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THESIS

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This is to certify that the

thesis entitled

THE USE OF FUNCTIONAL MAGNETIC RESONANCE IMAGING
IN THE STUDY OF DELAYED MUSCLE SORENESS

presented by

Roop Jayaraman

has been accepted towards fulfillment
of the requirements for

MS eein Physical
Edhcation ane Exercise Science

 

   
 

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1“ WM“

THE USE OF FUNCTIONAL MAGNETIC RESONANCE IMAGING IN THE
STUDY OF DELAYED MUSCLE SORENESS

by

Roop Jayaraman

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Physical Education and Exercise Science

1 997

ABSTRACT
THE USE OF FUNCTIONAL MAGNETIC RESONANCE IMAGING IN THE
STUDY OF DELAYED MUSCLE SORENESS
by

Roop Jayaraman

The purposes of this study were (1) to examine the acute T2 response to a bout
of concentric exercise performed before and after eccentric exercise had been
used to induce a delayed T2 increase and (2) to document the early time course
of the delayed T2 development. Eight non-weight trained subjects were imaged
before and after each exercise session, concentric #1, eccentric, and concentric
#2 at 24 h post eccentric. In addition, subjects were imaged at 1, 2, 4, and 6
hours following eccentric exercise. The acute AT2 following the first concentric
exercise was found not significantly different from the second bout. The delayed
T2 increase was observed as early as 4 and 6 h post eccentric exercise. The
findings of this study suggest that the underlying mechanism of the acute and

delayed T2 increase is the same and that the acute T2 has an upper limit.

DEDICATION

To my grandparents, Govindammal and Govindarajan, for a lifetime of

unconditional love, support and discipline.

ACKNOWLEDEMENTS

I wish to express my deep appreciation to Dr. Jeanne Foley, my advisor,
for her guidance and advice. Her thoughtful discussions on teaching, research,
and life in general have made the last three years a true learning experience.
Her understanding, patience, and willingness to help me improve as a
researcher and as an educator will not be forgotten. I would also like to thank
the members of my committee, for their timely and thoughtful suggestions: Dr.
James Pivamik and Dr. Eugene Brown. I would also like to express my deepest
gratitude to Dr. Ron Meyer for opening his laboratory to me and donating
valuable time. My profound thanks to Dr. Roger Haut for financially supporting
me early in my graduate studies and for your contribution to my learning
experience as a graduate student.

Finally, I would also like to thank my family, mom, dad and VIjay ( my best
friend) for their support and kind words of encouragement, and my friends,

especially Augustine Hernandez, for all those late night shoot arounds.

IV

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES .

LIST OF ABBREVATIONS.
Chapter

I. THE PROBLEM .

Need for the Study .
Purpose of the Study
Research Hypotheses
Research Plan
Significance of the Problem

ll. REVIEW OF RELATED LITERATURE

Tme Course of the Pathological Changes
Associated with DMS .
Morphological Changes Associated with DMS
Metabolic Changes . .
Mechanical Aspects of DMS . .
Force- Velocity Relationship In Eccentric Exercise .
Tension Imbalance Theories
Biochemical Aspects of DMS .
Muscle Damage Inferred from Serum Creatine Kinase Activity
Role of Calcium In DMS . . .
Chemically Induced Muscle Damage .
The Role of Calcium In Exercise Induced Muscle Damage.
A Comparative Analysis of the ‘Tom Tissue” Theory and Calcium
Induced Cell Damage Theory . .
Acute Inflammation as the Underlying Mechanism of DMS
Swelling . . .
Pain.

Page
VIII

000001

10
12

13
17
18
19
20

23
25
26
3O
32

37
38
40
42

Chapter Page

Cellular Infiltrates in DMS as well as Acute Inflammation . 43
The Effect of Anti-inflammatory Drugs 0n DMS. . 46
Effects of Aspirin on DMS after Performing Elbow Extensions 47
Effects of Diclofenac on DMS after Downhill Running . 49
Effects of Ibuprofen on DMS after Downhill Running. . 51
Effects of Flurbiprofen on DMS after Performing Eccentric
Exercise on a Motor-Driven Bicycle . 55

Effects of Therapeutic and Prophylactic Doses of Ibuprofen
on DMS after Performing a Negative Bench Stepping Exercise 56

Summary of the Effects of Anti-inflammatory Drugs on DMS 6O
Analgesic Effect Versus Anti-inflammatory Effect . 61
Use of Functional MRI to Study Muscle Use and Muscle Trauma
with Resistance Exercise . . . 63

Fundamental Principles of Magnetic Resonance Imaging . 64

Exercise-induced Acute T2 Increase: Qualitative Index of Muscle

Recruitment . 67

Exercise-induced Acute T2 Increase: Concentric vs. Eccentric

Exercise . 69

Proposed Mechanism of Exercise-induced Acute T2 Increase 73

Delayed T2 Increase: An Index of Muscle Damage. . 74

Exercise-induced Delayed T2 Increase: Concentric vs. Eccentric

Exercise . 76

Time Course of the Changes In T2 Relaxation “lime and Muscle

Cross-sectional Area Following Eccentric Exercise . . 79

Proposed Mechanism of the Delayed Increase in T2

Signal Intensity . . . . . . . 81

III. RESEARCH METHODS . . . . . . 85
Subjects . . . . 88
Exercise Protocol and Timing of MRI Scans . . . 88
MR Imaging Parameters . . . . . . 91
Images Analysis . . . . . . . 92
Statistical Analysis . . . . . . . 94

IV. RESULTS AND DISCUSSION . . . . . 95
Subject Characteristics . . . . . 95
Acute and Delayed T2 Responses . . . . 96

VI

Chapter

Muscle Cross-sectional Area
Muscle Soreness
Discussion

V. CONCLUSIONS, SIGNIFICANCE, AND RECOMMENDATIONS

Significance . .
Recommendations .
Summary

APPENDICES
Appendix A: Application for participation in exercise-induced
delayed muscle soreness research project

Appendix B: Informed consent
Appendix C: Muscle soreness questionnaire

LIST OF REFERENCES

VII

Page

102
103
106

113
115

116
120

121
122
123

124

LIST OF TABLES

Table I Page
1. Summary of the changes in muscle performance following

eccentric exercise . . . . . . . 16
2. Subject characteristics . . . . . _ . . 96
3. T2 data . . . . . . . . . 97

VIII

LIST OF FIGURES

Figure Page

1. Hypothesized changes in calculated T2 following concentric
exercise (Con1, A), elevated T2 at 24 h post-eccentric exercise
(Ecc, B), and concentric exercise at 24 h post-eccentric exercise

(Con2, C or D) . . . . . . . 9
2. Isometric strength loss after performing eccentric exercise 14
3. The classic force-velocity relationship curve. . . 20
4. Additive vs. non-additive T2 changes. . . . 87

5. The change in the acute and delayed T2 following exercise 98
6. Percent change in T2 from baseline (PreCon1) . . 99

7. Change from baseline (PreCon1) in T2 following eccentric
exercise . . . . . . . . 100

8. Percent change in acute and delayed T2 in each slice along
the arm . . . . . . . . 102

9. Mean muscle soreness ratings (1-no soreness to
10—extremely sore) at 24 h intervals following eccentric
exercise . . . . . . . . 104

10. Mean muscle soreness ratings (1-n0 soreness t0
10-extremely sore) vs. T2 at 24 h post eccentric exercise. 105

11. Mean muscle soreness ratings (1-n0 soreness t0
10-extennely sore) vs. T2 at 24 h post eccentric exercise. 105

IX

LIST OF ABBREVATIONS

. Con1 - the overall change in T2 from “normal” resting value immediately
following concentric exercise bout #1.

. Con2 - overall change in T2 from “normal” resting value immediately following
concentric exercise bout #2.

. Ecc24h - overall change in T2 from “normal” resting value at 24 hours post
eccentric exercise.

. PreCon1 - resting T2 value in “normal” muscle.
. PreCon2 - the elevated T2 value at 24 hours post eccentric exercise.

. PreEcc - the T2 value in muscle at the end of the 2 hour rest period following
a bout of eccentric exercise.

. PostCon1 - the acute elevation in T2 immediately following the concentric
exercise bout #1.

. PostCon2 - the acute elevation in T2 immediately following the concentric
exercise bout #2.

. PostEcc - the acute elevation in T2 immediately following the eccentric
exercise bout.

CHAPTER I

THE PROBLEM

Delayed muscle soreness (DMS) is the sensation of discomfort and/or
pain in skeletal muscles following unaccustomed muscular exercise. Needy
every adult has experienced DMS, most on numerous occasions.
Unaccustomed physical activity of even moderate intensity or duration will result
in the sensation of DMS, regardless of the individual’s general fitness level. The
most predictable method of inducing delayed muscle soreness is to subject
muscles to repetitive, strenuous eccentric or lengthening contractions (5.6.7.20).
Although, concentric or shortening contractions require more metabolic energy,
they generally induce little or no soreness. The high mechanical forces
produced during eccentric contractions have been shown to cause ultrastructural
muscle damage which develops gradually and has been implicated with the
sensation of DMS.

Muscles adapt to an initial bout of eccentric exercise, becoming resistant
to damage from subsequent bouts. Muscle soreness is often localized in the
distal portion of the muscle at the muscle/tendon junction and is accompanied by
reduced mobility (6.7.38). The sore muscles become extremely sensitive to

palpation and movement. Severity varies from slight stiffness to severe pain

2
which inhibits movement. Soreness is accompanied by swelling, decreased

muscular performance. and a reduced range of motion at the joint. Since muscle
soreness and swelling are only temporary, most DMS sufferers do not seek
medical attention. Studies using animal models suggest that injured muscles are
able to recuperate completely within 2 weeks (6,7).

A review of the literature to date will show that there is no firm consensus
on the pathophysiologiml mechanisms responsible for DMS. At present, there is
universal agreement that the muscle damage is initiated by the high mechanical
forces produced during muscular exercise, particularly in eccentric exercise
(6,20,36,60). Although the time course of the pathological changes associated
with DMS is well established. it remains to be determined how the sequence and
timing of DMS relate to a possible pathological mechanism. Thus far three
mechanisms have been proposed: (a) “torn tissue” theory. (b) calcium-induced
cell damage theory. and (c) acute inflammation theory (6).

Neither the ‘torn tissue” theory nor the calcium-induced cell damage
theory is able to fully explain the relation between tissue injury initiated during the
eccentric exercise bout and the soreness first experienced approximately 8 h
after exercise that reaches a peak at 24-48 h post-exercise (20). In an attempt
to better explain the unusual delay, several investigators have proposed that the
mechanism underlying DMS is acute inflammation, based on the similar time
course of DMS and inflammation (74). However, the time course data on the
accumulation of inflammatory markers and DMS do not correlate well. ruling out

acute inflammation as the sole mechanism of DMS.

3
The controversy over the mechanism(s) of DMS remains unresolved

partly due to the fact that two parameters most often used to measure or
document DMS. muscle performance and perceived muscle soreness. do not
provide a measure of intracellular activity. A promising path to resolving this
controversy is to accurately measure changes in intramuscular pressure, muscle
compartment cross-sectional area, muscle recruitment, and muscle damage
following eccentric exercise. Advances in the field of magnetic resonance
imaging (MRI) have allowed investigators to measure muscle recruitment,
muscle damage. and changes in muscle compartment cross-sectional area
following eccentric exercise.

MRI has quickly become the method of choice for medical imaging in
many orthopedic studies of soft tissue and skeletal muscle pathology. MRI
exploits the changes in the tissue’s hydrogen ion chemical environment (1 H-
NMR) that occur during normal activity. An NMR signal is the net magnetization
of all the hydrogen nuclei in a given sample when it is placed in a strong static
magnetic field. MRI is based on three different and independent fundamental
NMR parameters: spin density. T1 relaxation time, and T2 relaxation time (12).
A detailed explanation of each parameter can be found in Chapter II. Generally.
tissues and fluids with more free water have longer T2 relaxation times and
appear as brighter areas on T2-weighted images (3).

In 1988. Fleckenstein, Canby, Parkey, and Peshock published the first
report documenting increased signal intensity in T2-weighted spin echo images

of exercised muscles immediately following exercise (32). Soon thereafter,

4
Fisher. Meyer, Adams, Foley, and Potchen demonstrated that the extent of the

acute contrast change in T2-weighted images of exercised muscles is dependent
on the average force generated by the muscles during exercise (31). This acute
increase in T2 signal intensity decays at a relatively rapid rate following exercise
(tug < 20 min). More recent studies have shown that the acute increase in T2
signal intensity is a good qualitative index of muscle recruitment (2,49).

In addition, MRI is also able to document the changes associated with
exercise-induced muscle trauma that develop more gradually with DMS
(34.62.71 ,77,81). The change is seen as a delayed increase in the T2 signal
intensity that begins to be visible approximately 12 h post-exercise and peaks
24-48 h post-exercise, paralleling the development of muscle soreness and
damage associated with DMS. The strong correlation (r = 0.99) between T2
signal and ultrastructural damage was reported by Nurenberg et al. in 1992.
Their results suggest that the extent of the delayed T2 increase can be utilized
as an index of muscle damage.

Although there is universal agreement that the acute T2 change is an
index of muscle recruitment and delayed T2 elevation is an index of muscle
trauma, the mechanism of the contrast change remains unresolved. What is
known is that exercise-induced changes in MR images are related to complex
intracellular events and/or alterations in intracellular water concentration or
chemistry. Therefore, at present, acute and delayed T2 increases can only be

utilized as qualitative measures of muscle recruitment and trauma.

5
Need for the Study

The increase in T2 signal intensity following eccentric exercise has been
reported to follow a bimodal pattern (81 ). Immediately following exercise there is
a drastic increase in T2 signal intensity which returns to pre-exercise levels by
approximately 30-35 min post-exercise (31.49). Approximately 12 h post-
exercise, T2 signal intensity begins to increase again and reaches peak values
24-48 h following eccentric exercise.

Fleckenstein et al. proposed that the acute increase in T2 following
exercise is due to increased water movement into the extracellular muscle
compartment and that the acute increase in T2 reaches some upper limit with
increasing exercise (32). Fisher and coworkers found that the acute increase in
T2 signal following exercise increased as a function of exercise intensity (31). In
an attempt to examine the changes in extracellular volume. these investigators
used venous occlusion to increase extracellular volume. Fisher et al. reported
similar increases in muscle volume following low intensity exercise and venous
occlusion at rest (31). However. exercise during venous occlusion did not cause
a large increase in T2 signal. This led to the conclusion that exercise-induced
changes in MR images are probably related to alterations in intracellular water
concentration and/or chemistry (31.32.49). A later study using a new rapid-
scanning MR method called echo-planner imaging confirmed this result (84).

On the other hand, the second increase or delayed increase in T2 was
only observed in subjects experiencing DMS following eccentric exercise.

Shellock. F ukunaga. Mink, and Edgerton suggested that the delayed T2 increase

6
is the result of damaged muscle fibers (70). Direct evidence of this relationship

was reported by Nurenberg et al.. who reported a strong correlation between the
T2 signal intensity and ultrastructural damage (62). This study clearly showed
that the delayed T2 increase can be utilized as an index of muscle damage.

Based on the combination of these two studies, Fleckenstein et al.
proposed that the underlying mechanism of delayed T2 increase is edema (free
water) which occurs in DMS. The increase in free water in skeletal muscle
during DMS is likely due to the following factors: (a) damaged connective tissue,
(b) increased permeability of the capillaries, and (c) increased permeability of the
sarcolemma. As a result. the chemical environment of the water in damaged
muscle fibers will be different than in undamaged fibers. However, Shellock and
colleagues published another report in which delayed T2 signal intensity
remained elevated up to 80 days following exercised-induced muscle damage,
well after edema had subsided (71). Therefore, the delayed T2 increase
following eccentric exercise is not solely the result of edema.

