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THESIS

’LUUI

This is to certify that the

dissertation entitled

MAGNETIC RESONANCE IMAGING STUDIES OF
DELAYED MUSCLE SORENESS AND
THE REPEATED BOUT EFFECT

presented by

Roop Chand Jayaraman

has been accepted towards fulﬁllment
of the requirements for

Ph.D. degreein Kinesiology

 

 

   
 

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6101 C'JCIRC/DanDuo.p65-p.15

MAGNETIC RESONANCE IMAGING STUDIES or DELAYED MUSCLE
SORENESS AND THE REPEATED BOUT EFFECT
By

Roop Chand J ayaraman

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSPHOY

Department of Kinesiology

2001

ABSTRACT
MAGNETIC RESONANCE IMAGING STUDIES OF DELAYED MUSCLE
SORENESS AND THE REPEATED BOUT EFFECT
By

Roop Chand Jayaraman

The purposes of this study were, ﬁrst, to compare T2 relaxation computed by the
non-negative least squares algorithm (NN LS) from muscles aﬁer two bouts of damaging
eccentric exercise separated by 8 wk, and second, to examine the effect of eccentric
exercise-induced muscle damage on the acute T2 response to bouts of milder concentric
exercise. The ﬁrst aim was accomplished by obtaining conventional T2-weighted axial
spin-echo MRI images and echo-planar images of the upper arm before, and 1, 2, 4, 7, 12,
21, and 56 d after eccentric bout 1 (Eco 1), and at 2,4,7, and 15 d after eccentric bout 2
(Bee 2). Conventional spin-echo images were also obtained immediately before and after
concentric exercise bouts performed before and at 2 and 21 d after Bee 1. The prolonged
T2 increase measured by conventional imaging after Bee 1 (46.7 at 3.8 ms at 2 d, mean :1:
SE, n=6) was correlated with the presence of slow-relaxing, long T2 components (60-300
ms) in the NNLS T2 spectra. However, T2 remained elevated by 2.5 ms even after 56 (1,
when distinct slow-relaxing components were no longer detectable. The magnitude of
slow-relaxing components was less aﬁer Bee 2 compared to Bee 1, consistent with the
previously reported protective effect of prior eccentric exercise. The acute T2 increase

after Con 2 (1.5 :t 0.5 ms, mean :5 SE, n=6) was signiﬁcantly less (p < 0.05) compared to

Con 1 (3.2 :t 0.5 ms) and Con 3 (5.0 i 0.4 ms). The results conﬁrm that the prolonged T2

increase observed by conventional imaging methods after eccentric exercise is primarily
caused by edema, and show that this edema diminishes the acute T2 change measured
during exercise using conventional spin-echo imaging methods.

The initial aim of the second set of experiments was to monitor the effects of
topical heat and/or static stretch treatments on the recovery of muscle damage from
intense eccentric exercise. For this purpose, 32 untrained male subjects performed
intense eccentric knee extension exercise, followed by 2 wk of treatment (heat, stretch,
heat plus stretch) or no treatment (control, n =8/group). Isometric strength testing, pain
ratings, and multiecho MR images of the thigh were performed before and at 2, 3, 4, 8,
and 15 d post-exercise. Increased T2 relaxation time, muscle swelling, pain ratings, and
strength loss conﬁrmed signiﬁcant muscle damage during the post-exercise period. Pain
ratings and muscle volume recovered to baseline by 15 (1, although strength remained
lower (19 i 4%) and T2 values higher (12 :t 2%).

Treatment modality showed no effect on extent of recovery in any measured
parameter, allowing pooling of data for a follow-up study in which a subset of nine
subjects returned for testing 6 wk post exercise. Results showed a persistent elevation in
T2 (6 i 2%) and a small but signiﬁcant muscle volume loss (12 i 4%), conﬁrming a
previous report on arm muscle. These changes were accompanied by a persistence of
muscle weakness (17 i- 4% below baseline). These long term changes are consistent with
the hypothesis that intense eccentric exercise partially or totally destroys a small

subpopulation of mechanically weak ﬁbers.

To my parents, Drs. Gopal and Manimegalai Jayaraman

iv

ACKNOWLEDGEMENTS

First and foremoSt I wish to express my deep appreciation to Dr. Jeanne Foley,
my advisor, for her guidance and thoughtful critiques of my work through out my
graduate career. Our many discussions on teaching, research, and life in general have
contributed greatly to my development both as a scientist and person.

I would also like to thank Dr. Ronald Meyer, his many hours of technical advice
and guidance made my dissertation possible. He not only taught me the basic principles
of magnetic resonance imaging, but also gave me down to earth advice and helped me
navigate some of the rough waters during my graduate career.

I owe a special thank you to Dr. James Pivamik, who had a hand in guiding my
graduate career long before joining my committee. My interactions with Dr. Anthony
Paganini, guidance committee member, contributed greatly to my development both as a
scientist and an educator.

I would also like to express my deep appreciation to Dr. Laura McCabe for giving
me the opportunity to learn molecular biology techniques in her lab and for the timely
words of encouragement.

Thanks also to Mr. Rob Reid, and Dr. Barry Prior, fellow MRI laboratory
colleagues, whose help and friendship was invaluable. I would also like to thank Jo Ann
Janes, the graduate secretary, for all her help and encouragement during my graduate
career. I owe a special thank you to the UCIRHS staff and co-workers.

Finally, I wish to express by deepest gratitude to my entire family, especially,

mom and dad, my brother - Vijay, and my ﬁancée - Kathleen for their love,

encouragement, support and understanding as I pursed this goal. Special thanks to
Kathleen, for being understanding of my absence at numerous wedding and family

related activities.

vi

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
CHAPTER I - Introduction

CHAPTER II - Review of related literature.
Symptoms of DMS
Morphological Changes Associated with DMS
Biochemical Markers .
Repeated Bout Effect .

Mechanisms of DMS.
Initial and Autogenetic Stages of DMS
Phagocytic Stage .
Effects of Anti- Inﬂammatory Treatments on DMS .
Regeneration Stage .
Stress-Susceptible Fiber Theory and Repeated Bout Effect.
Neural Adaptations to a Bout of Eccentric Exercise.

Fundamental Principles of Magnetic Resonance Imaging
Exercise-Induced Acute T2 Increase.

Proposed Mechanism of the Acute T2 Increase After Exercise.

Delayed T2 Increase aﬁer Eccentric Exercise
Proposed Mechanism of the Delayed T2

Conclusions

vii

Page

xi

10
14
18

21
21
24
27
29
31
32

35
38
40
45

47

CHAPTER III - Effects of Exercise-Induced Damage on T2 Relaxation in
Skeletal Muscle

Abstract

Introduction

Methods
Subjects
Exercise Protocol

MRI Methods .
Statistical Analysis

Results

Discussion

CHPATER IV - MRI Evaluation of Heat and Stretch as Treatments for Muscle
Damage After Eccentric Exercise

Abstract
Introduction

Methods
Subjects
Exercise Protocol
MRI Methods .
Treatment Protocol

Statistical Analysis
Results
Discussion

Treatment Effect . .
Long term time course of muscle damage

viii

Page

48
49

52
52
52
54
55

56

61

67
68

71
71
72
73
74
75

76
86

86
89

Page

CHAPTER V - Summary and Future Studies . . . . 93
Future Studies . . . . . . 94

LIST OF REFERENCES . . . . . . . 97

ix

LIST OF TABLES

Page
1. Acute T2 increase after concentric exercise . . . . 61
2. Subject characteristics before exercise . . . . . 79

LIST OF FIGURES

Page

. Representative mid-brachial axial plane MR images from one subject before
(top let image; pre-exercise) and at each test interval during the 8 wk aﬁer
Bee 1. . . . . . . . . . 57

. NNLS spectra of one subject before and In the days following Ecc 1 (A)
and Ecc 2 (B). . . . . . 58

. Time course of knee extensor muscle volume and percent of the total signal
intensity attributable to components with T2 above 60 ms in 6 subjects for

8 wk after eccentric exercise bout 1 (Bee 1) and 2 wk after eccentric exercise
bout 2 (Eco 2) in 5 subjects. . . . . . . 60

. Representative mid-thigh axial plane MR images from one subject before
(top left image; pre-exercise) and at each test interval during the 6 wk after
the eccentric exercise bout. . . . . . . 78

. Peak percent change from baseline value (A) and percent change from baseline
value at 15 d post-eccentric exercise in T2, MVC, muscle volume, and pain

across the different treatment groups. . . . . . 8O

. Time course of muscle T2 relaxation times for 6 wk after eccentric exercise

(11 = 9). . . . . . . . . . 82
. Time course of muscle volume changes for 6 wk after eccentric exercise

(n = 9). . . . . . . . . . 83
. Time course of MVC changes for 6 wk after eccentric exercise (n = 9) . 84

. Time course of the pain ratings for 6 wk after eccentric exercise (11 = 9). 85

xi

CHAPTER I

IN TRODUCION

Delayed muscle soreness (DMS) is the sensation of discomfort and/or pain
experienced 24-48h following unaccustomed physical activity (6-8, 21, 45, 58, 77).
DMS occurs most often in individuals who engage in sporadic, vigorous physical
activity. Research has shown that eccentric or lengthening contractions cause structural
damage to the muscles. It is this muscle damage that results in the sensation of pain or
soreness that gradually develops in the ensuing days (6, 8, 9, 12, 46, 77).

Several studies have documented exercise-induced muscle damage after
lengthening contractions by tracking different indices, including serum levels of muscle
enzymes, inﬂammation markers, limb swelling, and decrements in muscle performance
(7, 12, 17, 21, 22, 44, 53, 64, 76, 78, 80, 85, 99, 101). In addition, it is well established
that performance of the same eccentric exercise 1-6 wk later produces more modest
changes in the indicators of damage and muscular performance (10, 17, 21, 22, 35, 76,
79, 80). In the literature, this protective effect of a single bout of eccentric exercise is
commonly referred to as the “repeated bout effect.”

A review of the literature will Show that several distinct hypotheses have been
proposed to explain the mechanism underlying DMS and the “repeated bout effect”.
Smith (99) has presented a plausible argument for acute inﬂammation as the sole
underlying mechanism of DMS. However, several key studies have failed to support
Smith’s hypothesis (8, 47, 95). On the other hand, Armstrong’s model (8) divides the

muscle injury process in DMS into four distinct stages: 1) initial, 2) autogenetic, 3)

phagocytic, and 4) regenerative. Armstrong’s group has primarily focused their research
on the ﬁrst two stages of the model. Although the experimental data failed to support
acute inﬂammation as the exclusive mechanism of DMS, the acute inﬂammatory
response ﬁts well with the phagocytic and regenerative stages proposed in Armstrong's
model of DMS. F urthermore, no single explanation shows more promise than
Armstrong’s theory combined with Smith’s acute inﬂammation model of DMS.

In an attempt to explain the “repeated bout effect”, Armstrong proposed the
“stress-susceptible” ﬁber theory. The "stress-susceptible" ﬁber model states that a sub-
population of ﬁbers in healthy muscles (<5%) nearing the end of their functional life are
more susceptible to irreversible ultrastructural damage during a bout of unaccustomed
eccentric exercise, which leads to degeneration of these cells in the days following the
exercise. In addition, microinjury occurs in the "normal" or non-susceptible muscle cells
that were recruited during the lengthening contractions but does not result in necrosis of
these entire ﬁbers. Accordingly, there is a prolonged decrease in muscle strength and
range of motion, muscle soreness, and a Signiﬁcant increase in the serum levels of
muscle proteins following the initial bout of eccentric exercise. During the degeneration-
regeneration process, necrotic ﬁbers are replaced with newly assembled ﬁbers and the
damaged ones are repaired. Therefore, the process of degeneration-regeneration should
result in fewer stress-susceptible ﬁbers to be destroyed by subsequent eccentric exercise
bouts.

Recent advances in the ﬁeld of magnetic resonance imaging (MRI) have allowed
in vivo measurements of gross muscle recruitment, muscle damage, and changes in

muscle compartment cross-sectional area during DMS (42, 43, 61, 81, 97, 103). Muscle

"functional" magnetic resonance imaging (fMRI) exploits the changes in tissue lH-NMR
properties that occur with exercise-induced muscle trauma and develop more gradually
with DMS (42, 43, 61, 81, 96, 103). Generally, tissues and ﬂuids with more free water
have longer T2 relaxation times and appear as brighter areas on T2-weighted images.
The damaged muscles appear bright in MR images compared to undamaged or normal
muscles. This contrast change in MR images is the result of increased T2 relaxation time
of damaged muscle, which occurs as early as 12 h and peaks 48-72 h, post-eccentric
exercise (61, 103). Several MRI studies have exploited this increase in eccentrically
damaged muscle T2 relaxation time to document the time course of muscle damage after
eccentric exercise, and the recovery of muscle damage in days following eccentric
exercise (42, 43, 61, 97, 103). In addition, application of MRI software has allowed
investigators to measure muscle volume changes during DMS (43, 61 , 90). Although
there is universal agreement that the increase in muscle T2 following eccentric exercise is
an index of exercise-induced muscle damage, the exact physiological mechanism of the
contrast change remains unresolved.

Recently, Foley et al. (43) reported a persistent elevation in muscle T2 at 56 d
post-eccentric exercise. These investigators also found the increase in muscle T2 is
signiﬁcantly reduced in the days following the second bout of eccentric exercise,
consistent with the repeated bout effect. Since edema has been reported to occur during
DMS, it is plausible that the smaller increase in muscle T2 after the second bout of
eccentric exercise is the result of more modest changes in the extracellular and/or
vascular ﬂuid compartments in the muscle. However, no studies to date have directly

tested this hypothesis.

Furthermore, these investigators reported a signiﬁcant decrease in muscle volume
after recovery from eccentric exercise. If this reported volume contraction is truly the
result of muscle loss, then a decrease in maximal isometric strength should also occur.
However, Foley et al. did not measure isometric strength changes in the eccentrically
damaged muscle.

In summary, three major questions remain unanswered related to the recent MRI
studies on DMS. First, how does muscle volume loss, detected using MRI, relate to
changes in functional capacity of the muscle? In our current study, we propose
experiments to answer this question by determining how well muscle strength loss
correlates with muscle volume loss following eccentric exercise. Secondly, no studies
have tested directly either the hypothesis that the increase in muscle T2 during DMS is
the result of exercise-induced edema or the hypothesis that the persistent elevation in
muscle T2 long after apparent recovery from eccentric-induced DMS is due to changes in
the intracellular compartment of the repaired and/or newly-synthesized muscle cells.
Finally, this work will also allow the testing of the hypothesis that the smaller increase in
muscle T2 after a repeated bout of eccentric exercise is due to reduced edema
development after the second bout. We propose using echo-planar imaging and non-
negative least squares algorithm to separate components of the multiexponential T2
decay in eccentrically damaged muscle. This analysis will allow us to localize the
exercise-induced edema and measure the contribution of the exercise-induced edema to

the well-documented increase in muscle T2 during DMS.

CHAPTER II

REVIEW OF RELATED LITERATURE

Nearly every adult has experienced delayed muscle soreness, especially those of
us who engage in sporadic physical activity. Delayed muscle soreness (DMS) is the
sensation of discomfort and/or pain following unaccustomed exercise. Regardless of the
level of ﬁtness, DMS in healthy adults occurs most commonly following repetitive,
strenuous eccentric or lengthening contractions (6-8, 21, 45, 58, 77). In contrast, during
an isomeric contraction the muscle length is relatively unchanged and during a concentric
contraction the muscle shortens. DMS is accompanied by variable muscle damage, and
the severity varies from slight stiffness to severe pain that inhibits movement. Additional
studies indicate that performance of the same exercise 1-6 wk later produces more
modest changes in the indicators of damage and muscular performance (10, 17, 21, 22,
35, 76, 79, 80). In the literature this effect is commonly referred to as the “repeated bout
effect.”

Numerous studies have documented the exercise-induced muscle damage
following lengthening contractions by tracking different indices, including serum levels
of muscle enzymes, markers of inﬂammation, swelling of limbs, and decrements in
muscle performance (7, 12, 17, 21, 22, 44, 53, 64, 76, 78, 80, 85, 99, 101). More
recently, several investigators have employed magnetic resonance imaging (MRI) to
quantify eccentrically induced muscle damage from the elevated T2 (transverse or spin-

spin) relaxation time in proton MR images (43, 81, 97, 103).

Although DMS has been studied extensively for over ﬁve decades, there is still
considerable controversy regarding the pathophysiological mechanism(s) responsible for
DMS. Many reports have been published regarding the time course of changes in various
pathological indices associated with DMS, and it is generally accepted that the pathology
associated with DMS is completely reversible. However, the relationship of the sequence
and timing of the symptoms of DMS to a possible pathological mechanism remains
unclear. In addition, the mechanism responsible for the repeated bout effect remains in
question. The ﬁrst section of this literature review discusses the symptoms of DMS. The
second section examines how the repeated bout effect alters the symptoms of DMS, and
the third addresses current concepts on the mechanism of DMS and the repeated bout
effect. The last section will discuss the use of MR parameters to study the underlying

mechanism(s) of DMS and the repeated bout effect.

Symptoms of DMS

The most commonly reported symptom of DMS is soreness in the exercised
muscle; however, soreness or pain is only temporary. Muscle soreness generally
increases in intensity in the ﬁrst 24 h after a bout of unaccustomed and/or eccentric
exercise (6, 14, 21, 22, 43, 45, 76, 79, 80). The soreness peaks at 24-72 h post-exercise
and does not completely subside until 8-10 (1 post-exercise. Lieber and F riden (68)
suggest that the muscle strain (A muscle length/initial muscle length) or lengthening
distance is the critical factor in determining the magnitude of muscle injury, which in
turn determines the intensity and time of peak soreness. On the other hand, McCully et

a1. (73) suggest that it is the number of lengthening contractions performed determines

the extent of muscle injury. More than likely, it is a combination of intensity and
duration parameters that determines the magnitude of the muscle damage, and it is this
damage to muscle that ultimately results in the sensation of soreness in the days
following eccentric exercise. Time of peak soreness and the intensity of the soreness
may vary between studies both because of the different exercise protocols employed on
different muscles (i.e. elbow ﬂexors versus knee ﬂexors) and because of the large
variability in the perception of pain between subjects.

A signiﬁcant and persistent decrease in isometric strength accompanies the
soreness following eccentric exercise (21, 22, 76, 80). The strength loss or fatigue
following concentric or isometric exercise recovers within minutes to tens of minutes
after the exercise. In contrast, a fraction of the strength loss following eccentric exercise
can persist for many hours to days after the fatiguing bout. This persistent loss of strength
may be the result of myoﬁbrillar damage caused by the lengthening contractions.
However, only a few studies have compared the long term time course of the recovery of
strength with other indices of muscle damage in human subjects.

