muscles of adult and old animals.
Contraction-induced injury is initiated by a mechanical
event, which involves focal damage to single sarcomeres
(27). During isometric contractions, the development of het-
erogeneity in sarcomere lengths (27,28) increases the likeli-
hood of large strains and injury of specific sarcomeres, even
during relatively small stretches (27,29–31). The damage
that occurs during the contractions can produce a delayed
secondary injury characterized by an inflammatory re-
sponse, free radical damage, phagocytosis, and ultimately
the degeneration of segments of severely damaged fibers
(1,6,32). The secondary injury, which is typically more se-
vere than the initial injury, peaks between 1 and 5 days later
(1,24). The adaptations responsible for protection from in-
jury provided by conditioning are not known (33). The im-
portance of the magnitude of strain and the degree of sar-
comere heterogeneity as predictors of the magnitude of the
damage suggests that the adaptation may involve changes
that prevent excessive stretch of small groups of sarcomeres
greater than the stretch imposed on the muscle (9,27,31).
Alternatively, numerous cellular adaptations could blunt the
free radical damage associated with the secondary injury
(6,32).
The rate and magnitude of the decline in force during a
lengthening contraction protocol are functions of both fa-
tigue and injury (1,6,8). Muscles of old mice were less able
to sustain force and power over time than muscles of young
mice (19). Consequently, the lack of any difference between
muscles in adult and old mice for either the isometric force
or the peak force developed during the stretch throughout
the first lengthening contraction protocol was surprising.
The metabolic requirements of exercise involving predomi-
The present experiments were not designed to distinguish
between effects on the initial and secondary injury, but the
significant increase between weeks 1 and 6 in the ability to
maintain isometric force during the contraction protocol
suggests a mechanism of the conditioning process on the

B170
BROOKS ET AL.
initiation of injury. The decrease in force attributable to fa-
tigue remained in the conditioned muscles, but the condi-
tioned muscles were able to maintain higher forces during
the stretches as a result of a decrease in the force deficit
from injury. Furthermore, precise experiments have been
directed to the specific roles of average and peak force,
strain, and work done to stretch a muscle fiber or muscle in
the initiation of injury (4,29,30,34,35). When muscle fibers
are maximally activated and the stretch is initiated from a
given initial fiber length, the relationship of the work done
during the stretch and the resultant force deficit has a coeffi-
cient of determination of greater than 80% (29). As a conse-
quence of the higher isometric and peak stretch forces main-
tained during week 6 compared with week 1, the average
forces developed and therefore the work inputs were greater
during the lengthening contraction protocol for conditioned
compared with unconditioned muscles. The observation of
greater work input’s resulting in a dramatically reduced
amount of damage during the contractions for conditioned
compared with unconditioned muscles also supports an ef-
fect of conditioning on the initial mechanical injury (4).
In the present study, the conditioning program resulted in
significant protection from contraction-induced injury for
muscles in both adult and old female mice, despite no sub-
stantial changes in muscle morphological properties. Taken
together, the data on muscle mass, single fiber CSA, fiber
length, and dry mass/wet mass from conditioned, uncondi-
tioned, and control muscles suggest that the conditioning
program did not cause hypertrophy or significant changes in
overall protein or water content. The elevated forces main-
tained throughout the contraction protocol by conditioned
muscles in the absence of any structural change is consistent
with the working hypothesis that conditioning affected the
intrinsic sarcomere strength within myofibrils. The process
of injury to weak sarcomeres and regeneration of stronger
sarcomeres is a potential mechanism by which conditioning
results in protection from injury. Newly regenerated fibers
in muscles of young and old animals are clearly more resis-
tant to contraction-induced injury (9). In contrast to the
present finding of conditioning in muscles of adult and old
female mice without any structural change, a similar 6-week
conditioning program resulted in hypertrophy of ف20% in
dorsiflexor muscles of young male mice (36). Whether the
mechanisms underlying the adaptations that allow muscles
to withstand lengthening contractions without injury are dif-
ferent in males and females is not known.
CK activity in the treated animals, force deficits of greater
than 50% and damage to more than 30% of the fibers in a
cross section were observed at 3 days in the muscles exposed
to the lengthening contractions (38). Similarly, marked dis-
crepancies have been reported between serum CK activity
and the magnitude of damage assessed through a direct
morphological examination of muscles in adult and old sub-
jects following a bout of exercise with lengthening contrac-
tions (5). Consequently, the use of CK activity as an indica-
tor of protection from damage should be viewed with
caution, particularly in the elderly population (5).
Our previous reports of a greater susceptibility to con-
traction-induced injury and a slower and impaired recovery
(6,8) were consistent with subjective, anecdotal, and experi-
mental (5) observations of muscle injuries to the elderly.