This led several investigators to speculate that the acute and delayed T2
changes might result from different mechanisms. A review of MRI literature
showed that no experiments have been reported which specifically tested this
hypothesis. In an attempt to address the mechanism of the T2 change. Sorichter
et al. in 1994. examined the effects of concentric contractions prior to and during
DMS in the quadriceps muscle (77). Subjects in the eccentric group performed a
single bout of seven heavy eccentric contraction and subjects in the

eccentric/concentric group performed additional concentric contractions before

7
and 2 h after eccentric exercise. and 1. 2. 3, 6. and 9 days after the initial

eccentric exercise. One leg was randomly assigned as the control limb
(unloaded leg) and the contralateral leg was used to perform the exercise
(loaded leg). Several inflammatory markers (e.g. circulating leukocyte and
neutrophil counts) and serum creatine kinase (CK) levels were measured before
eccentric exercise, 2 h after exercise. and 1. 2. 3, 6, and 9 days after exercise.
MR images were acquired from the loaded and unloaded legs of both groups at
3. 6. and 9 days after exercise.

Across all time points. CK levels were significantly higher in the
eccentric/concentric group than in the eccentric group, suggesting more damage
in the skeletal muscle. T2 relaxation times and inflammatory markers were not
significantly different between the two groups. These results suggest that the
elevated T2 signal might limit any additional increase in T2 signal during activity
of the damaged muscle. consistent with early findings of F leckenstein and
coworkers.

Investigators analyzed the calculated mean T2 relaxation times at
different time points using the student’s t-test to compare the two groups. A
more appropriate method would have been to look at the change in T2 relaxation
times (AT2). using a t-test on the difference scores which would have limited the
influence of subject heterogeneity. To measure the change in T2 relaxation
times from muscles experiencing DMS more accurately, the difference should
have been calculated by subtracting the T2 value measured before performing

the additional concentric exercise from the T2 value measured after performing

8
the concentric exercise. However, these investigators did not acquire pre-

exercise images from the leg that performed additional concentric contractions.
Both legs. loaded and unloaded, in the eccentric/concentric group, were imaged
after performing the additional concentric contractions.

Therefore, it is still unclear what influence, if any, an elevated T2 will have
on the acute T2 response to additional concentric contractions. Furthermore, as
in most other studies relaxation times were first measured 3 days post-exercise,
there is little information documenting the early time course of the development
of delayed T2 increase.

Pumose of the Study

The purpose of this investigation was to examine the acute T2 response
to a bout of light concentric exercise performed before (control) and after
eccentric exercise had been used to induce a delayed T2 increase. The
secondary or descriptive purpose of this study is to document the early time
course of the delayed T2 contrast development.

Research Hypotheses

The hypothesis tested in this study was that the mechanisms underlying
both acute and delayed T2 changes are the same. If this hypothesis holds, and
if acute T2 has an upper limit. as previously suggested. the combined T2 change
from damage plus concentric exercise (Con2) will not be different from the
response due to exercise alone in normal muscle (Con1).

The hypothesis leads to the prediction that concentric exercise performed

when muscle T2 is already elevated will n_ot result in a T2 response different from

9
that produced by the same concentric exercise performed in a baseline or resting

condition. If the mechanism of the T2 response is identical for the acute
exercise contrast change and for the delayed response to eccentric exercise, it is
likely that the combined effects of the two conditions would 1191 be additive. This
is illustrated by column C in Figure 1. where the T2 change after concentric
exercise in muscle already elevated in T2 from prior eccentric exercise is the
same as that seen in the resting condition (column A). If. however, the two
mechanisms are different. it is likely that the T2 changes from the different
conditions will be additive. This would produce a net change equal to the sum of

column A plus column B, as illustrated by column D.

 

Change

 

 

T2

 

 

 

 

or

 

 

 

     

 

 

Con1 Ecc24h Con2 Con2

Figure 1. Hypothesized Changes in Calculated T2 following concentric exercise
(Con1, A). elevated T2 at 24 h post-eccentric exercise (Ecc, B), and concentric
exercise at 24 h post-eccentric exercise (Con2, C or D).

1 0
Research Plan

Eight healthy non-weight trained adults between the ages of 21 and 40
years were recruited to participate in this study. Each subject will serve as
his/her own control. MR images of the biceps muscle will be acquired before
(PreCon1) and after (PostCon1) an initial bout of light concentric biceps curl
exercise to measure the acute T2 increase in “normal” muscle. Following a 2 h
rest period. muscles will also be imaged before (PreEcc), immediately after
(PostEcc), and every 2 h for the first 6 h following a single bout of eccentric
biceps curl exercise to document the acute T2 increase following eccentric
exercise and to document development of the delayed T2 increase. At 24 h
post-exercise, subjects will again be imaged before (PreCon2) and immediately
after (PostCon2) performing another bout of light concentric exercise.

Subjective muscle soreness scores will be collected using a questionnaire
(20) starting at 24 h after eccentric exercise and for seven days following the
eccentric exercise. Overall mean T2 value will be calculated for the biceps
brachii in each subject. Cross-sectional area of the biceps brachii muscle will be
calculated at 24 h post-eccentric exercise to measure muscle compartment
swelling.

Significance of the Problem

The results of this study will provide insight into the unresolved question of
whether a single mechanism is responsible for the increase in acute T2 and
delayed T2 signal intensity. In recent years, several investigators have

suggested the use of acute T2 increase as a qualitative measure of muscle

1 1
recruitment following exercise. and delayed T2 increase as qualitative measure

of muscle damage following exercise. However, it may not be appropriate to
employ the acute T2 increase as an index of muscle recruitment in muscles that
already have an elevated T2 induced by exercise-induced damage and/or
neuromuscular disease or trauma. If the research hypothesis is rejected, i.e. if
the acute T2 response to concentric exercise is larger when performed after
muscle T2 is already elevated due to eccentric damage, then the existence of
distinct mechanism is strongly supported. In addition, documenting the early
time course of the development of delayed T2 will fill gaps in the published data
on T2 time course, as well as provide data on swelling during this intermediate

period.

CHAPTER II
REVIEW OF RELATED LITERATURE

Delayed muscle soreness (DMS) is the sensation of discomfort and/or
pain in the skeletal muscles following unaccustomed muscular exercise. The
most predictable method of inducing delayed muscle soreness is to subject the
muscles to repetitive. strenuous eccentric or lengthening contractions (5.6.7.20).
Muscles adapt to an initial bout of exercise, becoming resistant to damage from
subsequent bouts. The muscle soreness is often localized in the distal portion of
the muscle at the muscle/tendon junction and is accompanied by a sense of
reduced mobility (6.7.38). The sore muscles become extremely sensitive to
palpation or movement. Severity varies from slight stiffness to severe pain
inhibiting movement.

There is no evidence to suggest that the pathology associated with DMS
is long term. Studies using animal models suggest that injured muscles are able
to recuperate completely within 2 weeks (6.7). A review of the literature to date
will show that there is no firm consensus on the pathophysiological mechanisms
responsible for delayed muscle soreness. Although the time course of the

pathological changes associated with DMS is well established. it remains to be

12

13
determined how the sequence and timing of DMS relate to a possible

pathological mechanism.

Time Course of the Pathological Changes Associated with DMS

Needy every adult has experienced delayed muscle soreness, most on
numerous occasions. Unaccustomed physical activity of even moderate
intensity or duration will result in the sensation of DMS. regardless of the
individual's general fitness level. Since the muscle soreness or pain is only
temporary. most people do not seek medical attention.

Muscle soreness usually increases in intensity in the first 24 h after
unaccustomed and/or eccentric exercise and peaks at 24—72 h post-exercise.
Soreness slowly dissipates and does not completely subside until 5-7 d post-
exercise according to most reports (6). Recently. Clarkson, Nosaka, and Braun
(1991) reported that following eccentric exercise of the forearm flexor muscles
the soreness did not completely subside until 8-10 d post-exercise (20). This
discrepancy might be due to the fact that the soreness data from 6-10 d post-
exercise was based on a small sample (15 subjects), while the soreness from
pre-exercise to 5 d post-exercise was based on 109 subjects.

Accompanying soreness, there was a dramatic decrease in muscular
performance following eccentric exercise (20). The greatest reduction in
isometric strength was reported to have occurred immediately post-exercise, a
loss of over 50%. refer to Figure 2. In the following days, strength was gradually

restored but a small deficit remained at 10 d post-exercise. On the other hand,

14
strength loss following concentric and isometric exercise has been shown to

recover within a few hours post-exercise (20).

 

18..

16..

14«»

120

Isometric Strength (kg)

10“

 

 

 

. A 4 A . A A
*7 Y v v 1 fi 7 Y v f

DaysAfhrEccantrlcExarclaa

Figure 2. Isometric strength loss after performing eccentric exercise (20). Note
the gradual recovery of strength over a period of days. At 10 d post-exercise,
the isometric strength has not yet completely recovered.

The prolonged decrease in performance following eccentric exercise is
thought to be the result of both a reluctance to use the sore muscles and a
reduction in the inherent capacity of the muscle to produce force (6.20).
However, the soreness is not likely the cause of the decrements in strength since

soreness peaks at 24-72 h after exercise. during which strength is gradually

15
recovering. To evaluate the reduction in the intrinsic ability of the muscles to

produce force, investigators used direct electrical stimulation, by-passing
voluntary control, which also resulted in a reduction in force generation (61).
Therefore. the prolonged reduction in strength following eccentric exercise is
more likely the result of a lowered inherent capacity of the muscle to produce
force. Another symptom of DMS is a reduction in the range of motion at the joint
or joints that are involved in the high-force eccentric exercise. Clarkson et al.
assessed the reduction in the range of motion about the elbow joint in two ways:
a) flexed elbow angle, and b) relaxed elbow angle (20).

Muscle shortening ability has been reported to decrease following
eccentric exercise (20). Clarkson et al. assessed muscle shortening ability by
measuring the subject’s ability to their elbow, flexed elbow angle. which is the
angle at the elbow after the subject has fully flexed the elbow, the arm being
fixed at the side. The flexed elbow angle increased dramatically immediately
after the eccentric exercise, indicating that the subjects were unable to fully flex
the elbow. The flexed elbow angle decreased gradually in the days following but
remained elevated above baseline at 10d post-exercise.

Along the same lines. Clarkson et al. observed that after eccentric
exercise muscles spontaneously shortened (20). They assessed spontaneous
muscle shortening by measuring the relaxed elbow angle. This angle was
measured at the elbow after the subjects were instructed to let their exercised
arm to hang freely. The relaxed elbow angle was dramatically decreased

immediately following the eccentric exercise. The relaxed elbow angle continued

to decrease over the next few days, reaching the maximal decrease at 3 d post-

exercise. At 10 d post-exercise the relaxed elbow angle had returned to

baseline.

Lastly, several investigators have documented swelling following eccentric
exercise (6.20). Most of the studies documented swelling by measuring the
changes in circumference of the limb involved in the eccentric exercise. Swelling
was reported to gradually increase in the days following eccentric exercise and
does not peak until 5 d post-exercise (20). Table 1, summaries the time course
changes in muscle soreness, strength. muscle shortening ability, spontaneous
muscle shortening and swelling associated with DMS. Based on the time course
changes in muscle function after performing eccentric exercise, several
investigators began to speculate that these changes in muscle function were the

result of damage, induced by eccentric contractions, at the cellular level.

Table 1. Summary of the changes in muscle performance following

eccentric exercise.

16

 

Exercise induced changes in
muscle performance

Time to peak following
eccentric exercise

 

Muscle Soreness

2-3 d

 

Isometric Strength Loss

Immediately following
exercise

 

Muscle Shortening Ability

Immediately following
exercise

 

Muscle Spontaneous Shortening

3d

 

Swelling

 

 

5d

 

 

1 7
Morphological Changes Associated with DMS

In 1902. Theodore Hough published the first report on exercise induced
muscle soreness and was the first to suggest that exercise induced muscle
soreness is the result of morphological changes at the cellular level. Subjects in
his study reported muscle soreness 8-10 h after performing rhythmical exercise
using the flexor muscles of the middle finger (45). Hough hypothesized that
DMS is the result of structural damage to muscle fibers or connective tissue
within the muscle during exercise.

Evidence favoring Hough’s “tom tissue ‘ theory has come from recent
studies that have looked at morphological changes associated with DMS.
Friden. Sjostrom. and Ekblom in 1981 found disorganization of the myofibrillar 2-
bands in human soleus muscles three days after downhill running (39). They
found that the ultrastructural damage resulting from eccentric exercise involves
streaming, broadening. and occasional disruption of Z-lines. In some cases A-
bands were out of register within affected sarcomeres, and in several cases the
thick filaments were completely absent. Armstrong. Ogilvie, and Schwane in
1983 also observed disruption of the striation pattern of rat slow-tvvitch extensor
muscles after downhill running (7).

In 1985. Ogilvie, Annstrong. Baird, and Bottoms documented three types
of lesions in rat soleus muscle following eccentric exercise: (a) Z-line dissolution,
(b) focal disruptions of the A-band, and (c) clotted fibers (63). In addition, they
found that 90% of the morphological changes were A-band disruptions.

Newham, McPhaiI, Mills. and Edwards conducted a study in 1983 to examine the

18
changes associated with eccentric exercise (60). They noted extensive

sarcomeric disruptions in biopsies taken from the human quadriceps muscles
following eccentric exercise. This same type of Z-Iine streaming has been
reported in control subjects by Fischman et al. and Meltzer and colleagues (36).
However. Friden and Lieber. in a recent review. suggest that Z-disk streaming is
primarily a myofibrillar response to high-tension physical exercise and altered
metabolic situations (36).

Evidence accumulated, thus far, clearly shows that, following eccentric
exercise, there are marked structural changes in the contractile and connective
tissues in both animals and humans. Using sophisticated analytical techniques.
several investigators have documented the different types of lesions that occur
as a result of eccentric contractions in both humans and animals. The sensation
of DMS appears to be related to this muscle and/or connective tissue disruption
(6.74). Although there is no proof. it is assumed that the morphological damage
observed in rodent muscle and DMS in humans are manifestations of the same

exercise - induced cellular events (7,74).

Metabolic Changes

Hough’s “torn tissue” theory was validated by Asmussen’s 1956 study on
concentric (positive) versus eccentric (negative) work (8). In this study.
Asmussen had volunteers perform hard negative work ( a step down exercise for
the quadriceps and a flexion exercise for the triceps muscle) and positive work

(step up exercise for the quadriceps and an extension exercise for the triceps)

19
until fatigue. In positive work, the muscles shorten during contraction and in

negative work the muscles lengthened during contraction.

In order to place the same amount of tension on the muscles during
positive and negative work the investigators instructed the volunteers to maintain
a steady speed of movement throughout the different exercise regimens. As
fatigue developed during positive work. some volunteers reported ischemic pain
which disappeared shortly after the cessation of work and did not reappear. On
the other hand, the volunteers performing negative work did not report any
sensation of pain during or immediately following the bout of exercise, but 12h to
36h post-exercise the muscles were extremely sore. Asmussen found that
fatigue always developed first in the muscles that performed positive work,
suggesting that the metabolic cost of positive work is much greater. Therefore.
this study effectively ruled out the possibility that DMS resulted from the

accumulation of metabolic by-products.

Mechanical Asgects of DMS
More recently, Bigland-Ritchie and Woods in 1976 reported that eccentric

contractions recruit fewer motor units than concentric contractions at a given
muscle load (10). Therefore, the force generated during the eccentric
contraction is distributed over a smaller cross-sectional area of muscle.
Armstrong suggested that this relatively increased tension in the active
contractile and elastic elements during eccentric contractions resulted in physical

damage to the structural components of the muscle fibers (6).