The prolonged decrease in isometric strength after eccentric exercise was once
thought to be simply the result of reluctance to use the sore muscles. However, this
appears unlikely, since strength actually recovers somewhat even before peak soreness
develops, typically at 24-72 h post-exercise. To determine if the loss in isometric
strength was due to sub-maximal activation of the sore muscles following eccentric
exercise; Newham et a1 (76) employed the twitch interpolation technique which
superimposes electrical stimulation on maximal voluntary contraction (MV C). Under

these conditions, additional force will only be generated if the MVC was sub-maximal.

Because force did not increase with twitch interpolation during MVC, Newham and
colleagues concluded that the subjects were able to fully activate their eccentrically
damaged muscle during MVC despite any soreness. More importantly, Newham and co-
workers suggest that the prolonged decrease in MV C after eccentric exercise is the result
of changes in the contractile elements.

Another symptom of DMS is a reduction in the range of motion at the joint or
joints that were involved in the eccentric exercise. Clarkson and others (21, 22, 80) have
used the following two criteria to measure the reduction in the range of motion at the
exercised joint. First, the muscle’s ability to fully shorten was assessed by measuring,
ﬂexed joint angle, the angle at the joint following full voluntary ﬂexion. The largest
change in the ﬂexed joint angle was reported immediately following eccentric exercise,
suggesting that the eccentrically exercised muscles were unable to fully ﬂex. The ﬂexed
joint angle gradually returns towards pre-exercise levels in the days following eccentric
exercise. However, the ﬂexed joint angle remained different from baseline at 10d post-
eccentric exercise. The decrease in muscular strength and the inability to fully shorten
muscles could be the result of over stretching of sarcomeres by the lengthening
contractions. Consequently, when the sarcomeres are stretched beyond their optimum
length the overlap between the actin and myosin ﬁlaments is reduced, which in turn
reduces the number of cross bridges that can be formed. Recent work by Talbot and
Morgan (104) supports this theory since greater muscle damage and decrement in
strength were seen after eccentric exercise at longer muscle lengths. In addition, there is
general agreement that increasing the lengthening distance of an eccentric contraction

causes more muscle damage.

The second criterion measure arose from Clarkson and co-workers observations
that muscles spontaneously shortened following eccentric exercise. To document this
phenomenon, they measured the relaxed joint angle, which was measured as the angle at
the exercised joint in the relaxed or resting position. Relaxed joint angle decreased (i.e.,
the joint was more ﬂexed) immediately following lengthening contractions and continued
to decrease in the next few days. The largest reduction in relaxed joint angle occurred at
3 d post-eccentric exercise. Unlike ﬂexed joint angle, the relaxed joint angle recovered
to baseline values by 10 d post-eccentric exercise.

Smith (99) suggests that the reduced range of motion may place the eccentrically
damaged muscles in a position/resting ﬁber length that is optimal for healing. Smith
refers to this idea as “voluntary “ immobilization, which was ﬁrst proposed by Clancy
and Clarkson (20). One possible mechanism for this change is increased electrical
activity in the damaged muscle. However, Bobbert et al. and Howell et al. (14, 57) found
no such increase in the resting EMG activity at 24, 48, and 73 h after eccentric exercise.
Therefore, the decrease in the relaxed joint angle is not the result of active muscle
shortening. Another possibility is increased intracellular calcium, such that myoﬁbrillar
activity is present in the absence of electrical activity. However, there is at present no
evidence that cytoplasmic calcium is sufﬁciently elevated in the damaged ﬁbers to result
in contraction of myoﬁbrils. It is also possible that the decrease in the relaxed joint angle
could be simply due to the mechanical effect of swelling, i.e. the muscle shortens in
length to accommodate the increase in volume.

Swelling of the exercised limb is another commonly reported symptom of DMS

following eccentric exercise. Numerous studies have reported an increase in the

circumference of the eccentrically exercised limb in the days following exercise (21, 22,
43, 80). Peak swelling occurred at 4-5 d post-eccentric exercise. The exercised limb
circumference typically returns to baseline values by 10 d post-exercise. It is important
to keep in mind that in many studies swelling was documented by measuring the
circumference of the exercised limb with a tape measure. Therefore, the sensitivity of
the instrument is relatively poor. In addition, this anthropometric technique was unable
to localize the swelling within the exercised muscle compartment (i.e. intracellular versus

extracellular ﬂuid).

Morphological Changes Associated with DMS

 

In the ﬁrst published report on DMS in 1902, Theodore Hough suggested that the
pain and decrements in muscular performance were the result of ultrastructural damage
induced by lengthening contractions (56). In order to obtain direct evidence of muscle
damage following eccentric exercise, many investigators have employed light and/or
electron microscopic techniques to examine changes in muscle biopsy samples. Most of
the studies have reported signiﬁcant morphological alterations in muscle at 24 to 48 hr
following eccentric contractions but not after concentric or isometric contractions in
humans and rats (8, 38, 44, 46, 47, 73, 77, 82). However, there are only a limited number
of studies that have reported signiﬁcant but minor cellular damage immediately
following eccentric exercise (8, 47, 77, 82). Therefore, the immediate muscle damage
following eccentric exercise is relatively minor compared to the damage observed in the
ensuing days. F riden et a1. (46) reported disorganization of myoﬁbrillar Z-bands in

human soleus m. 3 d after running down stairs. F riden and colleagues found Z-band

10

broadening, smearing, and in some ﬁbers, profound Z-band disruption. Adjacent Z-
bands appeared to be out of register and followed a zigzag pattern along the length of the
damaged ﬁbers. Friden and co-workers also observed that within a small percentage of
damaged sarcomeres the A-bands were out of register and the thick or myosin ﬁlaments
were missing.

Newham et al. (77) reported similar results in the eccentrically damaged human
quadriceps muscle at 24-48 h after a 20 min step exercise. No abnormalities were
reported in the human quadriceps muscle after concentric exercise. Moreover, Newham
et al. presented quantitative light microscope data on human biopsies taken at various
times after exercise. These researchers characterized myoﬁbrillar damage into three
distinct categories: 1) focal - areas of disruption affecting one or two adjacent myoﬁbrils
and one or two adjacent sarcomeres, 2) extensive - more than two adjacent sarcomeres
and myoﬁbrils with disruption, or more than ten focal areas within one muscle ﬁber, and
3) very extensive -- more than one extensive area of damage. Using these criterion,
Newham and co-workers found that immediately after the eccentric exercise, 16% of the
ﬁbers counted showed focal changes, 16% had extensive damage, 8% had very extensive
changes, and 58% of the total counted ﬁbers appeared normal. In the samples collected
at 30 h after eccentric exercise 6% showed focal changes, 23% had extensive disruptions,
28% had very extensive disruptions, and 45% of the total counted ﬁbers appeared
normal. Newham et al. suggested that the lesions observed immediately after the
eccentric exercise were precursors of the more severe damage reported in the days

following exercise. According to Newham and others, the initial injury exposes the

11

structural components to endogenous hydrolytic proteases, and these activated proteases
autolyze the damaged muscle cell.

In another study, Friden and co-workers (47) collected muscle biopsies from
human vastus lateralis m. before and at intervals after eccentric exercise on a modiﬁed
bicycle ergometer. This group reported that 12% of the ﬁbers counted showed focal
disruptions of the striated band pattern at 1 h, 52% on day 3, and 32% on day 6 post-
eccentric exercise. Friden et al. interpreted these results as evidence that the myoﬁbrillar
damage developed gradually in the days following eccentric exercise, and moreover, the
damaged ﬁbers had somewhat recovered by 6 d after eccentric exercise. However, some
investigators have questioned whether the needle biopsy sample (approximately 50 ﬁbers
per biopsy) is representative of the damage across the entire length of the muscle. As a
result, many investigators have used animal models, speciﬁcally the rat model, to
systematically sample the muscle to document the predominant lesion and its distribution
within the entire eccentrically exercised muscle.

Armstrong et al. (8) reported myoﬁbril damage immediately following eccentric
exercise, and progressively greater muscle degeneration at 24 and 48 h in approximately
less than 5% of the muscle ﬁbers. However, Armstrong et al. did not incorporate
systematic sampling to document the different type of lesions or their predominance.
This oversight was addressed in a later study by Ogilvie et al. (82). This group reported
three types of lesions in rat soleus m. 30 min after running downhill: 1) focal disruptions
of the A-band, 2) Z-line dissolution, and 3) clotted ﬁbers. In addition, they reported that
the 89% of lesions in the downhill running group and 97% of lesions in the level runner

group were A-band disruptions. Furthermore, these investigators found that the A-band

12

lesion density was signiﬁcantly higher in the soleus m. of the downhill runners compared
to the level runners. The highest incidence of A-band lesions were found in the distal
half of the soleus m. of downhill runners. On the other hand, in level runners the highest
incidence of A-band lesions was reported in the proximal half of the soleus m.

These investigators suggest that the A-band lesions are the initial observable
injury in muscle. However, this report failed to address the differential location of the A-
band lesions between the eccentrically exercised muscle (distal region of the soleus m.)
versus the concentrically exercised muscle (proximal region). This difference could be
the result of differential recruitment of motor units during a lengthening contraction
versus a shortening contraction. Another possibility is that the muscle ﬁbers near the
musculotendon junction experience greater active strain/stress during eccentric
contractions, which, in turns, leads to greater A-band lesions.

In addition, the dominance of A-band lesions in the rat soleus m. after downhill
running versus the dominance of Z-band streaming in the human vastus lateralis m. after
lengthening contractions could be the result of species difference in Skeletal muscle
architecture. Another possibility is that the differential damage in the rat muscle could be
due to the fact that the exercise intensity (downhill running) was much greater in the rat
experiments compared to the human exercise protocol.

McCully and Faulkner (72) found many ﬁbers in the rat extensor digitorum
longus m. that appeared abnormal at l d post eccentric exercise. However, the number of
ﬁbers per cross-sectional area in the eccentrically damaged muscle was not signiﬁcantly
different from control. These results suggest that initial damage immediately following

concentric and eccentric exercise was not different. However, two to four days after

13

lengthening contractions, McCully and Faulkner reported signiﬁcant macrophage
inﬁltration and muscle degeneration. Peak macrophage inﬁltration and muscle
degeneration were reported on day 3 and by day 4. There was also evidence of muscle
regeneration, such as the appearance of myoblasts and myotubes. Therefore, it appears
that in a small percentage of ﬁbers the initial damage leads to ﬁber necrosis.

In short, the histological evidence from human and rat studies suggest that
following the initial minor injury, further degradation of the injured ﬁbers occurs in the
ensuing hours (8, 46, 47, 73, 77). The sensation of DMS appears to be related to the
delayed muscle and/or connective tissue disruption that takes place in the days following
lengthening contractions (7, 99). Of particular interest is the fact that peak pain
correlates well with peak damage based on the histological changes; however, the strong
correlation between muscular strength loss and ultrastructural damage observed 24 - 48 h
post eccentric exercise begins to deteriorate after day 4 (1). Consequently, one could
argue that the persistent loss in muscular strength reﬂects incomplete regeneration and/or

re-enervation of the newly synthesized myocytes.

BiochemiciMm
In a continued attempt to characterize the initial damage immediately following
eccentric exercise versus the secondary damage in the ensuing days, many investigators
began to study biochemical markers of muscle damage. Serum concentrations of muscle
proteins such as creatine kinase (CK), myoglobin, and slow-twitch skeletal (cardiac beta-
type) myosin heavy chains (MHC) have been used as indirect markers of damage

following eccentric exercise (21, 64, 78, 85, 95, 101).

14

The serum level of several muscle proteins has been reported to increase after
exercise (21). The general consensus is that the increased serum levels of muscle
proteins reﬂect an increase in release and/or leakage from the damaged muscle cells. CK
is the most commonly studied muscle protein and is considered the " gold standard"
biochemical marker of skeletal muscle damage following eccentric exercise. CK is a
dimeric enzyme that catalyzes the reversible phosphorylation of ADP by creatine
phosphate to form ATP and free creatine (Cr). The two isoforrns of CK, M (muscle) and
B (brain) combine to form three principle dimeric cytoplasmic isoenzymes: CK-MM in
skeletal muscle, CK-MB in cardiac tissue, and CK-BB in the brain (55). Within skeletal
muscle, the majority of the CK is in the CK-MM isoforrn (approximately 90% of the
total CK), with the remaining activity being accounted for by the mitochondrial isozyme.
Furthermore, three subisoforrns of CK-MM have been identiﬁed: CK-MMl, CK-MM2,
and CK-MM3. CK-MMl is the only subisoforrn found within the muscle cell; the other
two isoforrns can be found in serum. The cleavage of either a lysine or arginine residue
from the carboxyl terminus on the M monomer of CK-MMl by carboxypeptidase N will
yield CK-MMZ. The additional release of either amino acid residue from the second M
monomer of CK-MMI yields CK-MM3.

Total CK activity in serum is the most commonly measured parameter for
documenting muscle damage following eccentric exercise. Intensity, duration, and the
type of exercise all inﬂuence the amount of CK released into the blood. After eccentric
exercise, CK is released into the circulatory system and remains elevated for several days
following the eccentric exercise. Generally, peak total CK activity occurs on the third or

fourth day following lengthening contractions, and remains elevated for 6-7 (1.

15

Clarkson and others (17, 21, 22, 59, 76, 78-80) have published numerous reports
using CK as an indirect measure of muscle damage following eccentric exercise. This
group found that, even after controlling for the exercise intensity, there was a large
intersubj ect variability in the total CK response following exercise. Furthermore, there
was no systematic relationship between the peak total CK response and the other indirect
indicators of muscle damage (i.e. muscle soreness, isometric strength, relaxed arm angle,
and ﬂexed arm angle). According to Clarkson et al. the low correlation between peak
CK response and other indirect indicators of damage could be due to the large variance
across all indirect measures of damage. In addition, serum activity of CK depends on the
rate of enzyme clearance by the reticuloendothelial system, not just on release from the
damaged muscle ﬁbers.

Hyatt et al. (59) found signiﬁcant leakage of the muscle speciﬁc CK into the
blood stream following eccentric exercise. In the hours following eccentric exercise,
CK-MMl is modiﬁed into the two other subisoforms: CK-MM2 and CK-MM3. These
investigators studied the ratio of CK-MMl :CK-MM3 to separate CK-MMl release from
clearance. Based on previous studies, they argue that if CK-MMl release and its
clearance are equal then the ratio will be equal to one, whereas ratios greater than 1
indicate that release is larger than clearance, and ratios less than 1 indicate that clearance
rate is faster than release rate. Haytt et al. reported a signiﬁcant increase in the CK-
MMl :CK-MM3 ratio in the hours following eccentric exercise, suggesting that release
exceeded clearance. Peak CK-MMl release occurred at 48 h post exercise. By day 4,
the ratio was less than one which indicates that the rate of clearance had overtaken the

rate of CK-MMl release from muscle. This report showed that there was a signiﬁcant

16

delayed release of CK-MMl from the eccentrically damaged muscles and that the rate of
clearance was not able to keep up with the release rate.

Myoglobin (Mb) is another biochemical marker of muscle damage. However, the
proﬁle of the Mb response after eccentric exercise is very different compared to the CK-
MM response. Mb level peaks within 4 h post eccentric exercise (10, 70). This earlier
appearance results from the fact that Mb is a smaller protein than CK-MM (17,200 vs.
41,300 Daltons). Thus it is able to exit the cell through smaller lesions in the
sarcolemma that are present immediately following eccentric exercise. The Mb response
is therefore much more sensitive to the initial damage after eccentric exercise.

Another protein that is present in very high concentration in striated muscle is
myosin. Myosin is a hexameric contractile protein made up of 4 light and 2 heavy chains.
After eccentric exercise, there is an increase in plasma concentration of slow-twitch
skeletal myosin heavy chain (MHC) fragments (70). The temporal proﬁle of the MHC
response is similar to the CK-MM response following eccentric exercise.

Despite the availability of these other serum protein markers, neither is used
nearly as often as CK in documenting exercise induced damage. There are several
reasons for this: 1) there is a large amount of variability in the Mb and MHC compared to
the CK response between subjects, 2) the correlation between the amount of damage and
the Mb and MHC response is poor, and 3) in the exercise science literature, CK is
considered the gold standard biochemical marker of muscle damage because the
biochemical technique required to measure serum enzyme activity is readily available
and is much easier compared to the techniques involved in measuring the speciﬁc protein

levels in serum (gel electrophoresis).

17

Repeated Bout Effect

The foregoing discussion has described reported discrepancies in the timing of the
development of muscle damage, soreness, CK release and other markers of exercise-
induced trauma. Researchers have also observed differences in the timing and magnitude
of the responses to subsequent bouts of eccentric exercise after an initial damaging bout.
A protective effect against muscle soreness has been noted for both concentric and
eccentric exercise training (12, 22); the Speciﬁc protective effect produced by a single
bout of eccentric exercise has been commonly referred to as the "repeated bout effect".

Nosaka et al. (80) examined the repeated bout effect by having the subjects
perform two bouts of eccentric exercise of the forearm ﬂexors. Subjects were randomly
assigned to two groups. Group One, the second bout of eccentric exercise was performed
6 wk after the ﬁrst bout. Subjects in Group Two performed the second bout of eccentric
exercise 10 wk after the ﬁrst bout. In addition, a subset of the subjects, three from each
group, performed the same eccentric exercise (bout 3) 6 months after bout 1.

All the subjects in the 6 wk group reported less peak soreness after bout 2
compared to bout 1, and the soreness rapidly subsided within 3-4 (1 after bout 2 compared
to 8-10 (I after boutl . However, peak soreness reached bout 1 levels after exercise bout 2
in the 10 wk group and after bout 3 in the 6 months group. Isometric strength loss
averaged about 40% after bout 1 and bout 2 in both groups. However, the 6 wk group
showed a more rapid recovery in isometric strength after bout 2 compared to bout 1. On
the other hand, the 10 wk group did not show any improvement in the rate of recovery

between bouts 1 and 2. Similar results have been reported by others following repeated

18

bouts of eccentric exercise (35, 76, 79). The patterns of isometric strength loss and
recovery after bout 3 (6 months after bout 1) did not differ signiﬁcantly from bout 1.

F lexed joint angle and relaxed joint angle were also measured after all bouts in
the above-mentioned study. These investigators found that the largest change in ﬂexed
joint angle occurred immediately after bouts l and 2 for both groups (6 and 10 wk).
Nonetheless, the 6 wk group showed a quicker recovery compared to bout 1 but no
difference between bouts in the 10 wk group. As expected, the relaxed joint angle
decreased signiﬁcantly immediately after the ﬁrst and second bout of eccentric exercise
in both groups (6 and 10 wk). However, the decrease in the relaxed joint angle was
signiﬁcantly less after the second bout compared to the ﬁrst bout in both groups. There
was no difference in the relaxed joint angle or the ﬂexed joint angle after bout 3 (6
months after bout 1) compared to bout 1.