The observation that during the lengthening contraction
protocols used in the present study there was no difference
in either the fatigability or induction of injury to fibers in
muscles of adult and old mice has major implications for the
design of conditioning programs for the elderly population.
Furthermore, despite the intensity of the contraction proto-
cols, in terms of the level of activation, the number of con-
tractions and the amount of damage induced relative to typi-
cal exercise programs for human beings, protective effects
were achieved in both adult and old mice. The successful
conditioning response with no evidence of permanent dam-
age in muscles of old female mice provides strong support
for the use of conditioning with lengthening contractions to
maintain and enhance the muscle strength of elderly men
and women.
Acknowledgments
This work was supported by the National Institute on Aging: Grant AG-
06157 and a Multidisciplinary Training in Research on Aging Grant, AG-
00114, which provided fellowship support to J. Opiteck.
We thank Robert Dennis and Stephanie Miller for their assistance with
the modifications to the in vivo apparatus and the technical support and
Cheryl Hassett and Krystyna Pasyk for their contributions to the morpho-
logical assessments.
The current address of J. Opiteck is Duke Clinical Research Institute,
Duke University Medical Center, Durham, NC 27713.
Address correspondence to Susan V. Brooks, PhD, Institute of Geron-
tology, 300 N. Ingalls, Room 961, University of Michigan, Ann Arbor, MI
48109-2007. E-mail: svbrooks@umich.edu
References
1. McCully KK, Faulkner JA. Injury to skeletal muscle fibers of mice
following lengthening contractions. J Appl Physiol. 1985;59:119–126.
2. Newham DJ, McPhail G, Mills DR, Edwards RHT. Ultrastructural
changes after concentric and eccentric contractions. J Neurol Sci.
1983;61:109–122.
3. Ogilvie RW, Armstrong RB, Baird KE, Bottoms CL. Lesions in the rat
soleus muscle following eccentrically biased exercise. Am J Anat.
1988;182:335–346.
4. Brooks SV, Faulkner JA. The magnitude of the initial injury induced
by stretches of maximally activated muscle fibres of mice and rats in-
creases in old age. J Physiol (London). 1996;497:573–580.
5. Manfredi TG, Fielding RA, O’Reilly KP, Meredith CN, Lee HY,
Evans WJ. Plasma creatine kinase activity and exercise-induced mus-
cle damage in older men. Med Sci Sports Exer. 1991;23:1028–1034.
6. Zerba E, Komorowski TE, Faulkner JA. Free radical injury to skeletal
muscles of young, adult, and old mice. Am J Physiol. 1990;258:C429–
C435.
Similar abilities of adult and old subjects to adapt to mod-
erate protocols of exercise have been reported based on se-
rum creatine kinase (CK) activity and subjective accounts
of pain (37). Although Clarkson and Dedrick (37) showed
comparable reductions in serum CK activity in adult and old
subjects following a second compared with the first bout of
exercise with lengthening contractions (37), CK activity is
often a poor marker of actual muscle damage. In rats treated
with vitamin E in an attempt to protect skeletal muscles
from contraction-induced injury, serum CK activity 3 hours
and 3 days following a protocol of repeated lengthening
contractions administered to EDL muscles in situ was not
different from that of nonexercised control animals (38).
Despite the lack of evidence of muscle damage based on

CONDITIONING OF SKELETAL MUSCLES IN MICE
B171
7. Faulkner JA, Brooks SV, Zerba E. Muscle atrophy and weakness with
aging: contraction-induced injury as an underlying mechanism. J Ger-
ontol Series A. 1995;50A(special issue):124–129.
8. Brooks SV, Faulkner JA. Contraction-induced injury: recovery of
skeletal muscles in young and old mice. Am J Physiol. 1990;258:
C436–C442.
muscle fiber types and sizes and muscle architectural properties in the
mouse hindlimb. J Morphol. 1994;221:177–190.
25. Bigland-Ritchie B, Woods JJ. Integrated electromyogram and oxygen
uptake during positive and negative work. J Physiol. 1976;260:267–
277.
26. Gosselin LE. Attenuation of force deficit after lengthening contrac-
tions in soleus muscle from trained rats. J Appl Physiol. 2000;88:
1254–1258.
27. Macpherson PCD, Dennis RG, Faulkner JA. Sarcomere dynamics and
contraction-induced injury to maximally activated single muscle fibres
from soleus muscles of rats. J Physiol (London). 1997;500:523–533.
28. Burton K, Zagotta WN, Baskin RJ. Sarcomere length behaviour along
single frog muscle fibres at different lengths during isometric tetani. J
Muscle Res Cell Motil. 1989;10:67–84.
29. Brooks SV, Zerba E, Faulkner JA. Injury to muscle fibres after single
stretches of passive and maximally stimulated muscles in mice. J
Physiol (London). 1995;488:459–469.