20
Force - Veloc Relationshi in Eccentric Exercise

An'nstrong’s hypothesis was based on the work done by Katz in 1939 on
the force-velocity relation in muscular contraction (51 ). Katz found that when
muscles shorten. the dependence of load (P0) or initial tension on the muscle
contraction speed was approximately 4%Pol % muscular velocity (V....,,). The
relationship between force and velocity in lengthening muscles is much more
drastic. A muscle lengthening at 1%VM could increase the tension placed on
the fibers by 50%. The classic force-velocity relationship curve (Figure 3)
demonstrates this 10-fold faster rise in tension for eccentric contractions than for

an equivalent velocity of concentric contractions.

 

Mime

  

150 Slope = 50%Pol%v
Slope = 4%Po/%Vmax

  

 

 

Muscle Valocity-‘inax

Figure 3. The classic force-velocity relationship curve (51). Note that for
lengthening velocities (velocities less than zero) force increases much more
rapidly than for shortening velocities (velocities greater than zero). Also note the
discontinuity in the force velocity relationship around zero velocity.

21
As previously noted, the high tensions associated with eccentric

contractions have been speculated to cause ultrastructural damage. Hough
found that the degree of perceived pain was directly related to peak forces
generated and to the rate of force development during the rhythmic contractions
used to induce the delayed soreness. According to Friden and Lieber,
discontinuity in the force-velocity relationship curve around zero velocity might
cause mismatches between adjacent sarcomeres in terms of the forces that are
placed on them, refer to Figure 3 (51).

Huxley and Peachey in 1961 showed that sarcomere length along the axis
of the muscle might vary by 1% (47). In 1983 Lieber and Baskin used isolated
frog muscle fibers to investigate sarcomere length as a function of time during
“fixed-end” contraction; in which isolated semitendinosus muscle fibers were held
at a constant length (54). They found that the sarcomeres near the ends of the
muscle fiber are shorter in length and shorten at a higher velocity. Approaching
the center of the muscle fiber. the starting sarcomere lengths become longer and
the shortening velocity decreased. At the center of the muscle fiber. the
sarcomere was fairly long and actually elongated (rather than shortening) at a
slow rate. The force-velocity relationship near zero velocity was such that a
small difference in muscle velocity during shortening caused minimal difference
in the force sustained by the adjacent sarcomeres (36). However. a muscle
lengthening at 1% Vm increased the tension by 50% Po on adjacent

sarcomeres.

22
Tension Imbalance Theories

If high tension is the principle cause of myofibrillar disruption, one would
expect to observe similar muscle damage in passive stretching. Lieber,
Woodbum, and Friden in 1991 found that cyclic passive stretching caused only a
10% increase in maximum tetanic tension (55). This prompted Friden and Lieber
to conduct an experiment in which they varied the strain ([L-LollLo) placed on
rabbit tibialis anterior muscle (36). One group was strained at 12.5% of fiber
length and the second group was strained at 25% of fiber length. The 12.5%
strain produced a 45% decrease in muscle load (Po). while 25% strain produced
a 65% decrease in muscle load. Although an earlier study by Lieber et al. had
dismissed strain as a possible cause of muscle damage. this differential
response to strain suggests that tension imbalance rather than absolute tension
might be responsible for the eccentric exercise induced muscle damage.

Reviewing these results. Friden and Lieber proposed that fiber stress
(load/cross-sectional area) rather than absolute tension might be determining
'the extent of muscle damage. Lieber and F riden conducted an experiment to test
this above hypothesis by imposing cyclic length changes on actively contracting
rabbit tibialis anterior muscle (TA) using a standard stretch magnitude of 20% of
TA fiber length and a constant strain rate of 60%Isecond. Two protocols were
used: (1) an early stretch (ES) group’s TA was stretched immediately following
muscle activation and (2) in the late stretch (LS) group, muscle stretch was
delayed by 200 ms. The delay in the LS group allowed the muscle to develop

tension and reach a peak force twofold higher than in the ES group. Despite the

23
fact that the LS group experienced over twice the total stress, maximum tetanic

tension for the two groups was almost identical. Although it is well accepted that
the muscle stress is high during eccentric contraction, these results indicate that

muscle damage induced by eccentric exercise might not be directly related to the
stress placed on the muscle.

F riden and Lieber propose that sarcomeres have a physical threshold for
damage that is more likely to be reached during an eccentric contraction. The
data presented thus far indicates that neither strain nor stress alone produces
ultrastructural damage associated with eccentric exercise. Friden and Lieber
suggested that active straining combined with increased tension per unit area
increases the likelihood of ultrastructural damage. The physical sensation of
DMS appears to be related to this ultrastructural muscle and/or connective tissue

disruption.

Biochemical Asggcta of DMS

Complementing these mechanical studies, evidence for ultrastructural
changes associated with DMS has come from biochemical studies that have
shown an increase in intramuscular enzymes and myoglobin in the blood
following eccentric exercise (27). Of these, the most frequently studied muscle
enzyme is creatine kinase (CK). There are several isoforrns of CK in the body.
This discussion will focus on the skeletal muscle isoforrn. It has been well
documented that increased skeletal muscle CK activity in the blood indicates

skeletal muscle injury. and increased cardiac muscle CK activity in the blood is

24
often observed after a myocardial infarction. The CK response varies

considerably with different forms of exercise; downhill running, marathon
running. eccentric and isometric exercise have been the modes most frequently
studied (20).

The increase in CK activity in the blood is significantly lower after downhill
running at a -10% grade for 30 minutes at a speed that elicited a heart rate of
170 beats per minute (approximately 300 units/liter) than with eccentric exercise
of the forearm flexors (about 2,500 U/L) (14,20). These results are particularly
interesting when one compares the amount of muscle mass involved in the two
different exercise regimens. Although the forearm flexors are very small
compared to the muscles involved in downhill running, the CK increase seen is
much greater than for intense leg exercise. In the downhill running condition, the
peak CK response occurred around 24 hours post-exercise. However, after
eccentric forearm exercise there was no notable increase in the CK levels in the
blood for two days after exercise. and peak CK values occurred around four days
post-exercise.

On the other hand. changes in blood CK levels following forearm flexion
isometric exercise were similar to the changes found following downhill running
(18.19). but CK activity in the blood after marathon running was considerably
higher. In addition to reaching higher levels, the CK level increased more
rapidly in marathon runners than after downhill running or eccentric exercise (4).

Blood levels of other muscle proteins, including lactate dehydrogenase,

25
asparate aminotransferase,_ myoglobin, and alanine aminotransferase. also

follow the same time course as CK.

Muscle Damage Inferred from Serum Creatine Kinase Activi_ty
It has been suggested that the extent of CK response represents the

magnitude of the injury induced by different forms of exercise. This would
explain the larger increases in CK levels in the blood following marathon running
and eccentric exercise. However. there is no explanation as to why there was no
relationship between CK response time course and other indicators of muscle
damage (e.g.. muscle soreness level. isometric strength, and relaxed arm angle).

Part of this problem was addressed in 1992 by Clarkson. Nosaka, and
Braun (20). They suggested that the studies looking at CK response after
exercise did not take into account the large inter—subject variability in CK
response and used too small a sample. To account for this, they conducted a
study in which they separated their subjects into three groups based upon the
amount of increase in CK following eccentric exercise of the foreman flexors. If
subjects had a peak CK response of over 2,000 UIL. they were classified as high
responders; peak CK response between 500 and 2,000 UIL was grouped as
medium responders; and peak CK response less than 500 UIL was the low
responders.

Significant differences were found between the low CK group and high CK
group on muscle soreness, isometric strength, and relaxed arm angle. The

medium CK group fell in between the low and high CK group on all indirect

26
indicators of muscle damage. Statistically there was no difference on muscle

soreness, isometric strength. or relaxed arm angle between the high and I
medium CK groups.

The authors believe that the low CK group might have been preadapted
resulting in less muscle damage. As to why there was no difference between the
medium and high CK groups, the authors quote Evans and Cannon (30): “the
post-exercise rise in circulating CK activity is a manifestation of skeletal muscle
damage but not a direct indicator of it.”

Another factor to consider when evaluating the CK response after
exercise is that the CK actiw‘ty in the blood is a steady state; dependent on the
amount of CK released by the muscle and the rate of CK clearance by the
reticuloendothelial system. Therefore, high levels of CK in the blood does not

necessarily indicate actual muscle damage.

Role of Calcium in DMS
Although the time course of DMS is well established, there is still
controversy over the mechanism. Various hypotheses have been put forward to
account for the unusual delay between the exercise. first soreness, and peak
soreness, no single explanation shows more promise than disturbance in
calcium (Caz’) homeostasis. In muscles Ca2+ plays a vital role in the regulation
of the contractile cycle and more importantly maintains the integrity of the

sarcolemma.

27

Muscle contraction is initiated by an increase in intracellular free calcium
ion concentration. Ca2+ is rapidly bound to troponin C and other cytosolic
calcium binding proteins and is removed by the ATPase pump of the
sarcoplasmic reticulum during relaxation. A low intracellular free Ca2+
concentration (0.1 to 0.2 M) is essential for normal cell function, but an
excessively high concentration of free Caz’ has been associated with cell
dysfunction and cell necrosis (i.e.. ischemia in the myocardium and specific
types of degenerative muscular dystrophies) (5).

The high mechanical forces generated during eccentric exercise have
been documented to cause structural damage to the sarcolemma and/or
alterations in permeability of the cell membrane. which results in a net influx of
Ca” from the interstitium into the muscle cell. In an attempt to buffer the high
cytosolic concentration of Ca”. mitochondria take up large amounts of the
excess Ca” ions. However, while reducing the potentially damaging effects of
high cytosolic Ca”, the uptake of Ca2+ impairs mitochondrial respiration and ATP
production. The attenuated levels of ATP could subsequently reduce Ca2+
extrusion from the cytoplasm via ATP-dependent Ca2+ pumps in the
sarcolemma, mitochondria. and sarcoplasmic reticulum. as well as limiting force
due to reduced ATP supply for the actin-myosin ATPase (5). However, this is
unlikely to occur in exercise employing eccentric contractions since the energy
(ATP) cost is relatively low.

The mechanism(s) by which Ca2+ is elevated in the muscle cells following

28
eccentric exercise is not known. Several possible mechanisms have been

suggested. First, the source of the Ca” could either be intracellular or
extracellular. The majority of the intramuscular Ca2+ is found in the sarcoplasmic
reticulum. It is entirely possible that the reuptake of the Ca2+ by the
sarcoplasmic reticulum was some how compromised following eccentric
exercise, resulting in the accumulation of Ca” in the cytosol (27).

However. if the source of Ca2+ is extracellular, two mechanisms of entry
have been proposed. First, the extracellular Ca2+ could gain entry into the
cytosol through the stretch-activated Ca” channels in the sarcolemma (27).
Ducan et al. , based on unpublished data, proposed that the increased active
strain during eccentric exercise stimulates the stretch-activated Ca2+ channels,
and Ca2+ moves through the sarcolemma down its concentration gradient. To
date, stretch-activated Ca2+ channels have not been described for human
skeletal muscles.

A second mechanism by which extracellular Ca2+ could gain entry into the
cell was proposed by Armstrong, Ogilive. and Schwane. Armstrong et al.
observed a marked elevation in creatine kinase and lactate dehydrogenase in
the plasma of sedentary rats after walking downhill (eccentric exercise) (7). They
interpreted these results as evidence that eccentric exercise caused disruption of
the sarcolemma. allowing intracellular proteins to exit down their concentration
gradient Based on these results. Armstrong et al. proposed that the ruptures in
the sarcolemma could allow the extracellular Caz+ to enter the cytosol down its

concentration gradient. In a more recent study, Ducan, Delp, Hayes, Delp, and

29
Armstrong noted that there was an accumulation of Ca2+ in injured rat muscles

immediately following downhill walking (27).

However. the relationship between the initial influx of Ca” and the
degradation of muscle fibers during the post-exercise period remains unclear. In
a 1984 review of DMS, Armstrong proposed the following as a possible
sequence of events in the production of DMS following the initial influx of Ca2+.
First, the elevated intracellular Ca2+ concentration increases the activation of
phospholipase A2, resulting in the production of Iysophospholipids, leukotrienes,
and prostaglandins; all of which could lead to degradation of cellular structures
(6). Also, high Ca2+ concentration activates proteases and stimulates production
of free radicals. resulting in the preoxidation of membrane lipids. As a result. the
sarcolemma becomes leaky (27). In addition, the calcium-dependent proteolytic
enzymes that preferentially degrade Z-discc, troponin, and tropomyosin are
activated by the abnormally high cytosolic Ca” concentration (6).

Second, the progressive deterioration of the sarcolemma in the post-
exercise period is accompanied by a diffusion of intracellular components into
the interstitium and plasma. Many of these substances. including
prostaglandins, attract neutrophils and monocytes that convert to macrophages.
In addition, these substances also activate mast cells and histocytes. As a result
of the active phagocytosis and cellular necrosis; histamine, kinnis. and
potassium could accumulate in the regions of group IV free nerve endings. This

would then activate the nocieptors and cause the sensation of DMS (6).

30
The question that merits further investigation is whether the elevated Ca

2+
in the muscle fibers is simply a symptom of DMS or whether Caz" is
mechanistically involved in the etiology of the DMS. As a result, two separate
experimental models have been developed to study the role of Ca2+ in DMS,

non-exercised induced muscle injury and exercise induced muscle injury.

Chemically Induced Muscle Damage

Non-exercise experimental muscle injury studies have contributed to our
understanding the function of Ca2+. Jackson, Jones, and Edwards in 1984
conducted a series of in vitro experiments on mouse muscle using dinitrophenol
(DNP)- an uncoupler of oxidation and phosphorylation in the mitochondria. In
the first series of experiments, they stimulated mouse muscle for 30 min with
both DNP and Ca2+ in the incubation medium (48). In order to significantly
impair mitochondrial respiration and ATP production, both DNP and Caz" were
included in the incubation medium. This resulted in the loss of lactate
dehydrogenase (LDH) into the medium during the period of stimulation and with
peak loss occurring within 30 min of the contractions. The loss of LDH into the
medium is an indirect measure of sarcolemmal damage.

However, when the incubation medium lacked Ca2+, LDH release was
significantly reduced both during the exercise and in the post-exercise period.
The role of DNP in the above scenario is not known, but has been speculated to

cause the release of mitochondrial Ca”. This is plausible, as it has also been

31
suggested that DNP’s mode of action is to make mitochondrial membranes

"leaky' to proton flux and possibly other ions.

In a second series of experiments, Jackson et al. examined the influence
of phospholipase A2 inhibitors on the release of LDH with DNP and Ca2+ in the
incubation medium. Phospholipase A2 has several deleterious effects on the
cell, in that it produces detergent fatty acids and Iysophospholipids which are
involved in cell-membrane degradation. Phospholipase A2 inhibitors
significantly reduced the loss of LDH, although various other protease inhibitors
were not able to produce the same result (48). These results suggest that
sarcolemmal damage following contractions in the presence of DNP involves a
Ca2+ mediated pathway. More importantly, they intimate that the underlying
mechanism of injury to membrane involves a Ca2+ activated phospholipase A2.

In 1987. Chang, Musser, and McGregor determined that phospholipase
A2 has a Ca2+ binding site and that the activity level increases as a function of
the Ca2+ concentration (17). In the same year Duncan and Jackson established
that inhibiting phospholipase A2 provides a protective effect to the cell
membrane but does not protect against myofibrillar damage (28). They noted
that the chemically induced muscle injury is much more rapid (5-30 min) than
muscle injury associated with DMS, and involves two distinctive Ca2+ mediated
pathways: (1) Ca” activated phospholipase A2 mediated injury to the
sarcolemma . and (2) ultrastructural damage to the myofilaments by an unknown

mechanism.