This group also measured total senun CK levels before, immediately after and
every day for 5 days after bout 1, 2, and 3. Both groups (6 and 10 wk) showed a similar
and signiﬁcant increase following the ﬁrst bout of eccentric exercise. The total CK
increase after bout 2 was signiﬁcantly less then the bout 1 change in both groups. Unlike
the other indirect markers of muscle damage, the CK-MM response after bout 3 (6
months) was still signiﬁcantly less than bout 1 but higher than after bout 2.

In another study, Hyatt et al. (59) separated CK-MMl release from clearance by
measuring the ratio of CK-MMl :CK-MM3 following a second bout of eccentric exercise
performed 6 d after bout 1. This group reported a decrease in CK-MMl release and an
increase in the clearance rate after the second bout of eccentric exercise compared to the

ﬁrst bout response. These investigators interpreted the decrease in CK-MMl release as

19

evidence that the injury induced by bout 2 was signiﬁcantly less severe compared to bout
1. Furthermore, Hyatt and co-workers found that the increased rate of clearance
contributed to the decrease in total CK-MM response after bout 2, suggesting an
adaptation in the clearance process. Lastly, Hyatt et al. documented swelling by
measuring the circumference of the exercised limb in the days following both bouts of
eccentric exercise. As expected, swelling was signiﬁcantly reduced after the second bout
of eccentric exercise compared to bout 1.

Recently, Nosaka and Clarkson (79) using the same methods from their earlier
study, plus ultrasound imaging, reported that repeated bouts of the same eccentric
exercise performed 3 and 6 d after the ﬁrst bout did not exacerbate the damage nor did it
affect the rate of recovery. Ultrasound imaging is a noninvasive technique that provided
these investigators with an in-vivo measurement of the exercise-induced muscle damage.
Ebbeling and Clarkson (35) reported similar results after repeated bouts of the same
eccentric exercise performed 5 and 14 d after the ﬁrst bout.

Taken together, these results suggest that an adaptation response had taken place
prior to full recovery of muscle function and ultrastructural damage after the initial bout.
Moreover, this adaptive response occurred as early as 3 d after the ﬁrst bout of eccentric
exercise. However, researchers studying the repeated bout effect have not shown direct
histological evidence of the reduced muscle damage after the subsequent bouts of
eccentric exercise.

In summary, a single bout of eccentric exercise results in temporary
ultrastructural muscle damage, delayed soreness, performance decrements, and an

increase in serum activity of CK-MM. Complete recovery of muscle damage and

20

ﬁrnction after a single bout of eccentric exercise can take up to 10 d or longer. During
the period of recovery, an adaptation or training response occurs providing a protective
effect against damage from a subsequent bout of eccentric exercise. Lastly, the
protective effect of a single bout of eccentric exercise, based on the reduced CK-MM
response, has been reported to last up to 6 months. A review of the literature will show
that several distinct hypotheses have been proposed to explain the mechanism underlying
DMS and the “repeated bout effect”. No single explanation shows more promise than

Armstrong’s model combined with Smith’s acute inﬂammation model of DMS (6).

Mechanisms of DMS

Armstrong’s model (8) divides the muscle injury process resulting from eccentric
exercise into four stages: 1) initial, 2) autogenetic, 3) phagocytic, and 4) regenerative.
Armstrong and co-workers have primarily focused their research on the ﬁrst two stages.
Conversely, Smith (99) has presented a convincing argument for acute inﬂammation as
the underlying mechanism of DMS. However, several key studies do not support
Smith’s hypothesis that acute inﬂammation is the sole underlying mechanism of DMS (8,
47, 95). On the other hand, the acute inﬂammatory response could explain the

phagocytic and regenerative stages proposed in Armstrong's model of DMS.

Initial and Autogenetic Stages of DMS
The most common belief in the lay exercise community is that DMS is the result
of lactic acid accumulation in the exercised muscle. However, this metabolic hypothesis

has been rejected by exercise scientist on the basis of two ﬁndings: 1) the metabolic cost

21

of lengthening contractions is signiﬁcantly less than for concentric contractions at the
same power output, and 2) eccentric exercise produced less lactic acid than concentric
exercise at the same power output (7). These ﬁndings are not consistent with the
common observation that eccentric exercise is much more likely than concentric exercise
to induce DMS.

During an eccentric contraction, fewer motor units are recruited to generate the
required force (13). As a result, the force generated during an eccentric contraction is
distributed across a smaller cross-sectional area of muscle compared to concentric
contractions. This increase in active stress/strain may be the primary cause of
ultrastructural damage in the muscle. Furthermore, Armstrong et al. (8) suggest that a
sub-population of ﬁbers (<5%) nearing the end of their functional life in healthy muscles
are more susceptible to this increased active strain (A muscle length/initial muscle length)
and/or stress (force generated/cross-sectional area of the active muscle). However,
McCully and Faulkner (72, 73) have shown that the eccentric exercise-induced injury is
not limited to a small population of susceptible ﬁbers. Following eccentric exercise,
McCully and Faulkner reported ultrastructural damage in 34% of the total muscle cross-
sectional area, of the extensor digitorum longus m. in mice, at 3 d after eccentric
exercise. However, the "susceptible" ﬁbers may be more likely to autolyze as a result of
the damage, whereas other ﬁbers recover.

In addition, Friden et al. (47) reported histological signs of damage in 52% of the
ﬁbers biopsied from the human vastus lateralis m., at 3 (I post eccentric exercise.
Histochemical analysis revealed that the ultrastructural damage occurs primarily in the

type IIb ﬁbers. Therefore, these results suggest that either more of the type IIb ﬁbers

22

were near the end of their functional life, or that type IIb ﬁbers were selectively recruited
during the eccentric exercise.

The increased active strain and stress results in physical disruption of the
sarcolemma, sarcoplasmic reticulum, myoﬁbrils, and causes the release of phospholipase
A2 onto the phospholipid bi-layer causing degradation of the structural components.
Moreover, the extracellular Ca+2 enters the cytosol down its concentration gradient
through the small lesions in the sarcolemma. Armstrong (6) suggests the following as a
possible sequence of events following the inﬂux of Ca+2 in the initial and autogenetic
stages of injury. The increase in the intracellular Ca+2 concentration further activates the
phospholipase A2 activity. The increased phospholipase A2 activity results in the
production of lysophospholipids, leukotrienes, and prostaglandins which, in turn, cause
the degradation of contractile and non-contractile proteins. In addition, increased
cytosolic Ca”2 concentration activates the endogenous proteases and stimulates the
synthesis of free radicals. The end result is the peroxidation of lipids in the cell
membrane. Further, the increase in cytosolic Ca+2 concentration also activates the Ca+2
dependent proteolytic enzymes that preferentially degrade Z-discs, troponin, and
tropomyosin.

The ﬁrst step in Armstrong's proposed sequence, however, is not supported by a
review of the chemically-induced muscle damage literature which suggests that the
increase in cytosolic Ca+2 concentration after eccentric exercise is well within the
physiological range (7, 32, 69). In addition, when the intracellular Ca+2 concentration
was increased using pharmacological agents, muscle cells were able to buffer the

chemically induced increase in cytosolic Ca+2 concentration. Therefore, it appears that

23

the increase in intracellular Ca’2 concentration following eccentric exercise is within the
physiological range and does not activate the endogenous Ca+2 mediated destructive
pathway.

However, it is entirely possible that the transient changes in the cytosolic Ca+2
concentration could initiate the Ca+2 mediated destructive pathway. Therefore, a
persistent increase in cytolosic Ca+2 may not be necessary to initiate the Ca+2 mediated
destructive pathway. Furthermore, the time resolution of the standard measurement
techniques might not be adequate to detect this rapid increase in cytosolic Ca+2
concentration. In addition, small lesions in the sarcolemma and the myoﬁbrils could
expose contractile and structural proteins to proteases located on the sarcotubular
network. These proteases would then hydrolyze the structural proteins, resulting in the
autolysis (l 8). The progressive deterioration of the sarcolemma leads to the diffusion of
intracellular components across the interstitial space and into the plasma. Many of these
intracellular components, especially prostaglandins, attract neutrophils and monocytes
that convert to macrophages. Lastly, the accumulation of macrophages is required for the

damaged muscle cell to proceed to the phagocytic stage ( 19).

Phagocytic Stage

Much of the evidence supporting the existence of a phagocytic stage was
proposed by Smith's acute inﬂammation model of DMS (99). The following section
describes the classic inﬂammatory response and the evidence relating this process to the

phagocytic stage in Armstrong's model of DMS.

24

The classic model of inﬂammation involves two separate but related processes: 1)
the vascular response, and 2) the cellular response. First, during the vascular response of
inﬂammation, the arterioles near the site of injury vasoconstrict for approximately 5-10
min following injury. After several hours, the vasoconstriction is followed by
vasodilatation. This vasodilatation results in increased blood ﬂow and vascular
permeability in the capillary bed surrounding the injury. The end result of this increase
in vascular permeability is edema and pain in the injured area.

Second, the cellular response aspect of inﬂammation involves the activation of
leukocytes. Neutrophils and monocytes are the two predominate leukocytes involved in
the inﬂammatory response. In the hours following injury, there is a dramatic increase in
the neutrophil count. The magnitude of the neutrophil response depends on the severity
of the damage. Generally, peak neutrophil concentration is reported 1-4 h post-injury
and declines rapidly in the ensuing hours. Neutrophils are the ﬁrst phagocytic cells to
arrive at the site of damage. Upon arrival, the neutorphils begin to digest necrotic cells
and cellular debris. Several hours later, monocytes exit the circulatory system and enter
the injured muscle cells. Monocyte concentration remains elevated for 48 h post-injury.
The monocytes mature into macrophages at the site of injury and digest dead cells and
damaged cell ﬁagments.

Swelling is a cardinal symptom of acute inﬂammation and is the result of
increased vascular permeability in the damaged tissue. Stauber et al. (102) have reported
increased permeability in the rat skeletal muscle following eccentric exercise. Other
studies have reported an increase in limb circumference at 24, 48, and 72 h after eccentric

exercise (21, 22, 43, 80). The time course of the development of swelling/edema during

25

DMS parallels the development of swelling during acute inﬂammation. Smith suggests
that the increased intramuscular pressure, due to swelling, mechanically stimulates
prostaglandin E series (PGEZ) — sensitive pain receptors. However, most studies have
reported that peak swelling occurs at a time when pain is recovering. For example, Foley
et al. (43) reported peak swelling at 4-7 (1 and peak pain at 48 h after eccentric exercise.
Many other studies have reported similar results (e. g., (21, 22, 80)).

Although, it is tempting to attribute the sensation of soreness to swelling, the data
does not support a causal relationship between swelling and pain. Alternatively, certain
chemicals can activate the pain afferents, mostly type III and IV nerve ﬁbers; for
example, histamine, acetlycholine, bradykinin, potassium, serotonin, and PGE2. The
inﬂammation literature suggests that the most likely candidate is PGE2. PGE2 per se does
not stimulate the pain afferents, rather it induces a sate of hyperalgesia in the nociceptors.
Smith and co-workers have reported a similar time course for the increase in PGE2 and
soreness after eccentric exercise: both peak at 24-48 h post eccentric exercise.
Interleukin-l, Ca”, and macrophages have been proposed as possible candidates for the
stimulation of local PGE2 production. The exercise-induced muscle damage literature
suggests that the accumulation of macrophages is most likely responsible for the
increased biosynthesis of PGE2.

Armstrong et al. (8) reported signiﬁcant macrophage accumulation in rodent
muscles aﬁer downhill running but found minimal neurtrophil inﬁltration. In contrast,
Kuipers et al. (65) found signiﬁcant margination and inﬁltration of neutrophils in the
soleus m. of the rat between 0-2 h post eccentric exercise. However, F riden et al. (47)

did not ﬁnd invasive macrophages in muscle biopsies taken from the vastus lateralis m.

26

of human subjects after eccentric exercise. In contrast, Jones and others have observed
the presence of invasive macrophages in the muscle of human subjects following
eccentric exercise (63, 94). This discrepancy between studies is partly due to the fact that
muscle biopsies require the removal of tissue from the belly of the muscle. Therefore, if
the damage was initiated in the myotendinous junction, the damage will only reach the
belly of the muscle at a later time (8). Furthermore, the above-mentioned studies
employed a variety of different eccentric exercise protocols, which could have
contributed to the discrepancy in the results. Smith et al. (100) reported signiﬁcant
increases in peripheral neutrophil count at 1 and 2 h after 40 minutes of downhill
running. These investigators propose that the increase in peripheral white blood cell
count is the result of the increased levels of circulating polymorphs (a type of neutrophil),
which has been reported to increase during acute inﬂammation. More recently, Croisier
ct al. (26) reported similar increases in polymorphonuclear neutrophil activation after

eccentric exercise.

Effects of Anti-Inflammatory Treatments on DMS

 

In a continued effort to determine the role of acute inﬂammation in the etiology
of DMS, several investigators studied the effect of non-steriodal anti-inﬂammatory drugs
on DMS. The data from these studies suggest that anti-inﬂammatory drugs reduced
DMS when the eccentric exercise recruited small muscle groups for a short duration of
time, i.e. lengthening contractions of the elbow ﬂexor muscle group. On the other hand,
anti-inﬂammatory drugs apparently do not reduce DMS following downhill running or

cycling (30, 31, 50, 52, 65). However, these studies did not measure the markers of

27

inﬂammation; rather, they measured the symptoms of DMS (i.e. soreness and muscular
performance).

Clinicians in sports medicine often use superﬁcial heat and cold in the treatment
of musculoskeletal injury. Heat and cold treatments have been shown to alter the
circulatory response and intravascular hydrostatic pressure in injured muscles, with the
overall desired effect being reduced accumulation of ﬂuid or swelling in injured muscles
(75). Immediate cold application is recommended during the acute stage of injury to
decrease bleeding, capillary effusion, pain, and inﬂammation. On the other hand, heat
application is recommend during the healing process to increase vascular and lymphatic
ﬂow. In addition, heat application improves the range of motion through increased
collagen extensibility. Like therapeutic cold application, heat also reduces pain through
sensory stimulation of the skin.

Most studies have found that cold application after eccentric exercise provided
temporary pain relief (60, 106). Kuligowski et al. (66) recently reported that cold
whirlpool and contrast therapy (alternating cycles of hot and cold), were more effective
than warm whirlpool and no treatment in alleviating the signs and/or symptoms of DMS.
In addition, Prentice showed that cold plus static stretching was superior to heat followed
by static stretching in reducing muscle pain and increased muscle relaxation (88). The
limited available evidence suggests that heat application does not alleviate the signs
and/or symptoms of DMS.

To date no studies have directly tested the effectiveness of therapeutic heat
application in alleviating the signs and/or symptoms of DMS. There are two main ﬂaws

in the experimental design of Kuligowski et al. and Prentice's study. First, the

28

application of heat immediately following the eccentric exercise has the potential to
promote the injury process by increasing capillary effusion and swelling. Second, the
application of heat (40°C) for 20-30 min might not be long enough to promote the
clearance of extracellular ﬂuid by the lymphatic circulatory system, which is much
slower than blood ﬂow. In addition, these studies did not include a measure of muscle
damage during the treatment period. Swelling was measured using limb circumference
measures, which is not sensitive to small changes in the extracellular ﬂuid volume.
Therefore, it was not possible to separate the temporary analgesic effect from the anti-
inﬁammatory effect of the different treatments on muscle damage.

In summary, the exact mechanism of the phagocytic stage in Armstrong's model
of DMS remains unclear. However, the evidence presented thus far suggests that the
edema and pain are partly related to the release of PGE2. Furthermore, several key
similarities exist between the proposed phagocytic stage and acute inﬂammation: 1) pain,
swelling, and loss of function, 2) similar cellular inﬁltrates, speciﬁcally macrophages, 3)
the presence of ﬁbroblasts, 4) increased lysosomal activity, 4) increase in the severity of
the lesions, and 5) signs of healing approximately 3 d after the initial injury. The
progression of the phagocytic stage through the steps of the inﬂammatory process
provides the only viable hypothesis to date for the delay between the initial injury and the

onset of the symptoms of DMS.

Regeneration Stage

In general, the purpose of acute inﬂammation is to promote tissue healing, which

clearly occurs in the days after eccentric exercise. Once the most severely damaged

29

regions of the ﬁber have been removed by autolysis, rapid regeneration of myoﬁbrils
begins within the existing basal lamina (19). The uninjured portions on either side of
necrotic zone begin to retract. The intact myoﬁbrils form a stump on either side of the
necrotic zone, which becomes covered by a newly synthesized cell membrane. The
invasion of macrophages into the necrotic area activates satellite cells, which are located
in the periphery within the basement membrane of the muscle ﬁber. The satellite cells
proliferate within the basement membrane and then migrate to the necrotic region of the
muscle ﬁber. At the necrotic region, these satellite cells differentiate into myoblasts,
these myoblasts fuse to form myotubes. Once the ends of the myotubes reach the
undamaged myoﬁbril stumps, the cell membrane on the myoﬁbril stumps dissolve and
union occurs.

However, if necrosis extends the length of the ﬁber, this will more than likely
result in cell death. Therefore, some unknown mechanism determines if the extent of the
necrosis is repairable or not. After the removal of the irreversibly damaged ﬁber,
regeneration begins with the activation of satellite cells located within the existing basal
lamina and follows a similar pathway as described above. Lastly, the newly formed
myotubes are reinnervated by motor axons.

Correspondingly, Darr and Schultz (27) found peak satellite cell activation at 72 h
after eccentric exercise in the rat soleus m.. These investigators reported that
approximately 3% of the ﬁbers exhibited degenerative changes under the light
microscope. Furthermore, Darr and Schultz showed that the satellite cell activation was
greater than expected for the repair/regeneration of the damaged ﬁbers. Therefore, these

results suggest that satellite cell activation occurred in far more cells than just those that

30

appeared to be damaged in the histological sections. In addition, McCully and Faulkner
(73) reported the presence of myoblasts and myotubes at 4 days after eccentric exercise
in the extensor digitorum longus m. of mice. These investigators found that not all ﬁbers
with local disruptions degenerated after eccentric exercise. McCully and Faulkner found
that maximum isometric strength and muscle mass remained below baseline values at 30
d post eccentric exercise. These studies support Armstrong's hypothesis that skeletal
muscles contain a small sub-population of muscle ﬁbers that are vulnerable to damage

during lengthening contractions.