30. Macpherson PCD, Schork MA, Faulkner JA. Contraction-induced in-
jury to single fiber segments from fast and slow muscles of rats by
single stretches. Am J Physiol. 1996;271:C1438–C1446.
31. Morgan DL. New insights into the behavior of muscle during active
lengthening. Biophys J. 1990;57:209–221.
32. McArdle A, van der Meulen JH, Catapano M, Symons MC, Faulkner
JA, Jackson MJ. Free radical activity following contraction-induced
injury to the extensor digitorum longus muscles of rats. Free Rad Biol
Med. 1999;26:1085–1091.
9. Devor ST, Faulkner JA. Regeneration of new fibers in muscles of old rats
reduces contraction-induced injury. J Appl Physiol. 1999;87:750–756.
10. Sacco P, Jones DA. The protective effect of damaging eccentric exer-
cise against repeated bouts of exercise in the mouse tibialis anterior
muscle. Exp Physiol. 1992;77:757–760.
11. Balnave CD, Thompson MW. Effect of training on eccentric exercise-
induced muscle damage. J Appl Physiol. 1993;75:1545–1551.
12. Byrnes WC, Clarkson PM, White JS, Hsieh SS, Frykman PN,
Maughan RJ. Delayed onset muscle soreness following repeated bouts
of downhill running. J Appl Physiol. 1985;59:710–715.
13. Clarkson PM, Trembly I. Exercise-induced muscle damage, repair,
and adaptation in humans. J Appl Physiol. 1988;65:1–6.
14. Mair J, Mayr M, Muller E, et al. Rapid adaptation to eccentric exer-
cise-induced muscle damage. Int J Sports Med. 1995;16:352–356.
15. Newham DJ, Jones DA, Clarkson PM. Repeated high-force eccentric
exercise: effects on muscle pain and damage. J Appl Physiol. 1987;63:
1381–1386.
16. Schwane JA, Williams JS, Sloan JH. Effects of training on delayed
muscle soreness and serum creatine kinase activity after running. Med
Sci Sports Exer. 1987;19:584–590.
17. Schwane JA, Armstrong RB. Effect of training on skeletal muscle in-
jury from downhill running in rats. J Appl Physiol. 1983;55:969–975.
18. Turturro A, Witt WW, Lewis S, Hass BS, Lipman RD, Hart RW.
Growth curves and survival characteristics of the animals used in the
biomarkers of aging program. J Gerontol Biol Sci. 1999;54A:B492–
B501.
19. Brooks SV, Faulkner JA. Maximum and sustained power of extensor
digitorum longus muscles from young, adult, and old mice. J Gerontol
Biol Sci. 1991;46:B28–B33.
20. Phillips SK, Bruce SA, Woledge RC. In mice, the muscle weakness
due to age is absent during stretching. J Physiol (London). 1991;437:
63–70.
21. Ashton-Miller JA, He Y, Kadhiresan VA, McCubbrey DA, Faulkner
JA. An apparatus to measure in vivo biomechanical behavior of dorsi-
and plantarflexors of mouse ankle. J Appl Physiol. 1992;72:1205–
1211.
33. McHugh MP, Connolly DA, Eston RG, Gleim GW. Exercise-induced
muscle damage and potential for the repeated bout effect. Sports Med.
1999;27:157–170.
34. Lieber RL, Fridén J. Muscle damage is not a function of muscle force
but active muscle strain. J Appl Physiol. 1993;74:520–526.
35. Warren GL, Hayes DA, Lowe DA, Armstrong RB. Mechanical factors
in the initiation of eccentric contraction-induced injury in rat soleus
muscle. J Physiol (London). 1993;464:457–475.
36. Faulkner JA, Opiteck JA, Brooks SV. Injury to skeletal muscle during
altitude training: induction and prevention. Int J Sports Med. 1992;13:
S160–S162.
37. Clarkson PM, Dedrick ME. Exercise-induced muscle damage, repair,
and adaptation in old and young subjects. J Gerontol Med Sci. 1988;
43:M91–M96.
38. Van der Meulen JH, McArdle A, Jackson MJ, Faulkner JA. Contrac-
tion-induced injury to the extensor digitorum longus muscles of rats:
the role of vitamin E. J Appl Physiol. 1997;83:817–823.
22. Miller SW, Dennis RG. A parametric model of muscle moment arm as
a function of joint angle: application to the dorsiflexor muscle group in
mice. J Biomech. 1996;29:1621–1624.
23. Faulkner JA, Jones DA, Round JM. Injury to skeletal muscles of mice
by forced lengthening during contractions. Q J Exp Physiol. 1989;74:
661–670.
Received July 19, 2000
Accepted October 31, 2000
24. Burkholder TJ, Fingado B, Baron S, Lieber RL. Relationship between
Decision Editor: Edward J. Masoro, PhD