32
The Role of Calcium in Exercise Induced Muscle Damage

Although studies of chemically induced muscle damage were not able to
explain the role of C32+ in DMS, these studies have set the stage for others to
look more critically at the exercise-induced sarcoplasmic reticulum dysfunction
and mitochondrial Caz+ concentration ([Cazjuno) as possible factors involved in
DMS etiology. [Cazjmo is relatively low compared to the calcium levels in the
sarcoplasmic reticulum in the resting muscle cell (5). As early as 1952. it was
established that [Cazjmo is an indirect indicator of cytosolic [Ca2*] in the muscle
cell (78). Sembrowich, Quintinskie, and Li reported in 1982 that Ca2+ uptake
kinetics of the mitochondria in slow twitch muscles are adequate to play a vital
role in regulation of Ca2+ concentration in the muscle fibers (69).

In fast twitch muscles. the cystolic Ca2+ concentration increased
considerably after maximal contraction, while the [Cazjmo remained fairly low
(76). In contrast. Tate, Bonner. and Leslie in 1978 exercised trained rats to
exhaustion and found that [Caz’hrro increased by 132% (83). The authors
suggested that the abnormally high cystolic Ca2+ levels resulting from exhaustive
exercise were buffered by the mitochondria.

These findings lead Duan et al. in 1990 to conduct a series of
experiments in order to evaluate the relationship between the [Cazjrvrrro and
muscle injury in rat soleus and vastus intennedius from eccentric exercise (27).
Duan et al. hypothesized there would be a direct relationship between [Caz‘hrro
and muscle injury. Several studies have reported that Ca2+ antagonists

attenuate the incidence of damage during the post-exercise period, suggesting

33
that Caz“ might have a direct causal role in DMS etiology.

Duan and co-workers addressed this issue by using three different Ca2+
antagonists - verapamil, EDTA (ethylenediaminetetraacetic acid). and EGTA
(ethylene glycol-bis-N,N.N',N-tetracetate) in their study. Verapamil blocks slow
Ca2+ channels. whereas EDTA and EGTA chelate Ca” and are excreted,
therefore lowering total body Ca” (27). Muscle injury was evaluated by
determining the number of undamaged fibers per unit area of muscle cross
section.

Results showed that [Cazjuno was inversely related to the number of
intact fibers per square millimeter of muscle cross-section. They reported that
verapamil reduced the increase in [Caz'lurro but did not reduce the occurrence of
injury. Verapamil results must be interpreted with care. because relatively high
concentrations can have harmful direct effects on the muscle fibers and the
dose-response relationships have not been completely established for this drug.
The investigators suggested that these side-effects of verapamil may have
contributed to the muscle injury, and thus might explain why verapamil reduced
the [cazfinno but did not lower the incidence of muscle injury (27).

The Caz+ chelators, EDTA and EGTA, significantly reduced the elevation
of [Caz’hmo and reduced the occurrence of injury but did not affect serum Ca2+
concentration. The mechanism by which chelators blocked Ca2+ from entering
the mitochondria is not completely clear. At two days after the first introduction
of chelator. serum Ca2+ concentration did not change. Therefore, the

extracellular-to— intracellular Ca2+ concentration gradient did not limit the influx of

34
Ca2+ into the cell. if it is assumed that serum Ca” concentration is equivalent to

interstitial Ca2+ concentration (27). These authors proposed that the chelators
may have accumulated within the affected muscle cells and restrictively bound
Ca” in some compartment within the muscle cell. thereby preventing it from
entering the mitochondria.

As an alternative explanation for the effects of Ca2+ chelators, it was
proposed that chelation might influence the recruitment of motor units in the
muscles. This is based on the fact that alpha-motor neuron’s ability to release
neurotransmitters is dependent on Ca2+ influx from the interstitium. If the release
of acteylcholine is limited from the initially recruited pool of motoneurons. then
additional motor units are recruited to produce the required force. In effect, this
will significantly reduce the active strain generated within the motor unit and
decrease the potential for mechanical damage to the constituent fibers. Since
the authors reported no change in the interstitial Caz“ concentration following
EDTA treatment. it is unlikely that the recruitment pattern in muscles changed as
a result of chelator treatment (27).

A very recent study focused on the natural ameliorative process that
attenuates the development of muscle injury and soreness (56). This work
demonstrated that following eccentric exercise mouse soleus muscles were able
to buffer the increased influx of extracellular Ca” and avoided the activation of
Ca2+ sensitive degradative pathways (56).

Lowe et al. conducted a series of experiments to examine the relationship

between extracellular Ca” concentration ([Ca2‘10) and the extent of muscle

35
injury. The investigators hypothesized that by increasing {Caz}. they could

increase the electrochemical gradient for Ca2+ across the cell membrane and
cause a greater influx of Ca2+ through damaged sarcolemma. In a normal
resting muscle. cell the Ca2+ concentration lies in the range of 0.1 to 0.2 pM and
in the extracellular fluids the concentration is about 1 mM (79). Lowel et al.
found that the extent of muscle injury following eccentric contractions was not
affected by [Caz‘]o in the range of 005-50 mM (56). The investigators chose
this range so as to avoid adverse effects on contractilitylforce production when
{Caz}, is either too low or too high.

Lowel et al. observed an initial decrement in force production following
their eccentric exercise protocol. The authors suggested that this decrease in
force production was due to the damaged force-producing and transmitting
structures and to a loss of excitation-contraction coupling. Interestingly. after
eccentric exercise, the total muscle Ca2+ concentration increased. although no
increase was seen in the protein degradation process. The muscles that
performed eccentric exercise showed neither a decrease in the contractile
properties nor was there an accumulation of LDH. leukotriene B4, prostaglandin
E2, or tyrosine in the incubation medium. Leukotriene B4, LDH and
prostaglandin E2 were measured because they are the end products of the
proposed Ca2+-mediated degradative processes (56).

More importantly, the muscles incubated in the higher [Caz’] solution did
not show a higher incidence of muscle injury than the muscles in the normal

[Ca2”] solution. The muscles that performed eccentric contractions had a higher

36
total muscle [Ca2"] than either muscles that performed isometric contractions or

control (unexercised muscles). This is consistent with other studies that have
compared total muscle [Caz‘] of normal muscles and damaged muscles (56).

It has been assumed that the critical variable that activates the proposed
Ca2+ mediated degradative pathways is intracellular Ca2+ concentration ([Ca2*]i).
Lowel et al. have cleady demonstrated that the [Caz‘]: in the injured muscle did
not increase into the pathological range of 10 - 50 W [Ca2*]i (15). The resting
contraction threshold for skeletal muscle was around 1 pM {Caz}. If the [Ca2+].
surpasses this value, the resting tension would automatically increase.

Since the resting tension did not change during the incubation period, it’s
unlikely that the injured muscle’s [Cab]. approached the pathological range. The
authors argued that the injured muscles were able to buffer the increased total
muscle [032+], since the [Ca2‘1i showed only a minimal increase (56). It has
been suggested by others that the mitochondria, sarcoplasmic reticulum. and
endoplasmic reticulum are primarily responsible for buffering the Ca2+ ions (10).
Unfortunately Lowel et al. did not measure any of the above organelle’s Ca2+
content.

The results of this study need to be interpreted with care since the
investigators incubated the muscle for only 120 minutes after the contraction
protocol prior to collecting the data. This time period may not be long enough for
the influx of Ca2+ to overcome the buffering capacity of the mitochondria and
activate the endogenous proteolytic pathways. Previous studies have clearly

demonstrated that the exercise induced muscle injury is more apparent two days

37
after exercise than immediately post-exercise (7). Armstrong et al. have shown

that local disruptions of the banding pattern in the injured fibers are visible
immediately post-exercise. However, two days after exercise, the regions of
degeneration are more apparent because they are marked by the accumulation
of macrophages.

Lowel et al. acknowledged the limitations of a short incubation period but
provided no explanation as to why they chose that particular incubation time
period, nor did they propose which of the injured muscle cell organelle(s) was
responsible for buffering the Ca” ions. Furthermore, there was no histochemical
analysis performed on the muscle samples following the incubation period to

document damage.

A Comparative Analysis of the “Tom Tissue” Theor_'y and Calcium Induced

Cell Damage Theory
Thus far. two hypotheses have been presented: (1) the "torn tissue"

theory, and (2) Calcium induced cell damage theory. Neither of the two fully
explains the relation between tissue injury initiated during the exercise bout. and
the soreness first experienced 8 h and reaching a peak at 24 h post-exercise. At
present, most investigators agree that the muscle damage is initiated as the
result of high mechanical forces produced during muscular exercise, particularly
in eccentric exercise.

Experimental research conducted over the past 90 years, since Hough’s

“torn tissue” theory was first published. generally have supported his contentions.

38
However, the studies focusing on the ultrastructural damage following eccentric

exercise are not able to elucidate why the tissue damage is not immediately
followed by the perception of pain, like other tissue lesions. Although with limited
success, the calcium induced cell damage theory predicts that the delay in the
sensation of pain is partly due to the slow influx of Ca2+ into the muscle cell. This
critical influx of Ca2+ into the cell is the result of the ultrastructural damage to the
sarcolemma and the contractile tissues following eccentric exercise. A review of
the Ca":+ literature suggests that increase in the intracellular calcium
concentration within the physiologically relevant range does not elicit the
indigenous calcium mediated destructive process. Yet to be resolved is the
mechanism and organelle(s) that are primarily responsible for buffering the
increased total muscle Ca2+ concentration following eccentric exercise.

In an attempt to better explain the unusual delay. several investigators
have proposed that the underlying mechanism of DMS is acute inflammation.
based on the similar time courses of the two conditions. The following sections
describe the mechanism of the inflammatory response and the evidence relating

this process to DMS.

Acute Inflammation as the Underlying Mechanism of DMS

Acute inflammation is a generalized response of the body to tissue injury
which confines the injury and promotes the healing process. The entire process
will generally last for approximately a week (74). The superficial characteristics

of inflammation include redness, swelling. heat, pain, and loss of function. The

39
inflammatory response in the classic sense is composed of two separate but

related processes: (1) the vascular response and (2) the cellular response.

First. the cellular component of inflammation involves predominately two
types of leukocytes: neutrophils and monocytes (74). Within a few hours of
injury, the number of circulating neutrophils increases dramatically. Neutrophil
concentration at the site of injury peaks between 1 and 4h post - injury,
depending on the intensity of the damage. and then declines rapidly.
Neutrophils are the first phagocytic leukocytes to arrive at the site of injury and
they participate in the digestion of necrotic cells and cellular debris. Several
hours following the neutrophil migration, the monocytes begin to accumulate at
the site of injury. Once the monocytes leave the blood and enter the injured
tissue compartment, they mature into macrophages. Unlike the neutrophil
concentration, the monocyte concentration at the site of injury increases
continually for 48 h. The monocytes. like the neutrophils, also digest dead cells
and cellular debris.

Second. the vascular component of inflammation involves the arterioles
near the site of injury constricting for approximately 5 - 10 minutes.
Vasoconstriction is followed several hours later by vasodilatation. which
increases blood flow to the injured tissue and increases the vascular permeability
in the surrounding blood vessels. This increased vascular permeability
continues throughout acute inflammation resulting in a continuous outward
leakage of blood cells and protein - rich plasma or exudate into injured tissues.

The end result of the increased vascular permeability is swelling and the

40
sensation of pain in the affected area. The following sections will evaluate the

evidence that suggests that similar changes also occur in association with DMS.

Swelling

Swelling is one cardinal symptom that is always present to some extent in
acute inflammation (74). The swelling or edema is a result of increased
permeability of the small blood vessels which allow the exudate to escape into
the damaged tissues. Evidence linking inflammation to DMS includes reports of
increased permeability in rat skeletal muscle following eccentric exercise (80).
Other studies reported increases in limb volume at 24 , 48, and 72 h after
eccentric exercise (45.79). More importantly. these investigators observed that
the increase in limb volume was specific to eccentric muscle action. The limb
volume did not change following isometric or concentric contractions.

This lead some investigators to speculate that swelling might play a
causal role in the sensation of DMS by increasing the intramuscular pressure.
Newham and Jones in 1985 reported no significant increases in intramuscular
pressure in the human biceps muscle following eccentric exercise (59). However.
a study conducted by Friden. Sfakianos, and Hargens in 1986 found significant
increases in intramuscular pressure (37).

It should be noted that Newham and Jones monitored intramuscular
prcssures in less restricted compartments and during rest. Smith suggested that
the largest increases in intramuscular pressure will only occur during a

contraction (74). In addition, it has been shown repeatedly by several

. 41
investigators that the sensation of pain in DMS is only experienced during

contraction or palpation and not during rest. Based on this, Smith proposed that
increases in intramuscular pressure during a contraction or palpation will provide
a mechanical stimulus for the PGE2 - sensitive receptors (pain receptors) (74).
This in turn results in the sensation of DMS.

The development of swelling / edema in the damaged tissue fits well with
the time course for the development of DMS. both showing marked increases at
24, 48 and 72 h after exercise. In contrast, a study in 1972 by Talag found the
greatest increases in limb volume occurred at 72 h after exercise, while peak
soreness occurred at 48 h (82). Smith makes use of the acute inflammation
model proposed by Ryan and Majno to explain the results reported by Talag
(82).

Ryan and Majno suggesed that during the early stages of inflammation
swelling is brought about by the accumulation of fluid in the tissue (67). During
later stages of inflammation. the increase in limb volume is primarily due to the
production of new connective tissue. Based on this model Smith suggested that
the increases in limb volume at 24 and 48 h after exercise is the result of fluid
accumulation. and the increases in limb volume at 72 h post - exercise (reported
by Talag) was primarily due to the synthesis of new connective tissue (82). In
addition, several investigators. including Smith. have suggested that the
adaptation observed in response to a single bout of eccentric exercise and
subsequent DMS is due to changes in the properties of connective tissue (74).

In summary, swelling is present in both acute inflammation and DMS.

42
The time course data on the development of swelling and sensation of pain in

DMS correlated very well, lending support to the notion that the underlying
mechanism of DMS might be acute inflammation. Unfortunately. the studies that
have examined the causal role of swelling in initiating the sensation of DMS by

increasing the intramuscular pressure are not conclusive.

Iggy;

Another cardinal symptom of DMS as well as acute inflammation and
tissue injury is pain (74). The sensation of pain involves the activation of pain
afferents, most likely Type III and IV nerve fibers. In addition. certain chemical
compounds are associated with the sensation of pain. Histamine, acetylcholine,
bradykinin, potassium, serotonin and prostaglandins of the E series (PGE) have
been proposed as possible candidates.

The non - exercise literature suggests that the most likely candidates are
the prostaglandins of the E series. PGE does not directly stimulate the
nociceptors but rather it produces a state of hyperalgesia ( increased sensitivity)
in the nociceptors. Therefore. a previously benign chemical, mechanical. or
thermal stimuli might be able to activate the afferent pain fibers. In particular, the
exercise induced muscle damage models have documented elevated levels of
PGE2 following a bout of eccentric exercise.

More importantly . Smith and colleagues have documented a similar time

course for the increase in PGE; and DMS 24 h after a bout of eccentric exercise.

Suggesting that the elevated levels of PGE; are responsible for the sensation of

43
DMS (74). The mechanism responsible for increasing the biosynthesis of P652

following eccentric exercise remains unresolved.

Interleukin - l . Ca2+, and macrophages have been shown to stimulate
local PGE; synthesis and have also been shown to increase after eccentric
exercise. However, a study conducted by Cannon and colleagues using human
subjects in 1989 reported that after unaccustomed eccentric exercise, the
increases in interleukin - I levels do not correspond with the time frame for PGEz
increases (16). As mentioned earlier. mitochondrial Ca2+ concentration
(Ca’zmo) was used as an indicator of muscle Ca2+ levels. [Ca‘zmo] has been
shown to reach peak levels 48 h after eccentric exercise in rodents (27), a time
when PGE; and muscle soreness are also significantly elevated in humans (74).