Stress-Susceptible Fiber Theory £111 Repeated Boﬁut Effect

This theory proposes that an unaccustomed bout of eccentric exercise causes
irreversible ultrastructural damage to the pool of stress-susceptible ﬁbers, which leads to
degeneration of these cells in the days following the exercise. Furthermore, microinjury
occurs in the "normal" or non-susceptible ﬁbers that were recruited during the
lengthening contractions but does not result in necrosis of these entire ﬁbers.
Accordingly, there is a prolonged decrease in muscle strength and range of motion,
muscle soreness, and a signiﬁcant increase in the serum levels of muscle proteins
following the bout of eccentric exercise. During the degeneration-regeneration process,
necrotic ﬁbers are replaced with newly assembled ﬁbers and the damaged ﬁbers are
repaired. Therefore, the process of degeneration-regeneration would result in fewer
stress-susceptible ﬁbers to be destroyed by subsequent bouts of eccentric exercise.

Histological evidence from human and rat skeletal muscle following repeated

bouts of eccentric exercise supports this prediction (8, 47). These studies reported a

31

decrease in the percentage of damaged ﬁbers after repeated bouts of eccentric exercise.
In addition, several studies have reported that alter a repeated bout of eccentric exercise,
the recovery of strength and range of motion is faster compared to the ﬁrst bout, soreness
development is less, and serum CK-MM activity peaks much earlier and is signiﬁcantly
blunted (43, 59, 76, 80).

In summary, available evidence seems to support the proposal that destruction of
a sues-vulnerable subpopulation of muscle ﬁbers produces the apparent protective
adaptation to a bout of eccentric exercise. After the initial damaging bout, fewer
vulnerable ﬁbers remain to be destroyed by subsequent bouts. Although the remaining
ﬁbers may again be partially damaged, the injury is less severe and should produce less

inﬂammation and pain.

Neural Adaptations M Bogt of Eccentric Exerci_sg

An alternative explanation for the repeated bout effect has been proposed by
investigators examining alterations in neural activation after damaging eccentric exercise.
A review of the relevant elctromyographic (EMG) literature will be provided followed by
an outline of the neural adaptation hypothesis. Several investigators have measured
EMG activity in the eccentrically damaged muscle during DMS (14, 57). These studies
reported no increase in the resting EMG activity of the damaged muscle. Grabiner et al.
(50) compared the maximum EMG activity after subjects performed either maximum
concentric or eccentric contractions with the knee extensor muscles on an isokentic

machine (30°/s). Speciﬁcally, the maximum EMG during maximal eccentric

contractions averaged 84 i 41 (SD)% of the value for the concentric contractions. This

32

result supports the hypothesis that muscle activation is incomplete during maximum
voluntary eccentric effort, i.e., that the same force is spread over fewer ﬁbers. In
addition, Grabiner and colleagues showed that when subjects expected to perform a
maximum concentric contraction, but the isokentic machine forced a lengthening
contraction, the initial EMG of the unexpected eccentric contraction averaged 104 i 40
(SD)% of that recorded in the maximum concentric effort. Grabiner and co-workers
interpreted these results as evidence that eccentric contractions require a unique motor
unit activation strategy from the central nervous system.

In addition, Owings and Grabiner (83) had subjects perform a single-leg exercise
protocol in which the knee extensor muscles either performed maximal concentric or
eccentric contractions to fatigue. Owings and Grabiner measured maximum eccentric
and concentric force in the uninvolved knee extensor muscles before and after the
fatiguing exercise. Aﬁer fatigue, for the subjects who performed concentric exercise, the
maximum concentric and eccentric forces were not different in the uninvolved knee
extensor muscles compared to before the fatiguing task. On the other hand, for the
subjects who performed eccentric exercise, the maximum eccentric force increased by
11% and the maximum concentric force remained unaltered in the uninvolved knee
extensor muscles aﬁer fatigue compared to before. These investigators suggest that the
increase in the maximum eccentric force in the non-exercised leg after fatiguing eccentric
exercise supports the hypothesis that unique neural strategies are involved during
repeated eccentric contractions.

Therefore, the evidence presented so far suggests that there is incomplete

activation of the muscle during eccentric contractions and that eccentric contractions,

33

through an unknown neural circuitry, control the homologous muscles in the unexercised
leg. Based on the ﬁnding that muscle activation is limited during voluntary eccentric
contractions, Golden and Dudley (49) argue that the subjects "learn" to activate motor
units that are not normally recruited during an eccentric contraction while recovering
from the ﬁrst bout. Therefore, the change in the recruitment pattern, sparing the
damaged ﬁbers, could explain the protective effect on performance and muscle damage
from a subsequent bout of eccentric exercise when the muscle has not fully recovered
ﬁ'om the initial bout.

The contention that "learning" can occur from a single bout of damage inducing
eccentric exercise is not unique. Bar et al. (12) suggested that the initial damage-
inducing eccentric exercise bout could be regarded as a one-trail learning session,
comparable to a passive avoidance test. In the avoidance test, rats receive a shock after
entering a dark cage. As a result of this unpleasant experience, rats do not reenter the
dark cage. This type of avoidance behavior continues even without additional training.
Bar and co-workers suggest that the central nervous system responds to the ﬁrst bout of
eccentric exercise, which results in pain and damage, by altering the muscle recruitment
pattern, such that the muscle is more resistant to damage from subsequent bouts.
However, to date there is no direct evidence to support the contention that the muscle
recruitment pattern is different after a bout of eccentric exercise.

In summary, the stress-susceptible ﬁber theory explains the protective effect after
a single bout of eccentric exercise from subsequent bouts performed several weeks later.
Golden and Dudley (49) have proposed a neural adaptation, which takes place within 48

h after the ﬁrst bout and alters the recruitment pattern in such a way that muscles are

34

resistant to damage from a repeated bout even prior to full recovery. Because of the
technical limitations of standard electrophysiological methods, it has not been possible to
measure the change in the recruitment pattern that Golden and Dudley have proposed.
Enoka and others (37) proposed using MR imaging to determine the distribution
of activation among muscle ﬁbers within the exercised muscle by quantify exercise-
induced changes in the spin-spin (T2) relaxation time. However, the spatial resolution of
MR imaging is not sensitive enough to detect the change in the recruitment pattern of
individual motor units within a muscle (89). On the other hand, MR imaging is a non-
invasive method that allows investigators to measure muscle damage, changes in muscle
compartment cross-sectional area, and exercise intensity. Furthermore, the muscular
changes that occur with DMS (i.e. edema in the extracellular compartment versus the
damage to the contractile machinery) provides an unique in vivo experimental
environment to study the determinants of contrast changes in MR images of muscle. The
following section will review the fundamental principles of MR imaging and the
literature on the application of MR imaging to study muscle damage after eccentric

exercise.

Fundamental Principles of Mngnetic Resonnnce Imngi_n_g
A complete discussion of the principles of MRI imaging is beyond the scope of
this review. However, a brief description of key points is needed. Tissue contrast in MR
images is based on three nuclear magnetic resonance (NMR) parameters: spin density, Tl

relaxation time, and T2 relaxation time (16).

35

Spin density depends on the concentration of the hydrogen nuclei in the tissue.
When a sample containing hydrogen compounds is placed in a strong static magnetic
ﬁeld, the hydrogen nuclei tend to align themselves with the external magnetic ﬁeld. This
alignment along the external magnetic ﬁeld produces a net magnetization vector, the
magnitude of which is proportional to the concentration of hydrogen nuclei in the
sample. The net magnetization vector can be measured (or mapped in space to form an
image) by exciting the sample with a pulse of energy at the appropriate radio frequency
(RF). In the absence of relaxation effects, the “brightness” of a tissue in an MR image
would simply depend on the net magnetization, and hence on hydrogen concentration, or
spin density. For example, the cortex of bone is dark in MR images largely because the
concentration of hydrogen nuclei in bone is less than in the surrounding soft tissues.

The effect of the excitation used to observe MRI signals is to change the
orientation of the net magnetization vector. This new magnetization state is unstable
because the hydrogen nuclei in the sample tend to realign themselves with the external
magnetic ﬁeld. This return to the original magnetization state is described by the two
relaxation parameters: T1 and T2.

T1 relaxation involves the dissipation of the energy that was absorbed from the
RF pulse. This energy must be lost to the surrounding “lattice” as the net magnetization
vector reorients with the static magnetic ﬁeld. On the other hand, T2 relaxation describes
the loss of phase coherence of the excited hydrogen nuclei following the exciting pulse.
Typically, this loss of phase coherence is faster than the energy transfer associated with
T1 relaxation. Therefore, T2 is typically much shorter than Tl. Two processes

contribute to the loss in phase-coherence: l) as the nuclei begin to interact with each

36

other, this creates local distortions in the magnetic ﬁeld around each nucleus, and 2)
heterogeneity of the static magnetic ﬁeld produces local differences in ﬁeld strength.
These two processes combined together contribute to the loss in phase-coherence.
However, generating "spin-echoes" can minimize the effect of static ﬁeld heterogeneity.
With heterogeneity effects minimized, measurement of the T2 time provides insight into
the molecular interactions of the observed nuclei. For example, if the sample contains
only freely mobile hydrogen nuclei, then the random interactions of the nuclei occur So
fast that they average each other out, thus preventing local distortions from persisting
long enough to affect phase coherence. In contrast, if the sample contains hydrogen
nuclei bound to other molecules (i.e. proteins), the random interactions occur at much
lower speed resulting in the faster loss of phase-coherence.

Image, contrast between tissues can be generated either using T1 or T2 relaxation
time differences. The details of various MRI pulse sequences are also beyond the scope
of this review. However, in brief, in a T1-weighted image (i.e., an image acquired with
short repetition time, TR), tissues with long T1 relaxation times will appear dark and
tissues with short Tl relaxation times will appear bright. In contrast, in a T2-weighted
image (i.e., long echo time, or TE), tissues with short T2 relaxation times will appear
dark and tissue with long T2 relaxation times will appear bright. For example, in a T2-
weighted image of the brain, cerebrospinal ﬂuid appears bright compared to the
surrounding, protein— and lipid-ﬁlled regions of brain tissue. Similarly, skeletal muscle
tissue (T2=30 ms) appears darker than adipose tissue (T2=50ms) in a T2-weighted

image.

37

Exercise-Induced Acnte T2 Increase

Over a decade ago, Fleckenstein et al. (41) ﬁrst reported an increase in T1 and T2
in exercised muscle. In addition, these investigators found a weak correlation between
exercise intensity and the signal intensity in T2-weighted images. More recent studies,
using a stronger external magnetic ﬁeld, found that the change in T2 was much greater
compared to the change in T1 in exercised muscle (2, 23). Fisher et al. (40) reported that
the acute increase in the T2 occurs during exercise and decays exponentially after the
exercise at a relatively slow rate (half time, t,,2 = 10 min). In this study, the exercise
intensity was altered by increasing the target force while maintaining a constant
contraction rate. These investigators reported a strong correlation between the increase
in T2 and muscle force generated during the exercise.

In a similar study, Jenner et al. (62) varied the exercise intensity by altering the
rate of contractions while maintaining a constant target force for each contraction.
Results again showed a strong correlation between exercise intensity and the increase in
signal intensity in T2-weighted images. Moreover, Jenner et al. reported an exponential
increase in image intensity at each exercise intensity and that the T2 change reached a
plateau after 3-5 min at all exercise intensities.

Shellock et al. (96) compared the exercise-induced acute T2 increase after
concentric versus eccentric exercise. They reported a larger increase in T2 of the elbow
ﬂexor muscles immediately after concentric compared to eccentric exercise. Since the
energy cost of an eccentric contraction is signiﬁcantly less compared to a concentric

contraction at the same load, and previous studies have shown that the increase in T2 is

38

related to exercise intensity, it is not surprising that Shellock et al. found signiﬁcantly
lower T2 values after eccentric exercise compared to concentric exercise.

In a continued effort to determine the relationship between muscle T2 and
exercise intensity, Adams et al. (2) compared the increase in T2 with EMG data after
graded concentric and eccentric exercise. These investigators found that at any given
exercise intensity the integrated root mean squared EMG (IEMG) of the biceps brachii
m. was signiﬁcantly less after eccentric contractions compared to concentric
contractions. Both IEMG and T2 increased as a function of relative exercise intensity.
Furthermore, linear regression analysis revealed a signiﬁcant correlation (r = 0.99, p <
0.05) between IEMG and the increase in T2 after both concentric and eccentric exercise.

Based on these studies, several investigators suggested that the increase in T2
could be used as a method for estimating the relative intensity of muscle recruitment.
Furthermore, recent studies have employed the exercise-induced T2 increase to map
regional variations in motor unit recruitment within a given muscle (3, 86). However, in
a recent study, Prior et al. (89) showed that the exercise-induced T2 increase on a pixel-
by-pixel basis could not be used to map recruitment of individual motor units within a
muscle. Nonetheless, Prior et al. reported a linear relationship between the mean T2 of
the entire exercised muscle and exercise intensity.

In summary, several studies have consistently reported a linear relationship
between exercise intensity and the acute T2 increase across the entire exercised muscle.
Therefore, the muscle T2 averaged across the whole muscle could serve as an index of

gross muscle activity.

39

The exact mechanism of the exercise-induced T2 increase remains unclear.
Several physiological changes associated with exercise could the cause in muscle T2,
including decreased intracellular pH, increased perfusion, and changes in the

extracellular and intracellular ﬂuid compartments.

Proposed Mechanism of the Acnte T2 Increase After Exercise

 

An increase in the extracellular water (H209 volume during exercise has been
well documented in the exercise literature (98). This increase in the extracelluar and/or
vascular ﬂuid volume is the most commonly cited explanation for the increase in muscle
T2 during exercise. Several studies have shown that the T2 relaxation is
multiexponential in isolated muscle preparations (24, 36, 48, 54, 67). Generally, three T2
components are resolved in isolated muscle: the ﬁrst component has a short T2 relaxation
time of less than 5ms, the second component has an intermediate T2 relaxation time in
the range of 25 to 40 ms, and the third component has a long T2 relaxation time greater
than 80ms. The components of T2 relaxation were resolved through the application of a
complex non-negative least squares (NN LS) algorithm on T2 relaxation curves collected
using echo planar imaging. Echo-planar imaging is a rapid imaging technique that is able
to collect the T2 relaxation or decay signal at several hundred echo times.

The short T2 component, which arises from interactions of water with
intracellular proteins, has been calculated to account for less than 5% of the total T2
signal intensity. On the other hand, the second component with an intermediate T2
accounts for more than 70% of the total T2 signal intensity and the long component

accounts for 10-20% of the total T2 signal intensity. The intermediate component of the

40

T2 relaxation has been suggested to represent the bulk of intracellular ﬂuid and the long
component has been proposed to represent the extracellular and/or vascular ﬂuid.
Therefore, the contribution of the slow-relaxing long component should increase after
exercise and other conditions that increase the extracellular and/or vascular component.

Ploutz-Snyder et al. (87) tested this hypothesis by comparing the T2 response to
exercise versus pressure-induced edema in the lower leg muscles. The temporary edema
in the lower leg muscles was produced by applying external negative leg pressure;
suction in this study was applied from the waist down. This type of negative leg pressure
has been shown to result in blood pooling in the compliant vessels of the legs. Continued
exposure to negative pressure results in increased capillary ﬁltration, which leads to
increased interstitial ﬂuid volume. The NNLS algorithm was used to resolve the
different components of the T2 response during exercise and edema.

Ploutz-Snyder et al. showed signiﬁcant swelling of the lower leg in all subjects
during negative leg pressure. Furthermore, these investigators showed that the T2
response during exercise was different compared to the T2 response during edema. First,
during edema, a slow-relaxing long T2 component (>80ms) was resolved which was not
present during rest or exercise. Second, appearance of the long T2 component did not
result in the shift of the faster relaxing component toward the longer component, which
occurs during exercise. Lastly, Ploutz-Snyder et al. estimated that the extracellular ﬂuid
volume would have to increase to over 30% to account for the T2 change during exercise.
However, extracellular ﬂuid volume has been measured in human muscle to increase

from 12% at rest to 18% during intense exercise (98). Therefore, these results suggest

41

that the increase in T2 during exercise is more than likely related to changes in the
intracellular ﬂuid.

Another well-documented physiological change that occurs with exercise is the
decrease in intracellular pH. However, pH recovers much faster than the T2 increase
after exercise (84). Nonetheless, there is a signiﬁcant increase in metabolic osmolites
during exercise due to the increase in the metabolic rate (i.e. inorganic phosphate and
lactate). Therefore, the accumulation of these metabolites could draw water into the
intercellular compartment and this increase in the intercellular ﬂuid volume could result
in altered T2 relaxation within the active muscle cell. In addition, the accumulation of
metabolites at a given rate of stimulation will be signiﬁcantly less in muscles with a high
aerobic capacity compared to muscles with low aerobic capacity. Recently, experiments
conducted in our laboratory found that the increase in muscle T2 of the human knee
extensor muscles depend on the exercise intensity relative to peak aerobic power and not
absolute power output (92).

In another set of experiments conducted in our laboratory, Prior et al. (91) studied
the T2 response in different muscles of the rat after three treatments that have been
documented to alter the net metabolite accumulation which, in turn, would alter the
intracellular ﬂuid volume. The three treatments are as follows: ischemia, inhibition of
lactic acid production with iodoacetate, and depletion of total creatine by feeding a
creatine analog, B-guanidinopropinate. Prior et al. showed that the increase in T2 during
exercise was signiﬁcantly less in muscles with a high aerobic capacity (triceps surare m.)
compared to muscles with a low aerobic capacity (white gastrocnemius m). Furthermore,

the replacement of phosphocreatine with the less labile analog diminished the increase in

42

T2 during exercise. The inhibition of lactic acid production by idoacetate also decreased
the T2 response during exercise. However, this decrease in the T2 response was not
signiﬁcant across all muscle groups. Prior et al. suggest that inhibiting glycolysis during
exercise caused an increase in the accumulation of phosphomonoesters which, in turn,
increased the osmotic load. This effect would counter balance the reduced osmotic load
caused by inhibiting lactate production.

During ischemic stimulation, Prior et al. reported an increase in the T2 response
with no change in the total muscle volume. Prior et al. interpreted these results as
evidence that the increase in T2 during exercise does not simply depend on the
intracellular volume. These investigators proposed that the increase in T2 during
ischemic stimulation could be the result of ﬂuid shits between intracellular sub-
compartrnents, i.e. the hydration layer surrounding the myoﬁbrillls. In these same
experiments, Prior et al. found an immediate increase in T2 when blood ﬂow was
reestablished.