On the other hand, elevation in [Ca‘zmo] was also seen immediately after
downhill running (27). Since PGEz levels were not measured, it is not known
whether PGE; levels were elevated immediately following downhill running. but
clearly the onset of DMS has not yet occurred. Therefore, it appears that, during
DMS. elevated levels of Ca2+ may not play a critical role in stimulating local PGE2

synthesis.

Cellular Infiltrates in DMS as well as Acute Inflammation

Of the three proposed stimulators of local PGE; synthesis, macrophages
show the most promise. Under inflammatory conditions macrophages begin
synthesizing and releasing large quantities of PGE2 within 30 min and continue

for at least 24h (22). It has been proposed by several investigators including

44
Smith . Tullson, and Armstrong that the presence of the macrophages at the site

of injury. during acute inflammation and after eccentric exercise in rodents, is
most likely responsible for the biosynthesis of PGE2 (74). In both conditions. the
macrophages are present in large numbers after 24 h and 48 h of tissue
damage. Conveniently. this is also the period when peak levels of soreness are
experienced. However. a study conducted in 1983 by Armstrong, Ogilvie, and
Schwane rarely found any neutrophils at the site of injury (7). The inflammation
literature suggests that the most important factor in acute inflammation in the
classic sense is the accumulation of neutrophils at the site of injury (74).
Accordingly, Armstrong et al. concluded that the absence of neutrophils indicated
that the pathology of DMS did not involve inflammation in the classic sense.

Kuipers, Drukker, Frederik, Geurten, and Kranenburg in 1983 reported
margination and infiltration of neutrophils in the soleus muscle of the rat between
0 and 2 h after eccentric exercise (52). This time course for the neutrophil
infiltration fits well with the acute inflammation scenario. In this study. the
eccentric exercise involved running at a 100 upward incline, which maintains the
soleus muscle in a lengthened position during the run. In addition, the muscle
damage observed in the soleus during incline running is similar to the damage
seen in downhill running.

In contrast. a study conducted in 1983 by Schwane, Johnson,
Vandenakker and Armstrong performed total and differential white blood cell
counts on blood samples from seven male volunteers taken before the exercise

and at 5 min and 24. 48. and 72 h following downhill mnning. The volunteers ran

45
on a -10% grade at 57% of their V02 max for 45 minutes. Schwane et al.

reported no significant increase in peripheral white blood cell count, a marker of
acute inflammation. following downhill running (68).

More recently Smith. McCammon, Smith. Chamness. and O'Brien
reported significant increases in the peripheral neutrophil count at 1 and 2 h
after 40 minutes of downhill jogging (75). Smith et al. suggest that the elevated
levels of peripheral white blood cells are the result of the increased levels of
circulating polymorphs (a type of neutrophil), which is also seen 1 to 2 h after
exercise. The acute inflammation literature has also reported elevated levels of
polymorphs during the same time period. The authors interpreted these results
as evidence to support the theory that acute inflammation is the underlying
cause of DMS.

However, a key study conducted by Friden, Sjostrom. and Ekblow in 1983
did not support the hypothesis that acute inflammation is the underlying cause of
DMS (40). Friden et al. obtained muscle biopsies from the vastus lateralis
muscles of human subjects following eccentric exercise. Although the muscle
biopsies exhibited the classic ultrastructural damage that is associated with
DMS, no invasive macrophages were present. Friden et al. concluded that the
absence of macrophages suggests that acute inflammation is not the underlying
mechanism for the sensation of DMS.

In contrast, two fairly recent studies have reported the presence of

invasive macrophages in the muscle of human subjects following eccentric

exercise (50,66). However. the time frame for the appearance of the

4e
macrophages was much later than what is usually seen during a classic

inflammatory response and was also much later than what is reported in the
exercise induced muscle damage literature using rodents. The discrepancy
might be explained by the fact that muscle biopsies require removal of tissue
from the belly of the muscle; whereas it is possible that the damage is initiated in
the myotendinous joint. and only later spreads to the belly of the muscle (7).
Therefore it is more than likely that the human muscle biopsy samples missed

the sites of injury especially during the early stages of DMS.

The Effect of Anti-inflammatogy Drugs on DMS

In a continued effort to determine the role of acute inflammation in the
etiology of DMS, several investigators have used anti-inflammatory drugs to
study the mechanism of DMS. The following section provides a critical review of
five studies that have employed non-steroidal anti-inflammatory agents to
evaluate their role on muscle soreness, muscle weakness, and muscle damage
inferred from serum enzyme activity changes that accompany DMS. Although
four out of the five studies employed different exercise protocols and used
different anti-inflammatory agents, their main hypothesis remained the same: If
the mechanism of DMS is acute inflammation, then administering anti-

inflammatory drugs will reduce DMS.

47
Effects of Aspirin on DMS after Performing Elbow Extensions

Francis and Hoobler in 1987 investigated whether the intensity of DMS
induced by eccentric exercise is attenuated by aspirin (35). Aspirin via a dose-
dependent mechanism inhibits prostaglandin synthesis which in turn results in a
decrease in white blood cell activation and inhibits release of lysosomal
enzymes. Twenty healthy female subjects were randomly assigned into either
the control group (n=10) or the aspirin group (n=10) which took 10 grains (650
mg) of aspirin 4 times for 48 h starting 4 h prior to the exercise (n=10). Pain
scores were measured at 24 h and 48 h after exercise using the Likert scale. 0
representing no soreness and 6 representing unbearable soreness. The
subjects were asked to verbally report the sensation of pain in the elbow flexor
muscle group. Elbow range of motion was measured at 24 h and 48 h using a
goniometer. Maximal elbow flexion troque was measured using the Cybex ll
isokentic dynamometer at 60°ls and 180°ls at both times.

The eccentric exercise was performed with the Nautilis upper body biceps
machine. The peak torque developed during the maximal elbow flexion at 60°ls
was used as the starting weight for the eccentric exercise. Each subject
performed as many repetitions as possible at this predetermined weight until the
subject was unable to lower the weight slowly in a controlled fashion. The weight
was then lowered by 5 pounds and the procedure was repeated at the new
weight. The weight was continually decreased until the final weight with which
the subject exercised was 5 pounds.

Francis and Hoobler found that muscle soreness was not significantly

43
different between the two groups at 24 h; whereas, the muscle soreness of the

aspirin group was approximately 25% (p<0.05) less than the control group at 48
h. Both groups exhibited a significant decrease in the range of motion at the
elbow at 24 h and 48 h compared to the pre—exercise values. The aspirin group
exhibited approximately 50% less change in the range of motion at the elbow
than the control group at 24 h and 48 h. Both groups showed a dramatic
decrease in maximal elbow flexor force at both time periods, but the change was
not significantly different when compared between groups. Based on these
findings, the investigators concluded that the reduced soreness and improved
elbow range of motion in the aspirin group was the result of aspirin inhibiting
prostaglandin synthesis and release.

There are several flaws with the Francis and Hoobler study that need to
be addressed before one can conclude that administering aspirin reduces the
sensation of DMS. First, the investigators did not screen the subjects for
resistance training prior to the study. It has been well established that exposure
to even a single bout of eccentric exercise causes a protective effect in the
muscles involved for up to 6-10 wk (14). Second, Francis and Hoobler collected
general soreness data which relates to the overall soreness of the muscle at
rest. It has been shown repeatedly by several investigators that the sensation of
pain in DMS is more pronounced during contraction or palpation and not during
rest (5.6.20.74). This effect is the result of increased production of
prostaglandins and swelling that accompanies DMS which in turn produces a

state of hyperalgesia (increased sensitivity) in the nociceptors. As a result, a

49
previously benign increase in intramuscular pressure might now be able to

activate the afferent pain fibers. Therefore, a more appropriate measure of
soreness would be one that documents the perception of pain during a
contraction or palpation.

In addition, the eccentric contraction rate was controlled using a verbal 3
count. This method allows for a great degree of variability between the subjects
in terms of total work performed. The investigators did not provide any data in
terms of the subjects’ height. weight, and pre-exercise maximal elbow flexor
force; and they did not report if there was any significant difference between the
two groups. Lastly, Francis and Hoobler in the methods section incorrectly
referred to the control group as the placebo. Therefore. the conclusions drawn

by Francis and Hoobler on the effects of aspirin are limited.

Effects of Diclofenac on DMS after Downhill Running
In an attempt to conduct a more controlled study. Donnelly. McCormick.

Maughan, Whitting and Clarkson used a double blind crossover design to
evaluate the effects of a non-steroidal anti-inflammatory drug on DMS (26).
They used 20 healthy. untrained subjects who were required to run downhill
(12°) for 45 min on two occasions separated by 10 wk. The running speed.
which was the same for the two running sessions, was set to elicit a heart rate
equivalent to 75% of the age-adjusted maximum heart rate (220-age). General
perceived soreness, serum activities of several enzymes, and soreness elicited

by the application of minor pressure over specific areas, were measured before

50
and at 6, 24. 48, and 72 h after the exercise.

Each subject took either 50mg of diclofenac or placebo before and for 72
h at 8-hour intervals after each run according to a randomized double blind
crossover design. Therefore, each subject took a total of 500mg of diclofenac
either during the first or second exercise bout. Diclofenac, like aspirin, is a non-
steroidal agent that possesses analgesic. antipyretic and anti-inflammatory
properties (41). Diclofenac inhibits cycoloxygenase and also appears to reduce
the intracellular concentrations of free arachidonate in leukocytes. Donnelly et
al. chose diclofenac because its potency is considerably greater than that of
other anti-inflammatory agents.

Donnelly et al. reported that administration of diclofenac did not
significantly reduce the magnitude of the enzyme response to exercise.
However. the serum enzyme response to the second run was reduced indicating
that a single bout of downhill running for 45 min. was able to produce a
protective effect lasting at least 10 wk. Diclofenac did not significantly reduce
total soreness at either period of the study nor did it consistently reduce probe-
induced soreness at any of the specific sites. It did. however, reduce the general
soreness in the front lower leg and back thigh and also reduced the probe-
induced soreness in the middle of the back thigh after the first exercise bout.

The researchers tried to explain the inconsistency in the soreness data by
suggesting that there might be some form of interaction between the action of
diclofenac and the protective effect of eccentric exercise. The possibility of such

an interaction was addressed by Evans (29). He suggested that since

51
prostaglandins increased muscle protein synthesis, inhibiting their production

would hinder muscle repair, making the muscle susceptible to further damage.
The results from the Donnelly et al. study seemed to indicate the opposite-
inhlbiting prostaglandin synthesis provided a protective effect against further
soreness and muscle damage. Another explanation for the difference in
soreness is that downhill running produces more muscle damage in certain
muscles of the lower leg. Its more than likely that the muscles of the lower leg
that did not change in soreness were contained in expandable compartments
where the exercise induced swelling did not produce a state of hyperalgesia.
Along the same line. it is entirely possible that the reason why diclofenac
did not reduce soreness is due to the fact that the 8 h interval between doses
might have been too long. Diclofenac extensively binds to plasma proteins and
has a half-life in plasma of about 1 to 2h. On the other hand. diclofenac has
been reported to accumulate in the synovial fluid, which might prolong the
therapeutic effects beyond the plasma half-life. Regardless of the mechanism.
therapeutic doses of diclofenac in a double blind crossover design did not reduce
serum enzyme changes but might reduce soreness at specific locations in the

lower leg.

Effects of Ibuprofen on DMS after Downhill Running

In a continued effort to determine the role of anti-inflammatory drugs on
muscle soreness. muscle weakness, and muscle damage inferred from serum

enzyme activity changes. Donnelly. Maughan, and Whitting in 1990 administered

52
ibuprofen or placebo to forty healthy untrained subjects (25). They had two

treatment groups: group 1 received ibuprofen at the first exercise bout and
placebo at the second exercise bout. and group 2 received placebo at the first
exercise session and received ibuprofen at the second exercise session. The
experimental design and exercise protocol were very similar to their pervious
study from 1987 with minor changes. Most obvious is that they used ibuprofen
rather than diclofenac in the current study. Second, the dose administered prior
to each exercise bout was increased to 1200mg of ibuprofen, which was then
followed by 600mg every 6 h up to 72 h post-exercise. Therefore, each subject
took a total of 84ng of ibuprofen. Third, Donnelly et al. measured isometric
endurance time at 50% of maximum voluntary contraction, which is an indicator
of the subject’s ability to sustain a muscle contraction. Fourth, the running was
decreased to elicit a heart rate equivalent to 70% of the age adjusted maximum.

Donnelly et al. found that the total muscle soreness. the sum of all 20
soreness sites, increased significantly after both downhill runs. However, there
was no difference in the soreness between the two treatment groups. The
soreness data was collected on a scale of 20 (no soreness) to 200 (very. very.
sore at all points). The highest reported mean soreness was 50 i 6 (mean i
SEM) for both treatment groups, suggesting that the damage induced by
downhill running was not severe. The changes reported in isometric strength
and 50% endurance time also support this notion that downhill running at the
prescribed speed did not cause extensive damage.

First, isometric strength decreased significantly after each exercise

53
session. However. ibuprofen did not significantly affect the loss in isometric

strength. The decline in strength was no more than 10% percent of the pre-
exercise isometric strength and was maximal at 24 h post-exercise for both
treatment groups. The decline in strength was not significantly different between
the first and second exercise sessions. More importantly, Donnelly et al.
reported that strength returned to normal level by 72 h post-exercise for both
treatment groups. In contrast, Clarkson et al. reported that following eccentric
arm exercise there was a 50% loss in isometric strength and strength was
gradually restored by 10 d post-exercise (20). Based on the assumption that
strength loss following eccentric exercise is related to muscle fiber damage. it
appears that the downhill running protocol employed by Donnelly et al. did not
cause needy as much muscle damage as the eccentric arm exercise employed
by Clarkson et al.

Second, the 50% endurance time decreased significantly following both
exercise sessions. The most drastic decrease occurred at 6 h post-exercise but
returned to pre-exercise levels by 72 h post-exercise. The authors suggested
that the reduced ability of the muscle to sustain a contraction following downhill
running was the result of myofibril damage. Although the decrease in 50%
endurance was significant. it was a relatively small decrease. Therefore, it is not
certain if the decrease in 50% endurance time reflects muscle fiber damage.

In contrast. the measured serum activities of creatine kinase. lactate
dehydrogenase. creatinine. urea. and aspartate transaminase before and after

each exercise session at 6, 24. 48, and 72 h suggest that muscle damage did

54
indeed occur after downhill running. Serum levels of creatine kinase and

aspartate transaminase increased to a maximum at 24 h post-exercise after both
exercise sessions. The lactate dehydrogenase, creatinine. and urea reached the
maximum at 6 h post-exercise after both exercise bouts. However, only creatine
kinase, lactate dehydrogenase, and aspartate transaminase were significantly
lower after the second exercise session. The investigators did not provide any
explanation as to why creatinine and urea did not show this decrease in serum
activity after the second downhill run. It is more than likely due to the fact that
creatinine and urea are less sensitive indicators of muscle damage.

More importantly. it was reported that the serum activity of creatine kinase
and serum urea level were higher in subjects receiving ibuprofen than in subjects
receiving placebo. The authors suggested that this cannot be interpreted to
indicate increased muscle damage in the subjects receiving ibuprofen, since the
other indicators of damage did not increase. In addition, Clarkson et al. reported
that serum creatine kinase activity after downhill running vanes a great
deal between subjects and did not necessarily represent the extent of muscle
injury (20).

Donnelly et al. suggested that the increase in serum creatine kinase and
urea concentration were the result of ibuprofen’s action on plasma protein and
plasma volume. since it has been reported that ibuprofen administered
immediately after exercise reduces plasma protein content. This in turn would
facilitate the movement of intracellular proteins down the concentration gradient

into the blood stream. Again. they were unable to explain why the other serum

55
proteins did not change in a similar fashion. Regardless. Donnelly et al.

concluded ibuprofen did not reduce DMS, since neither muscle soreness nor
strength loss were reduced. This is consistent with Donnelly et al.’s 1987 study

in which diclofenac was used to treat DMS following downhill running.

Effects of Flurbiprofen on DMS after Performing Eccentric Exercise on a

Motor-Driven Bicycle
Kuipers. Keizer, Versteppen, and Costill investigated whether the intensity

of DMS induced by eccentric exercise was significantly reduced by the anti-
inflammatory drug flurbiprofen (53). They used six trained cyclists to perform two
bouts of negative exercise on a motor-driven bicycle 24 h after ingesting a
placebo or a drug in a double blind, randomized crossover design, with 3 to 6 wk
intervening between trials. Kuipers et al. reported that the flurbiprofen did not
significantly reduce the intensity of the DMS and concluded that that sensation of
DMS may not be the result of an inflammatory response.

However, a more critical review of the results revealed that all subjects
experienced significantly less soreness after the second trial, suggesting an
order effect. This effect can be explained by the fact that a single bout of
negatively biased exercise can provide a protective effect for up to 10 wk
following the initial bout of eccentric exercise. In addition, the subjects were
trained cyclists. therefore, they were very likely to be preadapted to the eccentric

component of the cycling exercise.

56
Effects of Theramutlc and Prophylactic Doses of Ibuprofen on DMS after

Performing a Npgatlve Bench Stepping Exercise

More recently, Hasson. Daniels. Divine, Niebuhr, Richmond, Stein, and
Williams in 1993 reported that prophylactic and therapeutic doses of ibuprofen
significantly reduced muscle soreness perception. and significantly increased
isometric, concentdc. and eccentric torque at 48 h when compared with placebo
and control (42). Ibuprofen produces anti-inflammatory, analgesic, and
antipyretic effects. At doses greater than 400 mg q.i.d. (four times a day),
ibuprofen is a powerful anti-inflammatory drug that interferes with the metabolism
of arachidonic acid by inhibiting the enzyme cycloxygenese. This in turn inhibits
the production of endoperoxides and the inflammation mediating prostaglandins
PGE2 and PGFZA.

Hasson et al. employed four different treatment groups. each with five
randomly assigned healthy subjects (23.8 :I: 4.3 yr, 71.8 :I: 10.6 kg; 172.3 :I: 8.6
cm), to evaluate the effects of ibuprofen at 24 h and 48 h post-exercise.

Subjects in group one (prophylactic) were given three 400 mg doses of ibuprofen
in 24 h for a total of 1200 mg (400 mg-TID). The initial dose of ibuprofen was
given 4 h prior tothe exercise bout and was followed by placebo for the reminder
of the study. The subjects in group two were also given 400 mg-TID with the
initial ibuprofen dose given at 24 h after the exercise bout (therapeutic), but were
given placebo prior to the 24 h dose. In the third treatment group the subjects
received placebo throughout the entire experiment (placebo). The subjects in

the control group received no treatment during the experiment.

57
The investigators used powered sugar as the placebo. The drug and

placebo were packaged in gel capsules for two reasons: (a) so that the subjects
would not be able to taste the difference between the drug and placebo. and (b)
it made the drug and placebo almost identical in appearance. A double-blind
procedure was employed throughout the experiment. in which the investigators
providing the treatment and the subjects were unaware of the treetrnent protocol.
The muscle soreness perception was measured every day using the
following protocol. The entire quadriceps muscle was divided into 2cm x 2cm
sites using a transparent grid sheet. At each site, a gradually increasing force
was applied by a 2cm metal probe up to a maximum of 50 N. The subjects were
asked to verbally respond when the pressure changed to one of discomfort and
the force was recorded. If the subject did not report any discomfort up to 50 N,
soreness was not considered to be present. The intensity of the pain was
determined as the inverse of the amount of force applied to each site that elicited
discomfort. The mean intensity for each subject was determined by the
summation of all the forces that elicited discomfort divided by the number of
individual 2cm sites that were reported to be sore. The area of muscle soreness
was calculated by dividing the number of sites that were reported to be sore by
the total number of sites covering the entire quadriceps muscle. Muscle
soreness for each subject was reported as a product of intensity and area.
Hasson et al. measured the following variables prior to the ibuprofen!
placebo treatment and at 24 h and 48 h after exercise: (a) maximal isometric

force, peak eccentric and peak concentric torque production using the Lido

58
isokinetic dynamometer; (b) electromyography activity, documented during peak

torque production; (c) skeletal muscle creatine kinase levels in the blood; and (d)
muscle soreness. The exercise protocol employed by Hasson et al. was a 10
min. step exercise—stepping up on a bench with the right leg and lowering the
body weight down with the contralateral leg. The step frequency was set at 15
cycles per min. Bench height was set at 110% of the lower leg length (floor to
knee joint line) and the subjects carried an additional load of 10% body weight.

All four treatment groups showed a significant decrease in maximal
isometric force, peak concentric and eccentric torque at 24 h compared to the
baseline data (p<.005). At 48 h, maximal isometric force of the prophylactic and
therapeutic ibuprofen groups were not significantly different from the baseline or
between each other. However. the maximal isometric force at 48 h of the
placebo and control groups were significantly less compared to the prophylactic
and therapeutic ibuprofen groups.

At 24 h, the decrease in the prophylactic ibuprofen group’s peak eccentric
and concentric torque from baseline was significantly less compared to the
decrease from baseline in the other treatments, and at 48 h was significantly less
compared to the placebo and control. At 24 h. the decrease in the therapeutic
group’s peak eccentric and concentric torque from baseline was not significantly
different compared to the decrease from baseline in the other treatment groups
and at 48 h was significantly less compared to the placebo and control. These
results suggest that the prophylactic treatment improved muscular performance
at 24 h and up to 48 h.

59
The results from the creatine kinase analysis showed that there was a

significant increase in all groups at 24 h and 48 h compared to baseline. There
were no significant differences between groups at any time. Hasson et al.
interpreted these results as evidence that ibuprofen did not limit the extent of
muscle damage and that quantification of muscle damage could not be detected
through creatine kinase analysis.

All subjects consistently reported muscle soreness in the distal head of
the vastus medialis and proximal head of the vastus lateralis. The rectus femoris
was not affected by the stepping exercise. The muscle soreness perception was
significantly less for the prophylactic ibuprofen group than the therapeutic.
placebo or control groups at 24 h post exercise (p < 0.05). At 48 h post-exercise
the muscle soreness perception was significantly less for the prophylactic and
therapeutic ibuprofen groups than the placebo or control groups (p < 0.05).
However. muscle soreness perception at 24 h and 48 h post exercise was not
significantly different between the therapeutic and prophylactic ibuprofen groups.

Hasson et al. suggested that the mechanism behind the improved
muscular performance and decreased DMS at 48 h in the therapeutic group was
due to the analgesic effect of ibuprofen. On the other hand. Hasson et al.
suggested that the mechanism behind the decrease in DMS and improved
muScular performance at 48 h in the prophylactic group was less likely the effect
of ibuprofen. based on the assumption that the drug should have been
completely eliminated from the system by 48 h. Unfortunately, they did not

measure plasma levels of ibuprofen. As an alternative explanation the

60
investigators suggested that the improvement in muscular performance at 24 h

for the prophylactic and at 48 h for both groups, based on EMG data, was the
result of increased motor unit activation compared to the control and placebo
groups. However, it must be noted that the exact relationship between motor
unit activation and pain perception remains unresolved.

Cleady. therapeutic and prophylactic administration of ibuprofen was
effective in reducing DMS and improving muscular performance at 48h post-
exercise. As noted by the authors, this effect was partially due to the analgesic
response of ibuprofen. Since the analgesic effects of ibuprofen is closely related
to the anti-inflammatory effects, it was not possible to partition the effects of

each using the measurements made by Hasson et al.

Summag of the Effects of Anti-inflammatory Drug. s on DMS
Thus far, based on the two studies by Donnelly et al. and Kuipers et al., it

appears that treatment of DMS with anti-inflammatory drugs is not beneficial. In
contrast, Francis and Hoobler and Hasson et al. reported that administering anti-
inflammatory drugs reduced DMS. The conflicting findings might be the result of
the different exercises employed by the investigators to induce DMS. Francis
and Hoobler employed elbow extension exercise to induce muscle damage,
while Hasson et al. used an eccentric or negative bench stepping exercise. The
two studies by Donnelly and co-workers used downhill running. and Kuipers et al.
used a motor-driven cycle to induce muscle damage, both of which have limited

eccentric loading. therefore limit damage induced by eccentric contractions. This

61
is supported by the fact that the increase in serum creatine kinase activity was

considerably greater after eccentric arm exercise compared to downhill running
(20). In addition, isometric strength recovered much faster, 72 h post-exercise,
after downhill running compared to 10d post-exercise after eccentric arm
exercise.

In a recent review. Clarkson et al. suggested that the metabolic stress of
downhill running is considerably higher and the immune response to downhill
running is much different compared to an eccentric exercise that involves a
smaller muscle group, for example elbow flexors or quadriceps muscle (20). In
addition. people are generally more likely to be preadapted to eccentric
exercises like downhill running and cycling. Thus. it is not appropriate to
conclude that nonsteriodal anti-inflammatory drugs do not reduce DMS based on
the data from downhill running or cycling studies. On the other hand, it appears
that anti-inflammatory drugs reduce DMS when exercise involves eccentric
contractions of a small muscle group for a short duration so as not to drastically

affect metabolism or the immune system.

Analgesic Effect Versus Anti-Inflammatory Effect

Studies discussed thus far were unable to resolve the controversy of how
the sequence and timing of DMS relate to acute inflammation as a possible
pathological mechanism. This is primarily due to the fact that the parameters
most often used to measure or document DMS were muscle performance and

perceived muscle soreness, which cannot be used to separate the analgesic

62
effects of the drug from the anti-inflammatory effects. The best path to resolving

this controversy is to measure intramuscular pressure, muscle compartment
cross-sectional area. muscle recruitment. and muscle damage following

eccentric exercise.

Muscle damage and intramuscular pressure are seldom measured since
they require invasive procedures, for example muscle biopsies. There are
several disadvantages with using muscle biopsies. First, muscle biopsy itself
can cause muscle damage and is a painful procedure. Second. muscle biopsy
samples a small area of the muscle which might not be damaged by eccentric
exercise. Therefore, a noninvasive procedure that samples a larger area of the
muscle may provide more complete information.

Muscle recnritment has been measured using surface and intramuscular
electromyography (EMG) following eccentric exercise. Validity of EMG to
measure small changes in muscle recruitment has been questioned by several
investigators (2). However, the controversy regarding use of EMG to detect
changes in muscle recruitment is beyond the scope of this review of literature.
Muscle recruitment is an important variable because several investigators have
suggested that the improvement in muscle performance with anti-inflammatory
drugs is the result of increased motor recruitment.

Lastly, measuring muscle compartment cross-sectional area will allow
investigators to evaluate the role of swelling development at the cellular level and

whether or not anti-inflammatory drugs reduce swelling associated with DMS.

63
Thus far, swelling has been documented by measuring changes in limb

circumference, which gives no information on the distribution of swelling amongst
muscle compartments or between intra- and extra-cellular spaces. Therefore, a
noninvasive procedure to measure changes in muscle compartments will provide
addition information regarding the effects of anti-inflammatory drugs on swelling.
In the last 10 years, advances in the field of MRI have allowed
investigators to measure muscle recruitment. muscle damage. and changes in
muscle compartment cross-sectional area following eccentric exercise. The
following sections will provide a detailed description of the accuracy of MRI to
detect small changes in skeletal muscle function following exercise( i.e.. muscle

recruitment, damage, and swelling).

Use of Functional MRI to Stupy Muscle Use and Muscle Trauma with

Resistance Exercise.

Despite its relatively recent development, MRI has quickly become the
method of choice for medical imaging in many orthopedic studies of soft tissue
and skeletal muscle pathology. Functional MRI exploits the changes in tissue
1H-NMR properties that occur during norrnel activity; as a result tissues and
fluids with excess amounts of free water have long T2 relaxation times and
appear as bright areas on T2-weighted images (3).

In 1988, Fleckenstein, Canby, Parkey, and Peshock published the first
report documenting increased signal intensity in T2 -weighted spin-echo images

of exercised muscle immediately following exercise (32). Soon thereafter,

64
several studies demonstrated that the increase in T2 signal, which occurs in

exercised muscle, can be used to determine the extent of muscle recruitment for
a given exercise by assessing the magnitude of change in image brightness
(2.31.49). This change in image brightness or contrast is an acute change that
occurs immediately following exercise and even during exercise but begins to
wane within minutes. returning to resting values by approximately 40 to 45 min
post-exercise.

In addition, MRI is also able to document the changes associated with
exercise-induced muscle trauma which develops more gradually with DMS (71).
These change are seen as a delayed increase in T2 signal intensity that begins
to be visible approximately 12 hr post-exercise and peaks at 24-48 hr post-
exercise, paralleling the development of soreness and swelling. The precise
mechanism of acute and delayed contrast change following exercise is not yet

clear, but is known to be related to changes in muscle water chemistry (43).

Fundamental Principles of Magnetic Resonance Imaging
MR imaging is based on three different and independent fundamental

nuclear magnetic resonance parameters: spin density, T1 relaxation time, and
T2 relaxation time (12). The following section is a brief summary of the
fundamental principles of MRI, adapeted from Bushong’s textbook entitled
Magnetic Resonance lmaging-_-Phy_sical and Biolpgical Principles (12).

A sample containing hydrogen compounds placed in a strong static

magnetic field results in a net magnetization of all the nuclei. An NMR signal

65
generated after placing the sample within a strong static magnetic field depends

on two variables: (a) concentration of hydrogen nuclei, and (b) environment of
the hydrogen nuclei. Spin density is a measure of the concentration of free or
loosely bound hydrogen nuclei available to produce the NMR signal. The
relationship between MR image and spin density is as follows: a tissue with a
high spin density will result in a stronger NMR signal which, in turn. will result in
better resolution of the MR image.

T1 and T2 relaxation times are measured by placing samples within a
strong static magnetic field and then applying radio-frequency energy in a
pulsating fashion (2,3,43,). As a result. mobile hydrogen nuclei absorb the
energy and the net magnetization vector changes in both magnitude and
direction. This new magnetization state is a higher energy state, which produces
a detectable signal that declines over time by way of two coexisting processes
described as T1 and T2 relaxations, both of which are measured in milliseconds.
typically in the range of 10-500ms in most tissues. T1 relaxation is the result of
excited hydrogen nuclei’s retuming to the lower energy state or equilibrium,
which is accomplished by releasing energy to neighboring hydrogen nuclei within
the molecule or to neighboring molecules with similar resonant frequencies. The
relationship between the MR image pixel intensity and T1 relaxation time
involves a complex series of calculations. which is beyond the scope of this
literature review. In simple terms. on T1 -weighted images, the tissues with long
T1 relaxation times will appear dark and tissues with short T1 relaxation times

will appear bright.