In summary, the results of this recent study suggest that the T2 increase during
exercise is related to intracellular ﬂuid changes. Speciﬁcally, the accumulation of
metabolic osmolites could cause a shift in intracellular ﬂuid into a sub-compartment
within the muscle cell. This redistribution of the intracellular ﬂuid is hypothesized to be
primarily responsible for the T2 increase during exercise. In addition, the aerobic
capacity of the muscle determines the extent of the T2 increase during exercise and the

recovery of T2 following exercise.

43

Delayed T2 Increase after Eccentric Exercise

During eccentric exercise there is an immediate increase in muscle T2, as
described above, and this increase in muscle T2 retums'to baseline values 1-2 h after the
exercise (61). However, a delayed increase in muscle T2 following eccentric exercise
has also been reported, beginning at about 12 h post exercise (103). This delayed
increase in muscle T2 is not observed following concentric exercise (96).

Fleckenstein and colleagues were the ﬁrst to report on the delayed T2 increase
following eccentric exercise (42). These investigators measured MRI parameters, plasma
CK activity and subjective muscle soreness before, immediately after, and 24, 36, 48, and
72 h (n = 5) post-exercise and at 13, 23, and 48 (n=2) days after exercise. Pain and T2
signal intensity peaked at 24-48 h post-exercise. As expected, the CK release lagged
behind peak pain and T2 signal intensity. Peak CK release was reported at 72 h after the
eccentric exercise. This group reported a signiﬁcant positive correlation between CK
release and T2 during the ﬁrst 72 h post-exercise. They suggest that the underlying
mechanism of the delayed increase in T2 is related to muscle damage that develops with
DMS in the phagocytic stage of Armstrong's model of DMS.

Nurenberg et al. (81) collected muscle biopsy specimens from the soleus m.,
lateral gastrocnemius m., medial gastrocnemius m., tibialis anterior m., and peroneus
longus m. at 48 h following downhill running. These investigators collected muscle
biopsy samples from muscle regions that showed an elevated T2 at 48 h post-exercise.
Nurenberg et al. reported a strong correlation (r = -0.88, p < 0.05) between areas of high
T2 signal intensity and ultrastructural damage. In addition, Nurenberg et al. reported

poor correlation between the following pairs of variables: soreness and mean T2 increase,

soreness in the region of the biopsy and the degree of ultrastructural damage, and peak
soreness and peak CK levels. Furthermore, Nurenberg et al. showed direct histological
evidence correlating the increase in T2 signal intensity and ultrastructural muscle damage

after eccentric exercise.

Prrnrosed Mechanism of tne Delnyed T2

In a partial report of the results to be described in this thesis, Foley et al. (43)
examined the long-term time course of the delayed T2 increase and the effect of a
repeated bout of eccentric exercise on the delayed T2 increase. These researchers
acquired MR images, CK release data, and subjective pain ratings before, immediately
after, and 1, 2, 4, 7, 14, 21, and 56 d after the initial bout of eccentric exercise and at 2, 4,
7, and 14 d after bout 2. The delayed T2 increase peaked at day 7 (>60% increase
compared to rest) after bout 1 and remained elevated at day 56 (>3 5%). After bout 2,
peak T2 (<25% increase) was signiﬁcantly less compared to bout land T2 peaked earlier
after bout 2 (2 — 4 (1), T2 recovered more rapidly after bout 2 but still remained above
baseline values at day 14 (z 5%).

Overall arm swelling peaked at day 7 (z13%) after bout 1 and decreased to below
pre-exercise values at 56 d (z -7.0%). After bout 2, there was less swelling of the upper
arm, approximately a 5% increase, and swelling peaked earlier, at day 2, compared to
bout 1. Peak CK released was reported on day 7 (100-fold increase) and returned to

baseline values by day 14 after bout 1. After bout 2, CK release peaked at day 2 and was

negligible compared to bout 1. Peak pain was reported on day 2 after both bouts of

45

 

eccentric exercise. However, there was signiﬁcantly less pain reported after bout 2
compared to bout 1.

These results are consistent with pervious studies on the repeated bout effect.
However, Foley et al. were the ﬁrst to document the repeated bout effect on the delayed
T2 increase. In addition to overall arm swelling, Foley et al. measured the volume of the
elbow ﬂexor muscles in the imaged region. These results showed a decrease in muscle
volume, approximately 7%, at day 56 after bout 1. Muscle volume remained below pre-
exercise values at day 14 after bout 2. Foley et al. interpreted these results as evidence
supporting the stress-susceptible ﬁber model of the repeated bout effect (see earlier
discussion). If this reported decrease in the elbow ﬂexor muscle compartment is truly the
result to muscle loss, then there should be a decrease in MV C. However, Foley et al. did
not measure isometric strength loss in the eccentrically damaged muscle. On the other
hand, several studies have reported similar muscle loss in human and rat muscles after
eccentric exercise (69, 73).

In addition, these investigators proposed that the persistent elevation in muscle T2
at 56 d after bout 1 and 14 d after bout 2 might reﬂect a more permanent change in the
muscle. Based on the fact that overall upper arm swelling had subsided and elbow ﬂexor
muscle volume had actually decreased, it is very unlikely that residual edema caused the
elevated T2. Therefore, the persistent increase in T2 after a bout of eccentric exercise is
more than likely the result of changes in the intracellular water chemistry and/or

redistribution of the intracellular ﬂuid into sub-compartments within the cell.

46

Conclusions

This review of the DMS literature shows that the MR technique has already
contributed unique observation to the elucidation of the mechanisms underlying DMS.
In particular, the result of recent studies, including pilot work for this dissertation,
support the theory that destruction of stress-susceptible ﬁbers underlines both the
symptoms resulting from an initial bout of eccentric exercise and the reduction of
symptoms induced by subsequent bouts.

The review also reveals two major unanswered questions related to the outcomes
of these recently reported MR studies. First, how does the anatomical muscle volume
loss relate to changes in ﬁrnctional capacity of the muscle? In the subsequent chapter we
propose experiments to answer this question by determining how well muscle strength
loss correlates with muscle volume loss during the extended recovery period after
damaging eccentric exercise. Secondly, this review shows that no studies to date have
examined the separate components of the multiexponential T2 decay in eccentrically
damaged muscle. We propose additional studies employing echo-planar imaging and
NNLS analysis to resolve the short, intermediate, and long components of T2 relaxation F-
in such muscle. This work will allow testing of the hypothesis that the persistent
elevation in muscle T2 long after apparent recovery from eccentric-induced DMS is due

to changes in the intracellular compartment of the repaired and/or newly-synthesized

 

muscle cells.

47

CHAPTER 11]

Effects of Exercise-Induced Damage on T2 Relaxation in Skeletal Muscle

Abstract.

The purposes of this study were, ﬁrst, to compare T2 relaxation computed by the
non-negative least squares algorithm (NNLS) from muscles after two bouts of damaging
eccentric exercise (each 5 sets of 10 at 110% of lRM) separated by 8 wk, and second, to
examine the effect of eccentric exercise-induced muscle damage on the acute T2
response to bouts of milder concentric exercise (each 3 sets of 10 at 20% of 1 RM)
performed before [Corr 1] and at 2 [Con 2] and 21 (1 [Corr 3] after the ﬁrst eccentric
exercise. Conventional T2-weighted axial spin-echo MRI images (TE=30,6O ms) and
echo-planar images (64 TE's from 20 to 272 ms) of the upper arm were obtained before,
and l, 2, 4, 7, 12, 21, and 56 days after eccentric bout 1 (Eco 1), and at 2,4,7, and 15 days
after eccentric bout 2 (Ecc 2). Conventional spin-echo images were also obtained
immediately before and after each concentric exercise bout. The prolonged T2 increase
measured by conventional imaging after Ecc 1 (from 27.6 :I: 0.2 ms before to 46.7 :t 3.8
ms at 2 days, p < 0.05, mean 3: SE, n=6) was correlated with the presence of slow-
relaxing, long T2 components (60-300 ms) in the NNLS T2 spectra. However, T2
remained elevated by 2.5 ms even aﬁer 56 days, when distinct slow-relaxing components
were no longer detectable. The magnitude of slow-relaxing components was less after
Ecc 2 compared to Ecc 1, consistent with the previously-reported protective effect of

prior eccentric exercise. The acute T2 increase after Corr 2 (1.5 i 0.5 ms, mean 3: SE,

n=6) was signiﬁcantly less compared to Corr 1 (3.2 i 0.5 ms) and Corr 3 (5.0 :t 0.4 ms).

48

L010

 

The results conﬁrm that the prolonged T2 increase observed by conventional imaging
methods after eccentric exercise is primarily caused by edema, and Show that this edema
diminishes the acute T2 change measured during exercise using conventional spin-echo

imaging methods.

Introduction.

Exercise causes an increase in the 'H-NMR transverse relaxation time (T2) of
muscle water in active muscle (40, 41). This increase in T2 has been reported during the
ﬁrst few seconds of dynamic exercise (62), and continues to rise exponentially toward a
plateau that depends on exercise intensity. The acute increase in T2 decays relatively
quickly (tl,2 <20 min) once the exercise is discontinued. Adams et al. (2) reported a
strong correlation between mean T2 of exercised muscle and the relative intensity of
muscle recruitment measured using electromyography. Based on these and similar
results, the relationship between muscle T2 and exercise intensity is increasingly
exploited by "functional" muscle MRI (MRI) studies in order to map the recruitment
pattern (3, 86) and the relative intensity of muscle recruitment across different muscle
groups during a variety of exercises (86).

The biophysical mechanism underlying the acute T2 increase during exercise is
not entirely clear. Although muscle T2 relaxation is typically assumed to be a simple
monoexponential process in MRI studies, it is well-known that the T2 relaxation decay
of intact muscle can be decomposed into multiple components (24, 36). Generally, two
major T2 components are resolved in isolated muscles: a large component with

intermediate T2 (20-50 ms) and a typically smaller component with longer T2 (>60 ms).

49

 

 

The intermediate T2 component corresponds to the bulk of the intracellular ﬂuid while
the long component corresponds to the extracellular and/or vascular ﬂuids. Although an
increase in the extracellular water (HZOC) volume during exercise has been well
documented in the exercise literature (98), and is the most commonly cited explanation
for the increase in muscle T2 during exercise (24, 36, 48, 54, 67), more recent studies
Show that the T2 response to pressure-induced edema was different compared to the
exercise-induced T2 increase (25, 87). Furthermore, Prior et al. (91) showed that the
exercise-induced T2 increase in stimulated rat muscle is related to osmotically driven
changes in the distribution of intracellular ﬂuid within the cellular sub-compartments,
and does not simply depend on total tissue ﬂuid volume.

In addition to the acute T2 increase during exercise, MRI studies have reported a
more prolonged, delayed T2 increase in eccentrically damaged muscles. This delayed T2
increase coincides roughly with the onset of delayed muscle soreness (DMS), a syndrome
of pain or discomfort following unaccustomed eccentric exercise (42, 43, 61, 81, 96,
103). Numerous studies have documented the exercise-induced muscle damage
following eccentric contractions by tracking different indices, including serum levels of
muscle enzymes, inﬂammation markers, swelling, and decrements in muscle
performance (7, 12, 17, 21, 22, 44, 53, 64, 76, 78, 80, 85, 99, 101). Several recent studies
have shown that performance of a repeated bout of the same exercise 1-6 wk later
produced more modest changes in the indicators of damage and muscular performance
(10, 17, 21, 22, 35, 64, 76, 79, 80). This protective effect is commonly referred to in the
exercise literature as the "repeated bout effect". Foley et al. (43) reported a persistent

elevation in muscle T2 at 56 d post-eccentric exercise and showed that the repeated bout

50

effect extends to the delayed T2 increase, which is signiﬁcantly reduced following the
second bout of eccentric exercise.

Inasmuch as edema has been reported to occur during DMS, it is plausible that
the delayed T2 increase after eccentric exercise is related to changes in extracellular
and/or vascular ﬂuid compartments in muscle. The appearance of a slow-relaxing long
T2 component in NNLS computed T2 spectra would conﬁrm this increase in
extracellular and/or vascular ﬂuid (87). However, to date, no studies have directly tested
the hypothesis that the delayed T2 increase during DMS is the result of edema, or the
hypothesis that the smaller increase in the delayed T2 after a repeated bout of eccentric
exercise is due to reduced edema development after bout 2. Furthermore, the mechanism
underlying the recently reported persistent elevation in muscle T2 at 56 d post-eccentric
exercise remains unresolved.

The purposes of this study were: 1) to determine if the delayed T2 increase that
develops during DMS is associated with the accumulation of the slow-relaxing T2
components that are characteristic of muscle edema, 2) to test whether these slow
relaxing components persist for up to 8 weeks, and thus explain the elevated muscle T2
previously reported 8 weeks after eccentric exercise, 3) to determine if the protective
effect of a ﬁrst bout of eccentric exercise on the response to a second bout 8 weeks later
is also manifest by decreased magnitude of slow-relaxing T2 components, and 4) to
examine if the increase in muscle T2 caused by damaging eccentric exercise alters the

acute T2 response to a subsequent bout of mild concentric exercise.

51

Methods.
Subjects. Six male subjects (age = 22.3 i 1.5 years, mean i SD, height = 178 i 5

cm, weight = 84 i 5 kg, 1 RM = 20 :l; 3 kg) were recruited from the population at
Michigan State University. Subjects were screened for any medical and/or orthopedic
conditions that might preclude severe leg exercise or MR imaging procedures.
Furthermore, subjects who engaged in heavy lifting as part of their daily activities were
excluded from the study. The study was approved by the University Committee on
Research Involving Human Subjects and each subject gave informed, written consent
before participation.

Exercise Protocol. Each subject was tested for the one repetition maximtun
(lRM) for the standing concentric biceps curl of the right arm using a single weighted
dumbbell. The lRM lift was performed with the right elbow, starting from full extension
and proceeding to full ﬂexion. A trainer demonstrated the lift using proper technique and
subjects were coached to maintain proper form during the lRM testing. During these
tests, the trainer lowered the dumbbell on each repetition so subjects performed no
eccentric exercise. The initial weight load was set at 15% of body weight and was
increased in 2.5 lb. increments on subsequent trials until the subject failed to attain full
ﬂexion of the elbow with proper form (43). There were 90 seconds of rest between trials.
The lRM value was deﬁned as the ﬁnal load with which the subject was able to perform,
unassisted, a complete biceps curl. Approximately 4 to 5 trials were needed to reach the
lRM value. Following the lRM test, subjects were introduced to a muscle soreness scale

(21), wherein they were asked to rate their perceptions of overall soreness in the fully

52

extended and relaxed biceps brachii m. on a scale of 1 (no soreness) to 10 (extremely
sore).

Following a preliminary MRI scan, each subject performed concentric exercise
(Con 1) with the left arm at 20% of the lRM (43). The concentric lift started with the
left elbow fully extended. A trainer placed the weighted dumbbell in the subject's grip;
the subject then slowly ﬂexed the elbow to full ﬂexion to a 3 second count. A trainer
removed the weight at the top of the curl, and the subject returned the unweighted arm to
full elbow extension. Subjects repeated this movement for 3 sets of 10 repetitions at
approximately 1 repetition every 5 seconds. A 90 8 rest period separated each set of 10
repetitions. MR images were acquired immediately (< 2 min) following the completion
of the concentric exercise bout.

Later in the same day (3—4hr after Conl), muscle injury was induced by eccentric
biceps curls (Bee 1) at 110% of the concentric 1 RM (43). The eccentric contraction
began with the left elbow in the fully ﬂexed position. The trainer placed the weighted
dumbbell in the subject's grip; the subject then smoothly extended the elbow to full
extension. Subjects repeated this movement for 5 sets of 10 repetitions at approximately E—
1 repetition every 5 seconds. Subjects were verbally encouraged to follow strict form

(standing erect with feet shoulder-width apart) during each repetition. If the subject was

 

unable to complete the exercise protocol, the trainer provided minimal effort to help the
subject complete the exercise set. Muscle soreness measures and MRI scans were
repeated at the same time of day 2, 4, 7, 14, 21 d after Bee 1. At 2 (1 (Corr 2) and 21 d
(Corr 3) post-eccentric exercise, each subject was imaged before and after performing the

same concentric exercise bout (3 sets of 10 repetitions at 20% of lRM). At 56 d post-Ecc

53

1, each subject was imaged before and after performing the same eccentric exercise
protocol (Ecc 2). Pain ratings and MRI scans were taken 2, 4, 7, and 14 days after Ecc 2.
Subjects were instructed to refrain from taking any anti-inﬂammatory medications or
other therapeutic treatments to relieve pain.
MRI methods. Images were acquired using a linear extremity coil and a 1.5 T
Signa whole body imager (General Electric, Milwaukee, WI). Subjects were positioned
head ﬁrst and prone, with the left arm extended overhead. The coil housing was centered
on a mark inked 1/3 of the distance along a line ﬁ'om the middle of the antecubital fossa
to the acromion process of the scapula. Eight spin echo axial images 10mm thick and
5mm apart were acquired from a 12cm long region centered on the landmark. A 16 x 16
cm ﬁeld of view was used, with 2 echoes (TR/TE = 1500/30, 60 ms), a 256 x 128
acquisition matrix and 1 NEX. Echo-planar images were acquired at 64 TEs in
sequential order from 20—272 ms. The ﬁrst image (TE = 20 ms) was excluded from the
analysis to minimize saturation effects. The pre-exercise and post-exercise relaxation
curves at the different time points were analyzed using the non-negative least squares
(NNLS) algorithm with minimum energy constraints as described previously (87). i—
Mean T2 values were determined for the entire elbow ﬂexor muscle group in each i
of the 8 axial images collected with the Spin-echo pulse sequence. Data from the two
echoes of the fast spin echo imaging sequence were ﬁtted to a monoexponential decay
algorithm using the analysis software package "x-vessel" (40). Speciﬁcally, a region of
interest was deﬁned by manually tracing around the biceps brachii m. and brachialis m.,
excluding subcutaneous fat and skeletal tissues. Once the region of interest was

established, the software automatically calculated both the number of pixels within the

54

region and the mean T2 of those pixels. Volume weighted average T2 was calculated as

follows:

Volume weighted average T2 = 2 (Pixel count * mean T2 in each Slice)
2 (Pixel count in each slice)

In addition, "x-vessel" was used to measure the volumes of the whole imaged arm region
as well as the exercised (elbow ﬂexors) and unexercised (elbow extensors) muscle
compartments by the same manual tracing method. The volume data was calculated by
multiplying the pixel counts within the deﬁned region by the pixel area (ﬁeld of
view/matrix) and then multiplying by the interslice interval (2 cm). All MRI data
analysis was done by the same person, working with unmarked images which were later
decoded.

Statistical Analysis. To test the hypothesis that the mechanism underlying the
delayed T2 increase during DMS is edema, NNLS analysis was applied to the echo-
planar imaging data to separate the components of the multiexponential T2 decay in
eccentrically damaged muscle. The presence of a slow-relaxing long T2 component at
any time point conﬁrmed the contribution of edema to the elevated T2 value at that time.
Pearson product moment correlation coefﬁcients (r) were calculated to assess the F
relationship between the peak slow-relaxing long T2 component and peak increase in I -.

exercise-induced edema (i.e., swelling).

 

To test the hypothesis that the reported lower peak T2 elevation after Ecc 2 is the
result of reduced edema (i.e., repeated bout), paired t-tests were used to test for the
difference in the contribution of the slow-relaxing long T2 component to the overall
mean muscle T2 on the day peak swelling occurred after Ecc 1 versus Ecc 2. Finally, our

hypothesis that the edema-induced elevation in muscle T2 after eccentric exercise will

55

 

not abolish the acute exercise-induced T2 increase was tested using the t-statistic for a
one-tailed test on paired differences. Speciﬁcally, paired t-tests were used to test the
acute T2 increase, relative to the pre-exercise value, after a bout of light concentric
exercise performed before, 2 d and 21 d after eccentric exercise. Signiﬁcance levels for
all tests were set at p < 0.05. Data are reported as mean i SE, n = 6. A sample size of
six was determined to be required in order to detect a 10% difference in the contribution
of slow relaxing long T2 component to the overall T2 signal after Ecc2 versus Bee 1 for a
paired t-test with alpha set at 0.05, beta set at 0.20, and an estimated standard deviation
of 5%. To detect an acute T2 increase of > 3ms after exercise using a paired t-test, a
sample size of ﬁve was required for alpha set at 0.05 , beta set at 0.20 and an estimated

standard deviation of lms.

Results.

The image series in Figure 1 shows the progression of swelling and T2 in the
elbow ﬂexor muscles of one subject during the 8 wk period after Bee 1. At the start of
the experiment, NNLS analysis of the pre-exercise echo-planar data showed that the T2
relaxation is characterized by a single component of < 60 ms in all subjects. In contrast
to pre-exercise, there were components with T2 above 60 ms in the NNLS spectra in ﬁve
out of the six subjects at 2, 4, 7, 14, and 21 d after Ecc 1 (Figure 2A). The T2 spectrum
computed by NNLS at 56 d post-Ecc 1 showed that the T2 relaxation had reverted back
to a single component model in all subjects. On the other hand, after Bee 2, components
with T2 above 60 ms in the NNLS spectra were only observed on days 2 and 4 post-

exercise in all subjects (Figure ZB). The shape and peak T2 of these components varied

56

 

widely between different subjects. The magnitude plotted at 1000ms is the background
parameter, and is equal to the mean magnitude of the noise measured in a large region of

interest outside the arm.

Extensors

\

 

Figure 1. Representative mid-brachial axial plane MR images ﬁ'om one subject before
(top left image; pre-exercise) and at each test interval during the 8 wk after Ecc 1. Images
are oriented such that the anterior direction is to the right and the lateral direction is
upward.

57

250

 

 

Pre-Exercise
....... Day 2
—— Day 7
—-- Day 56

Eccl

150 -

100 -

Signal Intensity

 

 

 

 

10 100 1000

160

 

 

140 « Dayz

120 a
100 -l
80 e
60 ~

Signal Intensity

40.
20~

 

 

 

 

10 100 1000

Figure 2. NNLS spectra of one subject before and in the days following Ecc 1 (A) and
Bee 2 (B). The T2 relaxation is characterized by a single component at pre-exercise (A).
Note on days 2 and 7 after Ecc 1, the NNLS spectra is more complex, with variable
components at much higher T2. In comparison, the NNLS spectrum in the days
following Ecc 2 shows that the fraction of the overall T2 attributable to the slow relaxing
component is less on days 2 and 4.

Subjects reported peak pain on day 2 after both bouts of eccentric exercise.

However, subjective pain ratings were lower after Ecc 2 (5.6 i 0.5, soreness rating on a

58

1-10 scale) compared to Ecc 1 (8.0 :l: 0.4). Figure 3 shows the change in muscle volume
(Figure 3A) and the contribution of the long component to the overall T2 (Figure 3B) in
the days following Ecc 1 and Ecc 2. Muscle swelling peaked on day 2 (39 i 11%) and
remained swollen by nearly 23 i 6% on day 7 after Ecc 1 (Figure 3A). During the
second week after Bee 1, muscle volume decreased to a value < 90% of the baseline
volume (217 i 7 cm3 at 14 (1 vs. 245 i 10 cm3 preexercise; p < 0.05). This decrease in
muscle volume was maintained during the period from 3 to 8 wk after Ecc l (227 i 6 cm3
at 56 d). In contrast, peak swelling was only about one-third as much after Bee 2 (14 i
3%) on day 2 vs. Ecc 1. During recovery from Bee 2, the muscle volume reverted to the
reduced size that had been maintained at 2 to 8 wk after Ecc l.

The delayed T2 increase peaked on day 7 after Ecc 1 (46.7 i 3.8 ms) and
remained above pre-exercise values on day 56 (29.9 i 0.3 ms at 56 (1 vs. 27.6 i 0.2 ms
pre-exercise; p < 0.05). Peak delayed T2 increase after Ecc 2 was less and occurred
earlier compared to Ecc 1 (37.6 :1: 3.9 ms at 2 d, p < 0.05). The peak percent of the
overall T2 attributed to the long component (> 60 ms) after Ecc 1 occurred several days :2
after the peak swelling, 32 :l: 8% at 7 d (Figure 3B). The correlation between peak
muscle swelling on day 2 after Bee 1 and % total signal intensity attributed to the long I

component on the same day was not signiﬁcant (r = 0.741, p = 0.092). However,

 

Pearson's correlation test revealed a signiﬁcant strong correlation between peak % total
signal intensity attributed to the long component on day 7 and muscle swelling (r =
0.895, p < 0.05) on the same day. Peak % of the total signal intensity attributed to the
long component occurred earlier and was signiﬁcantly less after Ecc 2, (13 i 6% at 2 d, p

< 0.05). The correlation between muscle swelling and % total signal intensity attributed

59

to the long component was signiﬁcant on day 2 (r = 0.884, p < 0.05) and 4 (r = 0.977, p <

0.05) after Ecc 2.

>

 

% Change in muscle volume

 

 

 

 

 

 

 

 

B
E
a 50
\D
2 40 _ IfEccl Ecc2
I—
E t I
"3' 3o - #
3
E 20'i #e
O
I" #
°\ l0 1 I"
O
O
0 r 1 r r v {’1 r r 1
0 2 4 7 14 21 56=0 2 4 7 14

Days Post-exercise

Figure 3. Time courses of knee extensor muscle volume and percent of the total signal
intensity attributable to components with T2 above 60 ms in 6 subjects for 8 wk after
eccentric exercise bout 1 (Ecc 1) and 2 wk after eccentric exercise bout 2 (Ecc 2) in 5
subjects. Ecc 2 was performed after the scan done 56 d after Bee 1. All values are given
as percent change from baseline or pre-exercise values. Values are means 1 SEM.

# Signiﬁcantly different from pre-exercise or baseline, p < 0.05 ( Ecc 1, pre-exercise =
day 0; Ecc 2, pre-exercise = day 56).

* Signiﬁcantly different from Bee 1, p < 0.05.

A transient or acute T2 increase was observed in MR images taken immediately

after Corr 1, Corr 2, and Corr 3 (Table 1). An acute AT2 of 3.2 i 0.5 ms (change in T2

60

 

from pre-exercise) was observed immediately after Corr 1, the pre-injury condition. The
acute AT2 after Con 2 (1.5 i 0.5 ms, p < 0.05; 2 d after Eccl) was signiﬁcantly reduced
compared to C011 1. The acute AT2 after Corr 3, performed 21 d after Ecc 2 (5.0 i 0.4

ms), was somewhat higher compared to Corr 1 and signiﬁcantly higher compared to Con

2.

Table 1. Acute T2 increase after concentric exercise.

 

 

 

 

Pre-exercise T2 (ms) Post-exercise T2 (ms) Acute ATZ (ms)
Corr 1 27.8 i 0.1 31.0 i 0.5* 3.2 i 0.5
Corr 2 43.7 i 3.3 45.2 i 2.9“ 1.5 :t 0.5#
Con 3 34.0 r: 1.4 39.0 i 1.6* 5.0 i 0.4

 

 

 

 

 

 

Values are means 1: SE; n = 6 subjects.
* Signiﬁcantly different from pre-exercise, p < 0.05.
# Signiﬁcantly different from Corr 1 and Corr 3, p < 0.05.

Discussion.

The main results of this study are that the increase in muscle T2 during DMS is
primarily due to the eccentric exercise-induced edema, and that the persistent elevation in
T2 long after the apparent recovery from eccentric-induced DMS is not due to residual
edema. It is well known that eccentric exercise causes structural damage to the muscles
and it is this damage to muscles that results in the sensation of pain or soreness and
swelling that gradually develops in the ensuing days (7, 12, 17, 21, 22, 44, 53, 64, 77, 80,

85, 99, 101). In our study, the T2 spectra computed by NNLS at 2, 4, 7, l4, and 21 d

61

Ill

after Eccl showed components with T2 above 60 ms. This result is consistent with the
hypothesis that the T2 increase observed during DMS is caused by the exercise induced
edema (42, 43, 61, 81, 96, 103). Furthermore, the T2 spectra computed by NNLS at 56 d
post-Ecc 1 showed that the T2 relaxation had reverted back to a single component in all
subjects. Accordingly, this result is consistent with the hypothesis that the persistent
elevation in T2 long after recovery from eccentric exercise induced muscle damage may
be related to more permanent changes in the intracellular water chemistry and/or
redistribution of the intracellular ﬂuid into sub-compartments within the muscle cells (43,
91).

Peak swelling occurred on day 2 after Bee 1 and peak percent of the overall T2
signal attributed to the slow relaxing long T2 component occurred on day 7 after Ecc 1
(Figure 3). Consequently, the correlation between muscle swelling and the contribution
of the slow-relaxing long T2 component to the overall T2 on day 2 after Ecc l was poor.
However, on day 7 there was a. strong signiﬁcant correlation between muscle swelling
and the contribution of the long component to the overall T2 signal intensity. Note that
although peak swelling occurred on day 2, there was still signiﬁcant swelling on days 4
and 7 after Ecc l. The late peak in the slow relaxing long T2 component could be due to
the delayed gross heterogeneity of the extracellular ﬂuid (i.e., accumulation of muscle
proteins and circulating polymorphs) following eccentric exercise induced muscle
damage.

AS previously reported, there was a 7-10% loss in muscle volume on days 14 and
21 after Bee 1. Others have also reported similar loss in muscle mass in the days

following eccentric exercise (63, 70). The NNLS computed T2 spectra on days 14 and

62

 

21 after Ecc I consistently resolved a slow relaxing long T2 component. Consequently,
the MRI—determined muscle volume may have under-estimated the true loss in muscle
volume because of the residual edema present on days 14 and 21 after Ecc l. The T2
spectra computed by NNLS on day 56 after Ecc 1 and 14 d after Ecc2 showed that the T2
relaxation had reverted back to a single component model. However, one cannot
completely rule out the presence of residual edema at these later time points purely based
on the absence of the slow relaxing long T2 component. For example, although an
increase in extracellular water (H206) volume during exercise has been well-documented
in the exercise literature (98), Ploutz-Snyder et al. failed to resolve a slow relaxing long
T2 component in the tibialis anterior muscle after exercise (87). This result suggests that
small changes in the extracellular and/or vascular ﬂuid compartments (i.e., < 8-10%
increase in the extracellular ﬂuid volume) do not result in the appearance of long T2
components. However, there is no evidence, to date, that would suggest that the
eccentric exercise induced edema might be present at 8 wk post-exercise. Therefore, the
absence of the slow relaxing long T2 combined with the fact that this volume loss was
observed on two separate occasions, further increases the credibility of the MRI-
determined muscle volume loss observed in this study.

The time course of changes in the magnitude of the slow relaxing long T2
component did not lag behind the changes in muscle swelling after Ecc 2, as they did
after Ecc 1 (Figure 3). On the average, the magnitude of the slow relaxing long T2
component and muscle swelling was less after Ecc 2 compared to Ecc l. The peak
increase in swelling and the slow relaxing long T2 component occurred earlier after Bee

2, on day 2, and there was a strong signiﬁcant correlation between the two variables.

63

These results are consistent with the hypothesis that the repeated bout effect extends to
the delayed T2 increase, recently proposed by Foley and colleagues in 1999 (43). The
results from NNLS computed T2 spectra in the present study represent the ﬁrst
quantitative evidence that the smaller increase in the delayed T2 after the second bout of
eccentric exercise is the result of more modest changes in the extracellular and/or
vascular ﬂuid compartments in the eccentrically damaged muscle.

Furthermore, the observed muscle volume loss is also consistent with the
existence of a small population of "susceptible" or mechanically weak ﬁbers within a
muscle, a hypothesis originally proposed by Armstrong and colleagues (8). These
investigators reported necrosis in approximately 5% (of ﬁbers in muscles of rats after
down hill running. Other groups have also reported similar decreases in muscle mass in
humans after eccentric exercise (63, 70). Since the MRI-determined muscle volume data
was taken from only a portion of the entire muscle, it is possible that this decrease in
muscle volume was due to a partial destruction of ﬁbers and/or decrease in muscle
diameter rather than a decrease in the number of muscle ﬁbers.

Despite the exercise induced edema, we observed a signiﬁcant increase in the
acute T2 immediately following Corr 2, performed at 2 d after Ecc 1. This result is
consistent with the hypothesis that the exercise induced acute T2 increase is mainly
related to the osmotically driven changes in the distribution of intracellular ﬂuid within

the cellular sub-compartments (91). However, the acute AT2 after Corr 2 (1.5 i 0.5 ms)

was lower compared to Corr 1 (3.2 :l: 0.5 ms) and Con 3 (5.0 i 0.4 ms). Since the
subjects performed the same concentric exercise during Corr 1, Corr 2, and Corr 3, the

results of the current study suggests that in the presence of exercise induced edema (i.e.,

> ~20% of the total signal intensity attributable to the slow-relaxing long T2 component),
T2 calculated assuming a monoexponential decay from spin-echo images does not fully
reﬂect the degree of muscle activation during exercise.

Although the somewhat larger increase in acute AT2 aﬁer Corr 3, compared to
Con l, was not signiﬁcant, it is possible that this increase in acute AT2 might be due to
increased activation of the uninjured muscle ﬁbers in an attempt to counter the loss in
force production from the damaged and/or destroyed ﬁbers (i.e., decreased muscle
volume). However, in the absence of echo-planar data after Corr 3, this conclusion could
be criticized on the grounds that the residual edema might have contributed to the
somewhat larger increase in acute AT2 calculated assuming a monoexponential decay
from spin-echo images acquired immediately after Corr 3 rather than increased muscle
activation.

In summary, the main result of this study is that the delayed T2 observed in
eccentrically damaged muscles during DMS is mostly due to edema. On the other hand,
the persistent elevation in eccentrically damaged muscles long after recovery from
eccentric exercise is not due to residual edema. Therefore, the long-term elevation in
mascle T2 may reﬂect a more permanent change in the intracellular water chemistry
and/or redistribution of the intracellular ﬂuid into the sub-compartments within the
eccentrically exercised muscle. NNLS computed T2 spectra revealed that the smaller
increase in muscle T2 after Ecc 2 compared to Bee 1 is the result of more modest changes
in the extracellular and/or vascular ﬂuid compartments in the eccentrically damaged
muscle. This result is consistent with the hypothesis that the repeated bout effect extends

to the delayed T2 increase (43). Lastly, the eccentric exercise-induced edema diminishes

65

the acute T2 change measured during exercise using conventional spin-echo imaging
methods; therefore, this parameter should not be used an index of muscle activity in such

cases.

66

CHAPTER IV

MRI Evaluation of Heat and Stretch as Treatments for

Muscle Damage After Eccentric Exercise.

Abstract

The initial aim of this study was to monitor the effects of topical heat and/or static
stretch treatments on the recovery of muscle damage by eccentric exercise. For this
purpose, 32 untrained male subjects performed intense eccentric knee extension exercise,
followed by two weeks of treatment (heat, stretch, heat plus stretch) or no treatment
(control, n =8/ group). Isometric strength testing, pain ratings, and multi-echo magnetic
resonance imaging of the thigh were performed before and at 2, 3, 4, 8, and 15 (1
following the exercise. Increased T2 relaxation time, muscle swelling, pain ratings, and
strength loss conﬁrmed signiﬁcant muscle damage during the post-exercise period. Pain
ratings and muscle volume recovered to baseline by 15 (1, although muscle strength
remained lower (77 i 4 vs. 95 i 3 kg pre-exercise, p < 0.05, mean i SE) and T2 values
higher (32.2 i 0.8 vs. 28.6 i 0.2 ms pre-exercise).

Treatment modality showed no effect on extent of recovery in any measured
parameter, allowing pooling of data for a follow-up study in which a subset of nine
subjects returned for testing 6 wk post exercise. Results showed a persistent elevation in
T2 (31.0 :1: 1.3 at 6 wk vs. 29.2 i 0.3 ms pre-exercise) and a small but signiﬁcant muscle
volume loss (1340 i 180 at 6 wk vs. 1540 i 90 cm’ pre-exercise), conﬁrming a previous

report on arm muscle. These changes were accompanied by a persistence of muscle

67

 

weakness (17 :l: 4% below baseline strength). These long term changes are consistent
with the hypothesis that intense eccentric exercise partially or totally destroys a small
subpopulation of mechanically weak ﬁbers. The data also indicate that heat and/or static

stretching treatments are not effective in preventing or reversing this damage.

Introduction.

Delayed muscle soreness (DMS) is the sensation of pain and/or discomfort
experienced 24 - 48 hours after unaccustomed exercise (6-8, 21, 45, 58, 77). DMS
occurs most commonly in individuals who engage in sporadic, strenuous exercise or
abruptly increase the intensity of the training. Research has shown that eccentric or
lengthening contractions cause structural damage to the muscles, and it is this damage to
muscles that results in the sensation of soreness in the days following eccentric exercise
(6, 8, 9, 12, 46, 47, 77). Numerous studies have documented the exercise-induced
muscle damage following lengthening contractions by tracking different indices,
including serum levels of muscle enzymes, markers of inﬂammation, swelling, and
decrements in muscle performance (7, 12, 17, 21, 22, 44, 53, 64, 76, 78, 80, 85, 99, 101).