66
T2 relaxation involves the decay of the phase—coherent oscillation of

individual magnetic dipoles of excited hydrogen nuclei in the presence of a
strong static magnetic field. Immediately after the initial excitation pulse, all of
the individual nuclear magnetization vectors are rotating in synchrony, or “in
pluse.’ This coherence of phase then begins to break down as some individual
vectors begin to rotate slightly slower and some faster, producing vector
components that partially cancel one another. The two main processes that
contribute to the loss of phase-coherence are: (a) heterogeneity of the static
magnetic field and (b) interactions between neighboring magnetic dipoles. which
create local distortions of the field around each nucleus. Heterogeneity of the
static magnetic field can be minimized by the use of a process called “spin-
echoes.” Therefore, the main contributor to the loss of phase-coherence is the
interactions between neighboring magnetic dipoles.

In a pure water solution, rotational movements of the individual dipoles
occur so fast that they average each other out. However, if the free mobile
hydrogen nuclei are bound to proteins and/or other molecules. the rotational
movements of individual dipoles are slowed down for brief periods of time
(microseconds). As a result, these random interactions cause loss of phase-
coherence, and T2 relaxation time characterizes the rate of this decay of the
NMR signal following initial excitation. Therefore, a heterogeneous sample will

result in a relatively short T2 relaxation time.

67
Exercise-induced Acute T2 Increase: Qualitative Index of Muscle

Recruitment.

Using a low magnetic field (0.35T). Fleckenstein et al. reported increased
signal intensity in both the T1 and T2 weighted MR images of exercised muscle,
and that the changes in signal intensity correlated weakly with the level of
exertion (32). The weak correlation between signal intensity and level of exertion
was partly due to the low magnetic field utilized in the study. More recent studies
have shown that, at 1.5T. the change in T1 signal is minimal compared to T2
signal changes following exercise (2,21). Regardless, results of the Fleckenstein
et al. study cleady showed that, following exercise, changes in signal-intensity
can be utilized to determine which muscle groups were recruited.

It was not until 1990 that the relationship between T2 signal intensity and
exercise intensity was cleady established by Fisher. Meyer, Adams. Foley and
Potchen (31). Fisher et al. had 8 untrained subjects lying supine in the GE 1.5T
superconducting magnet perform three consecutive bouts of resisted ankle
dorsiflexion against graded loads (load 1 < load 2 < load 3). Each subject
dorsiflexed against each of the three loads for a total of 1.5 min at a constant
rate of 1Hz. The loads were presented to the subjects in a random order in an
attempt to minimize any load order effects that might affect T2 signal intensity.
The investigators allowed subjects at least 30 min between sequential exercise
bouts for recovery. Scans were collected before and immediately after each
exercise bout and during the recovery period (5,10,15,20. and 30 min).

Data from a subset of subjects was used to evaluate the effect of venous

68
occlusion on T2 signal intensity in resting and exercised muscles; venous

occlusion generally causes an increase in extracellular fluid volume. Three
subjects undenivent lower extremity venous occlusion for 5 min before, during,
and after one bout of ankle dorsiflexion against load 2 for 1.5 min at a rate of 1
Hz. Another subset of subjects (n=3) underwent lower extremity venous
occlusion for 20 min at rest.

These investigators reported that the mean forces generated against
loads 1, 2. and 3 were 3.9 i 0.4, 6.5 i 0.5, and 9.2 :I: 0.5 kg. T2 values of
exercised muscles increased significantly compared to resting muscles. The
extent of increase in T2 values of exercised muscles was dependent upon the
mean force generated during exercise. Fisher et al. were able to adequately
describe the relationship between force generated during exercise and T2 values
by the following first order equation: T2 = 29.6 + 0.9 X Force. T2 values returned
to near resting values by approximately 25 to 35 min, independent of mean force
development.

Venous occlusion during exercise resulted in larger T2 values compared
to T2 values without venous occlusion, although mean force generated was
slightly lower with venous occlusion. Additionally. venous occlusion during
exercise augmented the typical exercise-induced increase in muscle cross-
sectional area. Other studies have demonstrated that the increase in muscle
cross-sectional area following low-intensity exercise is primarily the result of
increased extracellular volume (73). Venous occlusion alone. however, resulted

in minimal increase in muscle T2 values. Therefore, a direct relationship could

69
not be established between the increases in T2 and muscle cross-sectional

areas (extracellular volume), resulting from venous occlusion. exercise. and
venous occlusion during exercise. Finally, venous occlusion did not affect the
postexercise T2 recovery time or rate.

Because the extent of the contrast change in T2-weighted images of
exercised muscles is dependent on average force generated by the muscles
during exercise, Fisher et al. suggested that this phenomenon can be utilized to

study muscle recruitment for a given exercise.

Exercise-induced Acute T2 Increase: Concentric vs Eccentric Exercise

In 1991 Shellock. Fukunaga. Mink. and Edgerton compared exercise-
induced acute T2 increase following concentric versus eccentric exercise (70).
Five subjects performed standing biceps cud exercise using a single dumbbell
weighted to a normalized percentage of each subject’s body weight (15% for
women and 20% for man). One arm was randomly selected to perform
concentric contraction (biceps curl) starting with the elbow in a fully extended
position and bending the elbow to full flexion. The weight was then passed to
the opposite arm by an assistant to perform the eccentric contraction. which was
accomplished by lowering the weight with the arm starting at full flexion and
lowering it to full extension. Throughout the entire range of movement the palm
was supinated in both eccentric and concentric movements. Subjects were
encouraged to maintain strict form and maintain a constant rate of eccentric and

concentric contractions.

70
Subjects performed isolated alternating eccentric and concentric

contractions until perceived exhaustion and “failure.” As a result. both eccentric
extremity and concentric extremity performed equal number of repetitions at
approximately the same rate; therefore. both eccentric and concentric actions
were performed at the same relative work level. MR images were acquired
before and immediately following exercise of both extremities.

Shellock et al. reported a larger increase in signal intensity of MR images
of the biceps and brachialis muscles immediately after performing concentric
contractions, compared to a minimal change in signal intensity of the biceps and
brachiallis immediately following eccentric contractions. Signal intensity of the
triceps muscle (nonactive muscle) appeared unchanged compared to pre-
exercise images in both extremities.

Shellock and coworkers noted that T2 relaxation times of the unexercised
triceps muscle were not significantly different between the concentric and
eccentric extremity. Significant increases in T2 relaxation times were reported
for the biceps muscles following concentric and eccentric contractions. More
importantly, the increase in T2 relaxation times for the biceps performing
concentric contractions were significantly higher than those performing eccentric
contractions. Since T2 values have been reported by Fisher et al. to be related
to exercise intensity, and because eccentric contractions require less of an
energy expenditure than concentric contractions at the same load. it is not
surprising that Shellock et al. found T2 values following eccentric exercise

significantly lower than T2 values following concentric exercise.

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In a continued effort to determine the relationship between T2 signal and

exercise intensity. in 1992 Adams, Duvoisin. and Dudley conducted a study to
compare contrast changes in MR images with EMG following graded concentric
and eccentric exercises(2). Prior to advances in the field of medical MR imaging.
EMG was the noninvasive method of choice for analyzing muscle activation
during exercise. Research has shown that there is a good correlation between
the integrated root mean squared EMG (IEMG) and force development during
exercise. Therefore, as pointed out by Adams et al.. increases in IEMG reflect
increased motor unit recruitment and not increased firing frequency of already
recruited motor units.

Adams et al. collected both MRI and EMG data on seven subjects who
performed eccentric and concentric exercisesof increasing intensity. Each
subject started the exercise session by performing five sets of 10 eccentric arm
cuds at 40% of their 10 repetition maximum for concentric cuds. There was a
1.5 min rest period between sets and a 30 min recovery between bouts. Surface
EMG signals were collected from both heads of the biceps brachii and the long
head of the triceps brachii muscles during exercise. Subjects were imaged
before and immediately after performing 5 sets of 10 repetitions. After the 30
min recovery period, the subject performed 5 sets of 10 concentric arm curls at
the same relative resistance on the opposite arm. Again, there was a 1.5 min of
rest between sets and 30 min of recovery between bouts, during which subjects
were again imaged. This exercise protocol was followed at three different

resistances of increasing magnitude: 60, 80. and 100% of the 10 repetition

72
maximum for concentric cuds.

Adams et al. reported that the IEMG of the biceps brachii muscle was
significantly less (p < 0.05) for eccentric than for concentric contractions at any
given exercise intensity. IEMG increased as a function of relative resistance in
both types of contractions. Rate of increase and absolute IEMG was significantly
higher (p <' 0.05) for concentric contractions, suggesting increased motor unit
recruitment.

T2 changes were uniform throughout the 10 regions that were sampled in
each image slice of the biceps brachii; as a result, values for the 10 regions
within each image plus the five image slices were averaged prior to analyzing the
T2 response to exercise. Shifts in T2 weighted MR images of the biceps brachii
after concentric and eccentric exercise increased as a function of the relative
resistance. However, rate of T2 increase and absolute T2 values were greater
following concentric exercise. These results reinforce the fact that muscles have
an inherent ability to generate greater force during eccentric actions with a given
number of active motor units. Finally. there was a strong significant correlation (r
= .99, p < 0.05) between IEMG and the shifts in T2 weighted MR images
following both concentric and eccentric contractions.

Theseresults were interpreted as evidence that the shifts in T2 weighted
MR images increase as a function of exercise intensity. However, use of
exercise-induced acute T2 contrast change as a quantitative index of muscle
recruitment is limited because the exact mechanism of the T2 increase is

unclear. It has been suggested that the exercise-induced T2 increase is related

73
to changes in muscle fiber water content.

Proposed Mechanism of Exercise-Induced Acute T2 Increase

The contrast in MR images depends primarily on spin density of the tissue
imaged. For example, bone, fat, and muscle have different spin densities which
result in markedly different contrasts. In addition, T1 and T2 relaxation
processes, independent of each other, have significant effects on the contrast of
the final MR image. This effect has been illustrated in the studies discussed thus
far. For instance, exercised muscles have longer T2 relaxation times than non-
exercised muscles. and subsequently appear brighter on T2-weighted images.
As Adams et al. noted, these changes in muscle T2 signals depend on the
molecular environment of protons of either water or lipid, since these contain the
largest spin density (2). Adams et al. reported that T2 of bone marrow did not
change following exercise. According to the researchers this was evidence that
there were no gross changes in lipid signal. Additionally, as suggested by the
researchers, it is very unlikely that these acute bouts of exercise caused any
changes in intramuscular fat content. in which case T2 signal intensity changes
must be the result of one or more of the water compartments in the muscle.

Recent research has shown that exercise causes rapid alterations in
skeletal muscle water content (72.73). Increase in water content of exercising
muscle is the result of increased water movement across the capillary bed.
Hydrostatic pressure in capillaries and osmotic forces in both capillaries and

interstitial fluid contribute to the increased water content of skeletal muscle.

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F urtherrnore. functional capillary surface area increases during exercise in active

muscles. resulting in increased capillary pressure. Elevated capillary pressure
then increases the rate of water filtration from the capillary bed into the interstitial
space.

Low intensity exercise has been shown to cause a drastic increase in
extracellular water volume (e.g., up to 100%) and only a minor increase in
intracellular water volume (e.g., close to 10%) (73). On the other hand. with high
intensity exercise majority of the increase in muscle water content is due to an
increases in intracellular water content (72). Adams et al. and Shellock et al.
reported a significant increase in T2 signal following high intensity concentric
exercise (2.70). Both groups of researchers suggested that the increase in T2
signal following high intensity exercise was the result of increased intracellular
water content

Fisher et al. reported similar increases in muscle volume following low
intensity exercise and venous occlusion. Venous occlusion generally causes an
increase in extracellular volume. However, venous occlusion alone and venous
occlusion during exercise had little additional effect on T2 signal intensity,
prompting Fisher et al. to conclude that increases in T2 signal intensity following
even low intensity exercise were related to intracellular water content. Therefore,
it is naive to assume that exercise-induced contrast enhancement is solely the
result of increased perfusion and extravascular movement of plasma water. The
Fisher et al. findings suggest that exercise-induced changes in MR images is

probably related to more complex intracellular events and/or intracellular water.

75
Delayed T2 Increase: An Index of Muscle Damage

Changes in T2 signal intensity discussed thus far have been acute T2
increases following exercise. Another observed phenomenon is a delayed
increase in T2 signal intensity following eccentric exercise but absent after
concentric exercise. Fleckenstein. Weatherall, Parkey, Payne, and Peshock
were the first to report on the delayed T2 increase following eccentric exercise
(34).

Fleckenstein et al. noted that all subjects (n = 6) reported muscle
soreness (DMS) after unaccustomed calf plantar-flexion exercise, which involves
concentric and eccentric loading. Two subjects were imaged before,
immediately after, and 24, 36, 48, and 72 hours post-exercise and at 13. 23. and
48 days post-exercise. These investigators also measured plasma creatine
kinase levels (CK), and collected subjective muscle soreness scores (on a scale
of 1 to 10). both of which have been documented to increase with DMS.

Results from the linear regression analysis showed a positive correlation
between the following pairs of variables: pain vs CK levels ( r = .59, p = 0.2), pain
vs T1 ( r = .87. p = .001), pain vs T2 (r = .67, p = .005). CK levels vs T1 (r = .50.
p = .05), CK levels vs T2 ( r = .76. p = .001). and T1 vs T2 (r = .73, p = .002). In
the first 72 hr post-exercise increase in CK levels lagged behind the increase in
pain and MR signal intensity. Although there is universal agreement that DMS
results in increased serum CK levels, several researchers have suggested that it
is inappropriate to interpret the increase in serum CK levels as a direct measure

of muscle damage. Signal intensity and pain peaked at 24—72 hr post-exercise.

76
Pain and serum CK levels returned to pre—exercise levels by approximately 2

weeks. However, signal intensity remained elevated for 48 days post-exercise.
Based on the positive correlations between pain, CK levels, and signal
intensity during the first 72 hr post-exercise. Fleckenstein et al. concluded that
the undedying mechanism of delayed increase in signal intensity is probably
related to the muscle damage that gradually develops with DMS. Fleckenstein et
al. were unable to provide an explanation as to why the signal intensity remained

elevated at 48 days post-exercise.

Exercise-Induced Delayed T2 Increase: Concentric vs Eccentric Exercise
Although, Fleckenstein et al. showed that the delayed increase in signal

intensity only occurred in subjects that reported DMS, the experimental design
was limited in terms of distinguishing damage resulting from concentric
contractions versus eccentric contractions. Since eccentric contractions are
most likely to cause DMS, Shellock, Fukunaga. Mink, and Edgerton conducted a
study to evaluate the delayed T2 increase following concentric versus eccentric
exercise (70).

Subjects (n = 5) performed isolated alternating eccentric and concentric
biceps cuds using a single dumbbell weighted to a normalized percentage of
each subject’s body weight ( 10% for women and 20% for men). One arm was
randomly selected to perform the isolated eccentric actions and the opposite arm
performed concentric actions. Subjects performed alternating eccentric and

concentric contractions at a rate of 1 repetition every 2 seconds until perceived

77
exhaustion. This same exercise protocol was employed by Shellock et al. in a

1991 study which documented acute T2 changes following eccentric and
concentric exercise (71). MR images were collected before, and 1 day, 3, 5, 10,
25, 40. 50. 60. and 80 days after exercise using a 1.5T 64-MHz unit and a
quadranture-driven. transmit/receive body coil. Subjective muscle soreness
scores and joint stiffness data were also measured before and on each day of
imaging.

Muscles that performed concentric exercise did not show a significant
increase in T2 signal intensity. pain. or joint stiffness on days 1-80 post-exercise
compared to pre-exercise values. The biceps muscle that performed eccentric
exercise had a significant increase in T2 signal intensity on days 1-80 post-
exercise compared to baseline measurements. Highest mean T2 signal
intensities were reported to occur on days 3 and 5 post-exercise and slowly
decreased towards baseline values by day 80. Muscle soreness and joint
stiffness following eccentric exercise returned to baseline values by day 10 post-
exercise.