In the lay exercise community, post-exercise stretching is often recommended as

 

a preventive measure against the development of DMS. However, there is little
experimental evidence to support these claims. Early studies by deVries and Abraham
reported that static or stationary stretching after eccentric exercise reduced muscle
soreness (l, 28). Recently, Buroker and Schwane found that post-exercise static

stretching did not alleviate the signs and/or symptoms of DMS (15).

68

Sports medicine practitioners often use superﬁcial heat and cold to treat
musculoskeletal injury. Immediate cold application is recommended during the acute
stage of injury to decrease bleeding, capillary effusion, pain, and inﬂammation (75). On
the other hand, heat application is recommended during the healing process to increase
vascular and lymphatic ﬂow. Heat application also improves the range of motion
through increased collagen extensibility. Like therapeutic cold application, heat also
reduces pain through sensory stimulation of the skin.

Most studies have found that cold application after eccentric exercise provided
temporary pain relief (60, 106). Kuligowski et a1. (66) recently reported that cold
whirlpool and contrast therapy (alternating cycles of hot and cold), were more effective
than warm whirlpool and no treatment in alleviating the signs and/or symptoms of DMS.
In addition, Prentice showed that cold plus static stretching was superior to heat followed
by static stretching in reducing muscle pain and increasing muscle relaxation (88). The
limited available evidence suggests that heat application does not alleviate the signs
and/or symptoms of DMS.

To date, no studies have tested directly the effectiveness of therapeutic heat
application in alleviating the signs and/or symptoms of DMS. There are two main ﬂaws
in the experimental design of Kuligowski et al. and Prentice's study. First, the
application of heat immediately following the eccentric exercise has the potential to
promote the injury process by increasing capillary effusion and swelling. Second, the
application of heat (40°C) for 20-30 min might not be long enough to promote the
clearance of extracellular ﬂuid by the lymphatic circulatory system, which is much

slower than blood ﬂow. In addition, these studies did not include a measure of muscle

69

 

damage during the treatment period. Swelling was measured using limb circumference
measures, which is not sensitive to small changes in the extracellular ﬂuid volume.
Therefore, it was not possible to separate the temporary analgesic effect from the anti-
inﬂammatory effect of the different treatments on muscle damage.

Recent advances in the ﬁeld of magnetic resonance imaging (MRI) have allowed
investigators to measure in vivo the extent of muscle damage and changes in muscle
compartment cross-sectional area during DMS (42, 43, 61, 81, 97 , 103). In addition to the
acute, temporary T2 increase which is observed in muscles even after non-damaging
exercise (e.g. Prior et a1.(91)), damaging eccentric exercise results in the appearance of a
delayed and much longer lasting T2 increase in muscle (42, 43, 61, 81, 96, 103). This
delayed T2 increase is correlated with the appearance of very slow relaxing T2
components in high resolution T2 relaxation spectra of the muscles (ref. Expt. 1), and
therefore is likely due to the accumulation of slow relaxing extracellular ﬂuid in the
damaged regions. Thus, the damaged regions of the muscles appear brighter in T2-
weighted MR images compared to undamaged or normal muscles. Several MRI studies

have exploited this increase in muscle T2 to document the time course of muscle damage

after eccentric exercise and the subsequent recovery in days following (42, 43, 61, 97,

103).

_ n- ”1&1“. J.

Recently, Foley et al. (43) reported a persistent elevation in T2 up to 8 wk after an

 

eccentric exercise bout. In addition, these investigators reported a 7-10% muscle volume
loss at 2 to 8 wk after the damaging eccentric exercise. To date no MR studies have been
published to conﬁrm the enduring muscle volume loss reported by Foley et al. (43).

Furthermore, if this reported decrease is truly the result of muscle loss, then a parallel

70

decrease in maximal isometric strength would be expected. However, Foley et al. did not
measure isometric strength loss in the eccentrically damaged muscle, an omission that
will be addressed in the current study.

The purposes of this study were: (1) to examine the effects of long term heat
application and/or static stretch on the recovery of muscle damage as indicated by the
delayed T2 response, recovery of isometric strength, dissipation of pain, and swelling, (2)
to test whether the volume loss reported by Foley et al. in the elbow ﬂexor muscle group
can be repeated in a different subject group using a different muscle group (knee extensor
muscle group) and exercise, and (3) if muscle volume loss occurs in our current study, to

test whether this is reﬂected by strength loss.

Methods.

Subjects. 32 non-weight trained male subjects were recruited from the university
community. Subjects were screened for any medical and/or orthopedic conditions that
might preclude severe leg exercise or MR imaging procedures. Furthermore, subjects
who engaged in strenuous leg exercise as part of their daily activity were excluded from i F
the study. The University Committee for Research Involving Human Subjects approved
procedures for the study, and all subjects gave informed written consent before

participation.

 

Subjects were randomly assigned to one of four groups. The three treatment
groups were heat (n=8), static stretching (n=8), and heat plus static stretching (n=8). The
fourth group (control, n=8) received no treatment. A randomly selected subset of twelve

subjects was asked to return at 6 wk after eccentric exercise for additional MR scans and

71

single-leg isometric maximal voluntary contraction force (MVC) testing. Out of these
twelve subjects, three were unable to return for additional testing, giving an n of nine for
the 6 wk tests.

Exercise Protocol. Following a preliminary MRI scan, subjects performed three
MVC trials with the quadriceps femoris muscle group in the seated position (hip at z 90°
and the knee at z 135°) using a standard cable tensiometer. Subjects were given 3 min
rest between each contraction and were verbally encouraged to give maximal effort on
each trial. Isometric strength was determined by averaging the MVC force from the three
trials. Upon completion of the MVC testing, there was a short rest period during which a
trainer demonstrated the single-leg eccentric knee extension exercise. The starting load
for the eccentric exercise was set at 100% of the MVC force. A trainer lifted the weight
to the starting position in which the knee was fully extended. The weight was lowered
smoothly to a 48 count to the ﬁnish position (z 60° knee angle). Subjects performed
eccentric knee extensions with maximal load in each set to failure (unable to lower the
weight in a controlled manner), with 3 min of rest between sets. On average, subjects
performed 6-8 sets of 5-10 repetitions. If a subject was unable to perform a minimum of
5 repetitions, the load was lowered by 5 kg so that he could complete the set. This
reduced load was used for the next set. The eccentric exercise continued until the
eccentric exercise load was below 50% of the MVC force.

Subjects were asked to rate perception of overall soreness in the quadriceps
femoris muscle by drawing a line on a linear pain scale of 1 (no soreness) to 100

(extremely sore)(33). The subjects were seated with the hip at z 90° and the test leg bent

72

"I

 

at z 90° before rating perception of pain. Pain ratings and MVC force measurements
were repeated at the same time of day 2, 3, 4, 6, 8, 15 d and after eccentric exercise.

MRI methods. Subjects were positioned feet ﬁrst and supine in a 1.5 T Signa
whole body imager (General Electric, Milwaukee, WI). The body coil was centered on a
mark inked 1/3 of the distance along a line from the top of the patella to the anterior iliac
crest. Twelve to fourteen T2-weighted Spin-echo axial MR images (TR/TE =
1500/30,60; 256x192 acquisition matrix; 10 mm thick; 10 mm gap; 32 cm FOV) of the
thigh region were acquired. The MR images were acquired at rest prior to MVC testing
and again at 2, 3, 4, 6, 8, 15 d after eccentric exercise. Images, pain ratings and MV C
measurements were also acquired in a subset of nine subjects 6 weeks after eccentric
exercise.

Mean T2 values were determined for the quadriceps muscle group from the ﬁrst
axial image below the scrotum and continuing through to the last image not showing the
quadriceps tendon. This criterion was used in order to measure the changes in the belly
of quadriceps muscle group across all subjects with varying heights. T2 images were
calculated from the images acquired at the two echo times assuming a monoexponential E
decay (40). A region of interest in each image was deﬁned by manually tracing around I
the entire quadriceps muscle group, excluding subcutaneous fat and skeletal tissues,

using the software package Xvessel (43). Once the region of interest was established, the

 

software automatically calculates the number of pixels within the region and the mean T2
according to the equation:

Volume weighted average T2 = 2 (Pixel count * menn T2 in ench slice)
2‘. (Pixel count in each slice)

73

In addition, "x-vessel" was used to measure the volume of quadriceps muscle
compartment by the same manual tracing method. Muscle volume was estimated by
multiplying the pixel counts within the deﬁned region by the pixel area (ﬁeld of
view/matrix) and then by the slice thickness plus interslice interval (2 cm). All MRI data
analysis was done by the same person, who was blinded to subject identity and treatment
group.

Treatment Protocol. At 24 h post-eccentric exercise, pain ratings, MRI scans, and
MVC force measurements were recorded in order to measure muscle status prior to
beginning any treatment. Subjects were then separated according to group assignments
and were given a brief training session on their speciﬁc treatment. In addition, subjects
were required to ﬁll out a daily log verifying treatment start and stop times. The subjects
were asked to refrain from discussing the treatment assignment with other subjects and
also with the investigators.

The heat treatment was applied using a heating pad (GAYMAR, Orchard Park,
NY) set at 41°C for 2 h starting at 36 h post-eccentric exercise to avoid the detrimental
effects of heat during the acute injury stage. The temperature and duration were chosen
to maximize the increase in vascular and lymphatic ﬂow that has been suggested to occur
with heat application, and to minimize any discomfort that might occur with prolonged

local hypertherrnia (75). The heating units were calibrated and the temperature was set at

 

41°C prior to distribution to the subjects. The heating pad (18"x24") was placed around
the leg to cover the belly of the quadriceps muscle group. The subjects were instructed to

apply heat for 2 hours every day at the same time of day until the soreness completely

subsided.

74

The static stretching treatment began at 36 hours post-eccentric exercise.
Stretches for the major muscle groups of the thigh were selected from two popular texts
(4, 5). A criterion for selection was that the stretches were easy and safe for an untrained
individual. Subjects warmed up the exercised leg by performing alternating leg lifts
while lying supine, 15 times without stopping. Two static stretches were performed for
each muscle group of the thigh: standing quadriceps stretch, prone quadriceps stretch,
standing hamstring stretch, and seated hamstring stretch. In addition, subjects performed
two additional static stretches: standing calf stretch and wall squat stretch. Subjects were
instructed to hold each static stretch for a 20 s count and each static stretch was
performed 3 times with a 30 5 rest period. Subjects were instructed to perform the
stretching program at the same time of the day until soreness subsided.

Subjects in the heat plus static stretching group were also instructed on the proper
use of the heating apparatus and the proper technique of the different stretches. Like the
other groups, heat plus stretching treatment began at 36 h post-eccentric exercise. The
subjects were instructed to ﬁrst apply the heat, which was then followed by the stretching
program.

All the subjects were instructed to refrain from taking any anti-inﬂammatory
medications and using other forms of therapeutic treatments (i.e. ice or message), other
than their assigned treatment.

Statistical Analysis. Data were analyzed using a repeated measures two way
analysis of variance (ANOVA) [heat (df =1) x stretch (df =1)] to assess the difference
between treatments in muscle soreness, muscle T2 relaxation time, MVC, and muscle

volume at 2, 3, 4, 6, 8, and 15 (1 following eccentric exercise. This analysis tests main

75

effects for treatment and time, and the interaction terms for heat x stretch and treatment-
by-time. If there appeared to be a difference in baseline values for a speciﬁc parameter, a
repeated measures two way analysis of covariance (AN COVA) was performed on the
time points after exercise, with the baseline preexercise values as a covariate to correct
for this difference. This type of statistical analysis allowed us to test the treatment effect
and the interaction between the two treatments in promoting recovery of the damaged
muscles.

Our hypothesis that the volume of the exercised muscle compartment will be
reduced after recovering from damaging eccentric exercise was tested using a two-tailed
paired t-test on the values for the control group for day 15 versus pre-exercise. If a
volume loss was detected, Pearson's product moment correlation coefﬁcient (r) was
calculated to assess the relationship between muscle volume loss and the decrease in
MVC in the control group at day 15. Initially, analysis of just the control group was
planned to test the previously reported persistent elevation in muscle T2 and muscle
volume loss, independent of treatment. However, our ﬁnding of no treatment effect
allowed pooling of the data and use of a repeated measures one-way ANOVA to assess
the difference across time on the data from the subset of subjects who returned for

additional testing at 42 days after the eccentric exercise. Signiﬁcance levels for all tests

7

 

were set at P < 0.05.

Results.
Figure 4 shows a representative series of MRI images of the thigh muscles of one

subject during the 6 wk following eccentric exercise. These images provided both

76

muscle volume and T2 relaxation time data (lighter contrast corresponds to longer T2
values). Analysis of pre-exercise images showed no signiﬁcant differences in muscle
volume or muscle T2 between subject groups (Table 2). Likewise, no between-group

differences were noted on any other recorded parameters prior to exercise.

77

Knee F. xtcnsors

Pre-exercise

 

Figure 4. Representative mid-thigh axial plane MR images from one subject before (top
left image; pre—exercise) and at each test interval during the 6 wk after the eccentric
exercise bout. Images are oriented such that the anterior direction is to the right and the
medial direction is upward.

78

Table 2. Subject characteristics before exercise.

 

 

 

 

 

 

Muscle Knee
Group Age Height Weight T2 extensor MVC Pain
(yr) (cm) (kg) (m8) Volume (kg) (0 -
(cm’) 100mm)
Control 20.8 i 179 i 79 i 28.7 i 1500 i 89.3 i 0.75 :t
0.8 1 5 0.3 90 6.9 0.2
Heat 20.5 i 179 i 85 i 28.7 i 1700 i 96.1 i 1.7 i
0.8 2 7 0.4 90 4.4 0.6
Stretch 20.8 i 180 i 83 i 28.5 i 1660 i: 97.3 i 4.9 i
0.4 3 4 0.4 90 4.5 1.8
Heat + 22.3 d: 178 i 80 i 28.4 :t 1450 :I: 98.1 :t 1.9 1:
Stretch 1.1 1.4 4 0.3 60 6.5 0.9
Pooled 21.1 i 179 i 82 i 28.6 :1: 1580 :1: 95.2 i 1.8 i
Mean 0.4 6 2 0.2 40 2.8 0.5

 

 

 

 

 

 

 

 

 

 

Values are means i SE.

Muscle T2 progressively increased in all groups in the days following eccentric
exercise and remained signiﬁcantly elevated at 15 (1 post eccentric exercise (Figure 5). A
signiﬁcant main effect for time was found in the ANOVA (p < 0.001), but the main
effects for heat (p = 0.829) and stretch (p = 0.911) were not signiﬁcant. Neither the heat-
by-stretch interaction (p = 0.745) nor the treatment-by-time interaction (p = 0.143) was

signiﬁcant.

79

 

 

40‘

20*

 

 

 

 

 

 

 

Peak % change from
baseline value
c

 

 

 

 

 

 

-20 ‘ :3 Control

Stretch
-40 . m Heat

{J Heat Plus Stretch
-60 . . . .
T2 MVC Muscle Volume Pain
B

30 -
20 -

10-

 

 

-10 _,

% change ﬁ'om baseline
value at 15 d
O

 

 

-20 -

 

 

 

 

-30 _

 

l l l I

T2 MV C Muscle Volume Pain

 

Figure 5. Peak percent change from baseline value (A) and percent change from baseline
value at 15 d post-eccentric exercise (B) in T2, MV C, muscle volume, and pain across
the different treatment groups.

80

Knee extensor muscle volume increased in the days following eccentric exercise
in all groups and returned to baseline values on day 15 (Figure 5). The main effect of
time (p < 0.001) and the heat-by-stretch interaction (p < 0.05) were found to be
signiﬁcant, but the main effect for treatment (heat, p = 0.605, stretch, p = 0.979) and the
treatment-by-time interaction (p = 0.888) were not signiﬁcant. Since the treatment-by-
time interaction was not signiﬁcant, we concluded that there was only a small heat-by-
stretch interaction across time.

In the days following eccentric exercise there was a signiﬁcant decrease in MVC
in all groups (Figure 5). A signiﬁcant main effect for time was found in the ANOVA (p
< 0.001), but the main effects for heat (p = 0.561) and stretch (p = 0.872) were not
signiﬁcant. Neither the heat-by-stretch interaction (p = 0.278) nor the treatment-by-time
interaction (p = 0.153) was signiﬁcant. MVC remained below baseline values on day 15
in all groups (p < 0.05).

All subjects reported an increase in pain in the days following eccentric exercise
(Figure 5). There was a signiﬁcant main effect of time (p < 0.001), but the main effect for
treatment (heat, p = 0.501, stretch, p = 0.712), treatment interaction (p = 0.719) and the
treatment-by-time interaction (p = 0.346) were not signiﬁcant. Pain ratings returned to
baseline values on day 6 in all groups.

A randomly selected subset of the subjects (n = 9) returned for additional testing
42 d after the eccentric exercise. Since neither main effect of treatment nor treatment-by-
time interaction was found in the repeated measures two way AN OVA, the data from this
subset of subjects was analyzed using a repeated measures one-way AN OVA to test for

signiﬁcance across time in the dependent variables.

81

Figure 6 shows the time course of the change in T2 in these nine subjects as a
percent change from pre-exercise or baseline. T2 relaxation times were signiﬁcantly
elevated compared to baseline at each time point after the eccentric exercise (p < 0.05).

Peak increase in muscle T2 occurred on day 8 (40.9 i 1.8 ms, mean i SE) and remained

signiﬁcantly elevated from baseline on day 42 (31.0 i 0.4 ms vs. 29.2 i 0.3, p < 0.05).

 

50
45 -
4o -
35 -

 

 

30‘

 

25- y
20-

% Increase in T2

154
10-
SJ

 

 

 

O IIj I I I r

0 234 6 8 15 42

Days Postexercise

Figure 6. Time course of muscle T2 relaxation times for 6 wk after eccentric exercise (n
= 9). All values are given as percent change from baseline muscle T2 measured before
the eccentric exercise bout. "' Signiﬁcantly different from baseline values, P < 0.05;

values are means :1: SE.

82

 

20 -

15"

 

 

'20 III I I I I
O 234 6 8 15 42

Days Postexercise

 

Figure 7. Time course of muscle volume changes for 6 wk after eccentric exercise (n =
9). All values are given as percent change from baseline muscle volume measured before
the eccentric exercise bout. * Signiﬁcantly different from baseline values, P < 0.05;
values are means 1 SE.

A signiﬁcant increase in the muscle volume was observed on days 2 through 8 (p
< 0.05) and peak swelling occurred on day 3 (17.2 i 3.5%) following eccentric exercise
(Figure 7). By day 15, muscle volume had not only returned to baseline, but also showed

a trend towards a slight volume loss (3 i 1%, p = 0.057). Muscle volume continued to

decrease in the weeks after eccentric exercise to a value approximately 10% below

83

baseline volume (1536 j: 89 cm3 at 42 days vs. 1339 i 60 cm3 baseline; p < 0.05). Both
T2 and muscle volume showed a rapid rate of recovery towards baseline values between

days 8 and 15, followed by a more gradual rate of recovery between days 15 and 42.

 

%MVCGiange
8 8

*-

*

 

 

 

40-

30-

20-

10-

0 HT I 1 I u
0 234 6 8 15 42

mysPostexa'cise

Figure 8. Time course of MVC changes for 6 wk after eccentric exercise (n = 9). All
values are given as percent change from baseline MVC measured before the eccentric
exercise bout. * Signiﬁcantly different from baseline values, P < 0.05; values are means
:1: SE.

MVC values were signiﬁcantly lower compared to baseline at each time point

after the eccentric exercise (p < 0.05). Peak decrease in MVC (39 :l: 4.4% loss in

isometric strength) was observed on day 2 (Figure 8). Despite the rapid recovery in

84

isometric strength from day 3 to 8, there was still a signiﬁcant deﬁcit in strength on days
15 and 42 (-37.4 i 5.0% at 15 days and 17 i 4.0% at 42 days, p < 0.05). Pearson's
correlation test revealed a signiﬁcant moderately high correlation between mean baseline
muscle volume and MVC (r = 0.719, p < 0.05). The correlation between muscle volume
and MVC on day 42 was not signiﬁcant (r = .511, p = .160). On the other hand, there
was a signiﬁcant moderate correlation between the change in muscle volume and the

change in MVC from baseline on day 42 (r = .683, p < 0.05).

 

70

60-

50‘

40‘

Pain Rating

30-

20-

10—

 

 

 

 

0.
023468 15 42

Days Postexercise

Figure 6. Time course of the pain ratings for 6 wk after eccentric exercise (n = 9).
Subjects rated their perception of pain by drawing a line on a linear pain scale of 1 (no
soreness) to 100 (extremely sore) in the seated position. "' Signiﬁcantly different from

baseline values, P < 0.05; values are means i SE.

85

On average, subjects reported peak pain on day 2 following eccentric exercise
with recovery by day 6 (Figure 6). The time courses of changes in the indexes of muscle
damage varied widely, with pain and isometric strength loss peaking earliest at 2 days
postexercise, followed by muscle swelling on day 3 and ﬁnally by T2 which peaked on

day 8.

Discussion.

Treatment eﬂect. The main result of this study is that therapeutic application of
superﬁcial heat and/or static stretching after intense eccentric exercise did not promote
the recovery ﬁ‘om muscle damage as indicated by the similar time course and magnitude
of the T2 changes after the eccentric exercise. Additionally, swelling, pain, and the
deﬁcit in strength on day 15 were not different between the treatment groups. It is well
known that eccentric contractions cause structural damage to the muscles, and it is this
damage to muscles that ultimately results in the sensation of soreness and the decrements
in muscular performance in the days following eccentric exercise. (6, 8, 9, 46, 47, 77).
The effectiveness of the eccentric exercise protocol in producing muscle damage is
evident from the large increase in T2 and muscle swelling, and from the decrease in
MV C in the ﬁrst few days after the exercise (Tables 1 through 4). Previous studies have
reported similar results in the days following eccentric exercise (21, 42, 43, 78, 97).

Although the exact mechanism of DMS is unknown at present, DMS ultimately
arises from a sequence of events that occur after the eccentric exercise. Armstrong's
model (7) divides the muscle injury process in DMS into four distinct stages: 1) initial, 2)

autogenetic, 3) phagocytic and 4) regenerative. Therapeutic application of heat and/or

86

static stretching could potentially affect the latter two stages of the model. Application of
topical heat increases local vascular and lymphatic ﬂow, which, in turn, may enhance the
removal and repair aspects of the healing process (75). Possible mechanisms by which
static stretching post-eccentric exercise could alleviate the sensation of pain are as
follows: 1) disperse the excess ﬂuid that accumulates from muscle damage (14), 2)
reduce muscle spasms that may occur during DMS (28, 56), and 3) alter the response of
the afferent group IV nerve ﬁbers, which conduct pain impulses to central nervous
system (7). Accordingly, heat combined with static stretching should be effective in
reducing the sensation of pain as well as enhancing the healing process.

In our study, the peak increase in T2 and the persistent elevation in T2 at day 15
were similar across the different groups, which suggests that heat and/or static stretching
did not enhance the healing process. As expected, there was signiﬁcant swelling in the
exercised muscle compartment during the ﬁrst week after the eccentric exercise (21, 22,
43, 58, 78, 80). Heat and/or static stretching did not reduce the extent of swelling in the
exercised muscle compartment, nor did any treatment expedite the recovery from edema.

Accompanying the increase in T2 and muscle swelling, there was a signiﬁcant
decrease in MVC in the days following eccentric exercise in all groups. The decrease in
MVC and subjective pain ratings were not different between the treatment groups across
time. The decrease in MVC on days 2 and 3 may be due at least in part to the reluctance
of subjects to voluntarily produce maximal force in sore muscles. However, soreness
cannot explain the long term decrements in strength, because strength was still reduced

when soreness was no longer apparent.

87

Our study results are consistent with other reports that have shown that post-
exercise static stretching did not alleviate the signs and/or symptoms of DMS (15, 74,
105). On the other hand, deVries (29) and Abraham (28) reported a signiﬁcant chronic
reduction in soreness with repeated stretching after eccentric exercise. However, a
comparison of deVries ﬁndings with ours may not be valid because of the wide variety of
muscle disorders among his subjects.

A more appropriate comparison can be drawn between our results and the
ﬁndings of Buroker and Schwane. Speciﬁcally, Buroker and Schwane found that post-
exercise static stretching did not alleviate the signs and/or symptoms of DMS as
indicated by the creatine kinase (CK) release (an indirect marker of muscle damage),
muscle strength and limb girth (an indirect measure of muscle swelling) (15). The lack
of a difference in the T2 response from our study conﬁrms the interpretation from
Buroker and Schwane's study that the cellular processes of muscle recovery were not
affected by the static stretching regimen.

Therapists often use superﬁcial heating modalities to increase tissue extensibility
to allow for increased efﬁcacy of stretching techniques (75). Furthermore, this
decreased tension, along with increased clearance of metabolites could hypothetically aid
recovery in the later stages of DMS (phagocytic and regenerative stages of Armstrong's
model of DMS), during which the muscles are stiff and painful, but not acutely inﬂamed.
Our results show that neither heat alone nor heat plus static stretching reduced the
sensation of pain. These results are consistent with previous reports that have shown that
heat by itself and heat combined with static stretching were not effective in alleviating

the subjective symptoms of DMS (51, 66, 88).

88

The MRI technique employed in the present study provides the ﬁrst objective
evidence that heat and/or static stretching does not enhance the recovery of damaged
muscle ﬁbers after eccentric exercise. Despite the widespread belief that heat and/or
static stretching alleviates DMS, our results indicate that heat and/or static stretching
does not consistently reduce soreness, swelling or muscle damage. The practical
implication of these results is that clinicians should be aware that prescribing heat and/or
static stretching following intense eccentric exercise will not enhance the recovery of
damaged muscles.

Long term time courses of muscle damage. In the subset of subjects who returned
for additional testing, T2 increased signiﬁcantly in the days following eccentric exercise
and peaked on day 8, (40.9 i 1.8 ms, Figure 6). Although there was a gradual decline in
T2 over time, T2 remained elevated by ~6% on day 42 in the present study. Based on the
fact that muscle volume had decreased, it is unlikely that residual edema caused the
elevated T2 on day 42. Therefore, the persistent increase in T2 after a bout of eccentric
exercise is more than likely the result of more permanent changes in the intracellular
water chemistry and/or redistribution of the intracellular ﬂuid into sub-compartments
within the cell.

Foley and co—workers reported a much larger mean peak T2 value of 46.7 i 3.8
ms on day 7 in the elbow ﬂexor muscles, which suggests that the exercise induced
muscle damage was more severe in their study, despite similar intensity of the eccentric
exercise protocols between the two studies. Unlike the arm muscles, the leg muscles are
more likely to perform lengthening contractions during daily activities (i.e., walking

downhill or stairs). Therefore, it is possible that subjects in the current study may have

89

been somewhat "pre-adapted" which, in turn, could make them less susceptible to the
eccentric exercise induced muscle damage (21).

During the ﬁrst week after eccentric exercise, we observed signiﬁcant swelling in
the exercised muscle compartment (Figures 4, 7). These results are consistent with
previous reports in which the investigators tracked swelling by measuring the change in
the circumference of the exercised limb (21, 58, 93). In particular, Foley et al. recently
reported a similar time course of changes in the MR measurements of the elbow ﬂexor
muscle group after eccentric exercise (43). Foley et al. reported a ~40% increase in the
exercised muscle compartment on day 2 following eccentric exercise. We observed peak
swelling on day 3 but this increase (19.0 i 3.2%) was not nearly as high as the previous
report. This is consistent with the smaller peak T2 increase noted above, again
suggesting some degree of pre-adaptation in the leg muscles examined in the current
study.

During the second week after the eccentric exercise, swelling subsided and a
trend towards a small decrease in muscle volume was observed on day 15 (2.7 i 1.2%)
(Figure 7). Muscle volume continued to decrease in the weeks after the exercise to a
value <90% of baseline volume on day 42 (Figure 7). Other groups have also reported a
decrease in muscle mass after eccentric exercise in humans (63, 70). Recently, Foley et
al. assessed muscle volume in MR images after eccentric exercise and found a 7-10%
decrease in the elbow ﬂexor muscle compartment 2 to 8 wk post-exercise (43). The
MRI-determined muscle volume loss found in our study conﬁrms the volume loss
previously reported by Foley and co-workers. Since the MRI-determined muscle volume

data was taken from only a portion of the entire muscle, it is possible that this decrease in

90

muscle volume was due to a partial destruction of ﬁbers and/or decrease in muscle
diameter rather than a decrease in the number of muscle ﬁbers.

A unique aspect of this study was that we measured both MVC force and muscle
volume in the days following eccentric exercise (Figure 8). Pearson's product moment
correlation test revealed a signiﬁcant moderate correlation between mean muscle volume
and MVC (r = 0.719, p < 0.05) before the start of the eccentric exercise. Others have
reported similar correlations between uninjured muscle mass/volume and MV C (34, 71).
Recently, Bamman et al. reported that the correlation between MRI-determined muscle
volume of the uninjured triceps surae muscle and MV C (r = 0.649) was better than whole
limb antrhopometric (r = 0.584) and dual-energy x-ray absoprtiometry (DEXA) (r =
0.381) estimates of muscle size (11).

In the present study, the correlation between mean muscle volume and MVC 42
days after the eccentric exercise was not signiﬁcant, mostly due to the large variance in
these measures. In an attempt to reduce the variance in these measures, we calculated
delta (A) scores for muscle volume and MVC (i.e., the change in muscle volume and
MVC from baseline values) which, in turn, resulted in a signiﬁcant correlation between A
muscle volume and A MVC on day 42 (r = 0.683, p < 0.05). To date no studies have
examined the relationship between MVC and MRI-determined volume of eccentrically
damaged muscle. This persistent deﬁcit in MVC weeks after the eccentric exercise is
mostly likely the functional consequence of the chronic loss in muscle volume observed
in this study.

The persistent decrease in muscle volume on day 42 is consistent with the "stress

susceptible" ﬁber theory proposed by Armstrong and co-workers (8). Armstrong et al.

91

suggest that a sub-population of ﬁbers (~5%) nearing the end of their functional life in
healthy muscles are more susceptible to the increased active strain and/or stress produced
during lengthening contractions. This increase in active stress/strain is the primary cause
of the irreversible ultrastructural damage to the pool of stress-susceptible ﬁbers, which
leads to degeneration of these cells in the days following the exercise. Accordingly,
there is prolonged decrease in muscle strength and mass, and a signiﬁcant increase in
muscle soreness and serum levels of muscle proteins after eccentric exercise. We
interpret the persistent loss in muscle volume and the decrease in MV C observed in this
study weeks after eccentric exercise as evidence in favor of the existence of a
"susceptible" or "vulnerable" subpopulation of muscle ﬁbers and that these ﬁbers may
fail to repair and subsequently undergo degeneration.

In summary, we interpret the lack of a treatment effect across time as evidence
that heat and/or static stretching did not enhance the cellular processes involved in the
phagocytic and regeneration stages of DMS. Despite the widespread belief in the lay
exercise community that heat and/or stretching post-eccentric exercise alleviates DMS,
our results indicate that these modalities do not consistently reduce soreness. The 11%
loss in MRI-determined muscle volume in the weeks after the eccentric exercise conﬁrms
the previously reported results in arm muscle by Foley and co-workers (43). This
volume loss combined with the persistent deﬁcit in MVC may be related to the partial or
total destruction of a small subpopulation of mechanically weak ﬁbers in days following

intense eccentric exercise.

92

CHAPTER V

SUMMARY AND FUTURE STUDIES

The ﬁrst set of experiments in this study demonstrated that the delayed T2
increase in eccentrically damaged muscles during DMS is primarily due to edema in the
early post-exercise period, as indicated by the presence of the slow relaxing long T2
component and muscle swelling. However, the T2 parameter remains elevated long after
recovery from edema. This persistent elevation in muscle T2 may thus reﬂect a more
permanent change in the intracellular water chemistry and/or redistribution of the
intracellular ﬂuid into the sub-compartments within the eccentrically exercised muscle.

The application of the echo planar MRI method in the current study also
demonstrated that the attenuation of the acute T2 increase after a second eccentric bout is
the result of more modest changes in the extracellular and/or vascular ﬂuid compartments
in the previously damaged muscle. This result conﬁrms and extends a previous report
that the repeated bout effect extends to the delayed T2 increase (43).

Although several investigators have suggested that the increase in T2 could be
used as a method for estimating the relative intensity of muscle recruitment (2, 3, 40, 86,
87), the current study shows that this is not true under all circumstances. In the current
study, the acute AT2 after repeated bouts of the same concentric exercise performed
before and 2 and 21 d after eccentric exercise was different. This result suggests that the
acute T2 change calculated from standard spin-echo images in the presence of eccentric

exercise-induced edema might not accurately reﬂect the relative intensity of muscle

93

recruitment during exercise. This ﬁnding also supports a recommendation that caution
be used in applying the T2-based method of estimating muscle recruitment in other
populations with damaged or abnormal muscle tissue.

The initial aim of the second set of experiments was to monitor the effects of
topical heat and/or static stretch treatments on the recovery of muscle damage from
intense eccentric exercise. Increased T2 relaxation time, muscle swelling, pain ratings,
and strength loss conﬁrmed signiﬁcant muscle damage during the post-exercise period.
Pain ratings and muscle volume recovered to baseline by 15 (1, although muscle strength
remained lower and T2 values higher. Treatment modality showed no effect on extent of
recovery in any measured parameter, allowing pooling of data for a follow-up study in
which a subset of nine subjects returned for testing 6 wk post exercise. Results showed a
persistent elevation in T2 and a small but signiﬁcant muscle volume loss, conﬁrming a
previous report on arm muscle. These changes were accompanied by a persistence of
muscle weakness. These long term changes are consistent with the hypothesis that
intense eccentric exercise partially or totally destroys a small subpopulation of
mechanically weak ﬁbers.

In general, the current study shows that MRI provides investigators a new
technique to study, in vivo, the time course of the development and recovery of muscle
damage and swelling during DMS. Furthermore, MRI promises to be a valuable tool in
clinical rehabilitation research in evaluating the efﬁcacy of different treatment modalities
in promoting the recovery of damaged muscles. 3

Future Studies. The following are offered as potential experiments that could

build on the ﬁndings of our current study applying MRI to the study of muscle damage.

94

A review of the MRI literature shows that all of the MR studies documenting
muscle damage following eccentric exercise have used healthy, young subjects. To date,
no in vivo measurements of muscle damage have been reported following eccentric
exercise in the elderly population. This may be due to the fact that previously used
methods of measuring damage are not very sensitive, requiring the induction of
signiﬁcant eccentric damage to produce a reliably measurable change. The sensitivity of
the MR T2 measurement may allow studies of more moderate eccentric injury, suitable
to application in an elderly population.

Faulkner et al. (3 9) suggest that age-related development of muscle atrophy,
weakness, and loss of power in the elderly are due to intrinsic age-related changes in the
muscles that render them mechanically weak. Furthermore, these investigators suggest
that the repeated exposure to lengthening contractions from daily activities could cause
selective atrophy and/or degeneration of the large fast twitch ﬁbers in the old, thus
explaining the age-related decrease in muscle mass and power. However, no studies to
date have examined the time course of the exercise-induced muscle damage and swelling
during DMS at the start of a strength training program in the elderly population.
Furthermore, no studies have examined the long term effects of strength training on
muscle volume in the elderly.

We propose to use MRI to document the muscle damage and swelling, and
measure isometric strength loss after a bout of light eccentric knee extension exercise in
older subjects. Based on the ﬁndings of our current study and the available evidence on
the effects of aging on skeletal muscle, we expect that the muscle volume loss, swelling,

and strength loss will be greater in the older subjects compared to the young. Moreover,

95

due to the decrease in the ability of muscle ﬁbers to regenerate in older hosts, we expect
to observe deﬁcits in strength and muscle volume for a longer period of time following
eccentric exercise in the older individuals. The practical implication of this study is that
it will provide insight into the design of strength training programs for the elderly that
minimize the negative effects of eccentric contractions while maximizing the positive,
hypertrophic effects of eccentric contractions, by manipulating the rest period between
training sessions and the intensity of the eccentric or lengthening contractions.

In another set of experiments, we propose to follow up on our ﬁnding that the
acute AT2 response to identical bouts of concentric exercise is altered by the presence of
edema or stress-induced adaptations in the muscle. In the current study, the T2 change
was calculated using the standard method of ﬁtting a monoexponential decay to data
from several spin-echo images. Our echo-planar results on damaged muscles at rest have
shown that this assumption of monoexponential T2 decay does not apply in acutely
damaged muscle. However, in the current study we did not collect echo-planar images in
the concentrically exercised muscles. Repeating this experiment using the echo planar
method should clarify the source of the apparent differences in the acute T2 response in
normal, damaged, and recovered muscle.

Our hypothesis is that echo-planar analysis will show that the T2 decay in
edematous muscle follows a double rather than single exponential time course. If this
hypothesis holds up, then application of a double exponential model to standard spin
echo data should result in a T2 estimate that more accurately reﬂects the extent of muscle

activity during the exercise.

96

10.

11.

12.

13.

14.

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