These investigators suggested that the time course of the increase in T2
signal intensity are consistent with other studies that have documented a similar
time course for other indirect measures of muscle damage (e.g. pain, CK levels.
and swelling) following eccentric exercise. Therefore, Shellock et al. suggested
that the delayed increase in T2 signal intensity reported was most likely the
result of muscle damage induced by eccentric contractions. Increase in T2

signal intensity in days 1-5 was interpreted as evidence that muscle damage

78
progressively increased in days following eccentric exercise, consistent with the

acute inflammation theory of DMS.

Studies presented thus far have only suggested that the delayed increase
in T2 signal intensity is related to the muscle damage associated with DMS. In
1992, Nurenberg, Giddings, Stray-Gundersen. Fleckenstein, Gonyea, and
Peshock conducted a study to examine if there was a correlation between the
degree of delayed increase in signal intensity following eccentric exercise and
amount of ultrastructural damage and DMS (62). Nine untrained subjects ran on
a treadmill set at a negative 8% grade for 30 min at a speed of 8kmlh. MR
images were collected using a 0.35T imager at 48 and 96 hr post-exercise.
Muscle biopsy samples were taken from muscle regions that showed an
elevated T2 at 48 hr post-exercise. Structural features of muscle fibers were
assessed using electron microscopy.

Peak soreness was generally reported between 12-36 hr after exercise.
Linear regression analysis showed a strong correlation between the degree of
increase in T2 signal intensity at 48 hr post-exercise and the degree of
ultrastructural injury. Highest correlations were reported between the signal
intensity increases with the T1 -weighted and spin-density pulse sequences and
the degree of ultrastructural injury. As pointed out by Nurenberg et al.. these
results are not consistent with the findings of Fleckenstein et al. and Shellock et
al., who showed a significantly larger increase in T2-weighted images than T1-
weighted images.

Nurenberg et al. observed that the peak CK levels was considerably

79
greater in the study conducted by F leckenstein et al.. Lower peak CK levels was

interpreted as evidence that the damage induced by downhill running was less
compared to the more concentrated eccentric exercises employed by
Fleckenstein et al. and Shellock et al. Since. more severe muscle damage
generally results in more edema (free water), and free water plays a significant
role in the increase in T2-weighted signal intensity; Nurenberg et al. concluded
that this was the cause of the large increases in T2-weighted images that were
reported by Feckenstein et al. and Shellock et al.. Two flaws were identified with
this reasoning: (a) increase in serum CK level is a poor indicator of muscle
damage, and (b) more recent studies have shown that using a low magnetic field
generally results in a more pronounced T1 signal than T2 signal (2.20.21).

In addition. there was poor correlation between perceived peak soreness
and peak CK level. and between perceived peak soreness in the region of biopsy
and degree of ultrastructural damage. The most significant finding of this study
was that it showed a high correlation between areas with increased signal
intensity and ultrastructural injury. These results suggest that the magnitude of
delayed increase in T2 signal intensity is an index of the extent of muscle

damage.

Time Course of the Changes In T2 Relaxation Time and Muscle Cross-

sectional Area Following Eccentric Exercise
Majority of studies that have evaluated the delayed increase in T2

signal intensity associated with DMS have not documented the eady time course

80
changes in the T2 signal intensity following the transient acute increase in the T2

signal. Takahashi, Kuno. Miyamoto. Yoshioka. lnaki. Akime. Ketsuta, Anno. and
ltai conducted a study in 1994 to examine the eady time course data on the
changes in T2 relaxation time and muscle cross-sectional area following
eccentric exercise (81).

Six healthy male subjects were imaged before, immediately after. and at
7, 15, 20, 30. and 60 min and 12, 24, 36. 48, 72. and 168 hr post-exercise.
Subjective muscle soreness scores were collected every day for 6 days after
exercise. Plasma CK levels were measured before and at 5 and 60 min then 24
and 48 hr post-exercise. Subjects began the exercise in a seated position on a
chair 42cm high. then stood up on both legs. From the standing position, left leg
was slightly raised of die ground so that all the body weight was placed on the
right leg. While the left leg was slightly raised, the subjects slowly crouched back
down to the seated position without lowering the left leg. This movement was
completed in a total of 4 seconds and was repeated 300 times by all the
subjects. Takahashi et al. pointed out that during this type of movement the right
quadriceps muscle undergoes eccentric contraction as the subject advances
from the standing position to the sitting position. However, the investigators
failed to recognize that the right quadriceps muscle also undergoes concentric
loading as the subject goes from sitting position to standing position using both
legs.

In most subjects peak soreness occurred 1-2 days after eccentric

exercise. CK levels remained unchanged for the first 24 hr and then increased

81
significantly at 48 hr post-exercise. Immediately following the eccentric exercise

there was a significant increase in the T2 signal intensity and muscle cross-
sectional area of the quadriceps muscle as a whole, both muscle cross-sectional
area and signal intensity returned to baseline values by 60 min post-exercise. At
12 hr post-exercise signal intensity increased significantly, reaching peak values
at 24-36 hr after exercise. Cross-sectional area of the quadriceps muscle as a
whole increased significantly at 12 hr post-exercise and reached peak values at
12-24 hr after exercise.

Muscle cross-sectional area and T2 signal following eccentric exercise
appears to follow a bimodal response with the first peak occurring immediately
after the exercise, and the second peak sometime after 12 hr post-exercise.
These investigators also found that subjects with the lowest increase in T2 and
muscle cross-sectional area at 12 hr post-exercise exhibited a faster decrease in
muscle soreness. The bimodal response of muscle cross-sectional area and T2
signal was interpreted as evidence that two different mechanisms are
responsible for the acute and delayed increase in T2 signal intensity, and muscle

cross-sectional area following eccentric exercise.

Proposed Mechanism of the Delayed Increase in T2 Signal lntensl_ty

Several investigators have suggested that the delayed increase in T2
signal intensity is the result of increased edema associated with DMS (34.62.81).
The increase in edema in skeletal muscle during DMS is mainly due to the

following factors: (a) damaged connective tissue, (b) increased permeability of

82
the capillaries, and (c) increased permeability of the sarcolema. As discussed by

Takahashi et al., ultrastructural damage in muscle fibers results in accumulation
of degraded proteins and release of protein bound ions. Therefore, the chemical
environment of the water in damaged muscle fibers will be different compared to
undamaged fibers. However, it is not clear as to which of the above mentioned
changes is most responsible for the delayed T2 increase.

Takahashi and co-workers have suggested that the combination of
changes in proton concentration and chemical environment of muscle water
contribute to the increase in T2 signal intensity after 12 hr post-exercise. These
investigators also found that subjects with the lowest increase in T2 and muscle
cross-sectional area at 12 hr post-exercise emibited a faster decrease in muscle
soreness. In addition, Nurenberg et al. showed a good correlation between
increases in signal intensity and extent of ultrastructural damage following
eccentric exercise. Based on these findings, Takahashi and co-workers
suggested that the extent of muscle damage and rate of soreness
disappearance can be assessed by the magnitude of the delayed increase in T2
signal intensity.

According to this proposed mechanism of delayed T2, any additional
damage during the recovery period should result in an additional increase in the
delayed T2 signal intensity. Sorichter, Koller, Haid, Vlficke, Judmaier, Werner,
and Raas examined the consequences of a single bout of eccentric exercise with
and without additional concentric exercises during recovery (77). One leg was

randomly assigned as the control limb (unloaded leg) and the contralateral leg

83
was used to perform the exercise (loaded leg). Eccentric exercise was

performed using a specially designed exercise equipment that forced the
quadriceps femoris into a lengthening contraction. The eccentric load was set at
110% of the maximum voluntarily generated force. Subjects in the eccentric
group performed a single bout of seven eccentric contractions, at a rate of 1
repetition every 1-2 seconds with 15 seconds of rest between each repetition.

Subjects in the eccentric/concentric group performed addition concentric
contractions before and 2 h after exercise, and 1, 2, 3, 6, and 9 days after
eccentric exercise. Subjects performed two voluntary maximum knee extensions
at angular velocities of 30°ls and 90°ls and three voluntary maximum knee
extensions at 180°ls. Therefore, it is misleading to use the term light concentric
in describing the addition concentric contractions performed by the
eccentric/concentric group.

Several inflammatory markers and serum CK levels were measured
before eccentric exercise, 2h after exercise, and 1, 2, 3, 6, and 9 days after
exercise. MR images were acquired from the loaded and unloaded legs of both
groups starting at 3, 6, and 9 days after exercise. The timing was chosen based
on a pervious study in which the investigators on day 3 post-exercise found the
first significant increase in signal intensity.

Over time, inflammatory markers did not increase above the reference
range. Additional concentric contractions caused a five fold increase in the
serum CK levels. T2 weighted relaxation times for the eccentric and

eccentric/concentric group were not significantly different, over time. Sorichter et

84
al. noted that the maximum mean T2 values of the affected regions of muscle

from the eccentric group and concentric group were 19.65 ms and 36.44 ms. It
is possible that the variance in the data, indicated by the large standard
deviation, contributed to the non-significant results.

Investigators analyzed the calculated mean T2 relaxation times at
different time points using the student’s t-test to compare the two groups. A
more appropriate method would have been to look at the change in T2 relaxation
times ( AT2 ) using a t-test which would have limited the influence of variance in
the data set. To measure the change in T2 relaxation times from quadriceps
muscles experiencing DMS more accurately, the difference should have been
calculated by subtracting the T2 value measured before performing the
additional concentric exercise from the T2 value measured after performing the
concentric exercise. However, these investigators did not acquire pre-exercise
images from the leg that performed additional concentric contractions. Both
legs, loaded and unloaded, in the eccentric/concentric group were imaged after
performing the additional concentric contractions.

In any event, it is unclear what influence, if any, an elevated T2 will have
on the acute T2 response to additional concentric exercise. Furthermore, a
review of the MRI literature to date reveled that most of the studies acquired the
first images starting at 3 days post-exercise, there is little information

documenting the early time course of the development of delayed T2 increase.

 

CHAPTER III

RESEARCH METHODS

As discussed in the two preceding chapters, the precise mechanism of
acute and delayed T2 contrast changes in MR images following exercise is not
yet clear; but, it is known to be related to changes in the intracellular water
chemistry (64). A review of the MRI literature to date has shown universal
agreement that the extent of the acute T2 elevation following exercise is a good
qualitative index of motor recruitment (2,31 ,49,21 .70). Furthermore, the acute
increase in T2 reaches some upper limit with increasing exercise (33). Several
studies, focusing on the delayed T2 elevation following exercise-induced muscle
trauma, have reported a strong correlation between delayed increase in T2
signal intensity, ultastructural damage, and sensation of DMS (2.31.62).
Although acute and delayed T2 changes might result from different mechanisms,
no experiments have been reported which specially test this claim.

The primary objective of the current study was to compare the changes in
acute T2 following a bout of concentric exercise performed before vs. 24 _h after
eccentric exercise. Eccentric exercise has been shown to induce: (1) both an
acute T2 change, which fades within an hour similar to the acute T2 elevation

due to concentric exercise, and (2) delayed T2 change which peaks at 24-48 h

85

 

86
and then is sustained at an elevated level for a period of days to weeks

(2,31,62).

Specifically, the present design tested whether or not the acute T2
response to concentric exercise is larger with an elevated resting T2 resulting
from the exercise-induced muscle trauma. Based on the hypothesis that the
undedying mechanism of the acute T2 and delayed T2 is the same and acute T2
has an upper limit at a given exercise intensity, one would expect that a standard
bout of concentric exercise would cause the same total T2 response whether
starting from a normal or an elevated baseline T2. In this scenario, acute
concentric and delayed eccentric T2 changes occur via the same mechanism, so
concentric exercise on top of an eccentrically—elevated baseline T2 will reach the
same absolute T2 “saturation” or “plateau” level as a concentric bout performed
from a normal baseline. This is illustrated by column C in Figure 4, where the T2
effects of concentric exercise after eccentric exercise is the same as for
concentric exercise alone.

Alternatively, if the acute concentric and delayed eccentric T2 changes
occur via different mechanisms, it is very likely that the effects would be additive.
In this case, a concentric bout after T2 elevation due to eccentric exercise would
be expected to produce an acute T2 change greater than the initial concentric
exercise, and the difference should be approximately equal to the eccentrically-

induced T2 elevation. This situation is depicted by column D of Figure 4.

 

87

 

D
Change
in A C
T2 ....... “mm, .................................................... -m..---. ...............................................
or
4 B _

 

 

 

 

 

 

 

     

 

 

 

Con1 Ecc-24h Con2 Con2
' non-additive additive

Figure 4. Additive vs. non-additive T2 changes. Column A represents acute T2
elevation following a single concentric exercise bout (Con1). Column B depicts
the delayed T2 increase at 24 h after eccentric exercise (Eco-24h). The
predicted effect of a second concentric bout after eccentrically-induced T2
elevation is represented by column C if the mechanisms are identical (Con2,
non-additive) or by column D if the mechanisms are different (Con2, additive).

A secondary purpose of this study is to examine the early time course of
development of the T2 change following eccentric exercise. For this purpose, T2
data will be collected at 1, 2, 4, and 6 h following the bout of eccentric exercise to
document the early time course of the acute T2 following eccentric exercise and
the development of the delayed T2 increase. Although changes in this time span
are unlikely to be large and perhaps not even discernible from resting values,

documentation of these time points will fill gaps in the published data on T2 time

course following eccentric exercise.

88
Subjects

Four males and four females between the ages 21-40 were recruited from
the student population at Michigan State University. During the initial recruiting
process, subjects were interviewed to ensure that they had not participated in
any type of resistance training for a minimum of six months prior to the study.
Furthermore, subjects who engaged in heavy lifting as part of their daily activity
were also excluded from the study. Non-resistance trained subjects were used
in an attempt to minimize any prior adaptation that might reduce the
development of DMS and attenuate the T2 signal response. Subjects were also
screened for any acute or chronic diseases or injuries that might interfere with
their exercise performance (see Appendix A). Subjects with orthopedic implants
and/or pacemakers were also excluded from the study. The strong magnetic
field generated by the MRI magnet has been shown to interfere with pacemakers
and orthopedic implants. A week before the scheduled experiment, the subjects
met with the investigator on an individual basis to review the experimental
procedures, including possible risks. All subjects signed an informed consent
approved by the Michigan State University Committee on Research Involving

Human Subjects (see Appendix B).

Exercise Protocol and Timing of MRI Scans

Each subject was imaged at rest to obtain baseline data on “normal”
muscle and again before and after each exercise session. Therefore, the study

was conducted as a repeated measures design. This design allowed each

 

89
subject to serve as his/her own control. After completion of the baseline image

sets, subjects performed an isolated concentric standing biceps curl exercise
with the left arm using a single dumbbell weighted to a normalized percentage of
body weight (15% for women and 20% for men) (70).

Light concentric exercise began with the subject’s elbow in a fully
extended position. An assistant placed the dumbbell in the subject’s grip; the
subject then slowly flexed the elbow to full flexion. To control the rate of elbow
flexion, subjects followed the investigator's verbal count of “one-one thousand,
two—one thousand, thme-one thousand.” The weight was then removed from
subject’s grip so slhe could fully re-extend the elbow without external load.
Subjects repeated this movement for 3 sets of 10 repetitions at approximately 1
repetition every 5 seconds. There was a 1.5 min. rest period between sets.
Subjects were verbally encouraged to follow strict form (standing erect with feet
shoulder-width apart) (9). Subjects were also encouraged to maintain a constant
rate and keep the arms stationary throughout the entire range of motion. Palms
remained supinated throughout the full range of motion to ensure full activation
of the biceps brachii musde (9). MR imaging was performed immediately ( i.e.,
within a minute) following the completion of the concentric exercise bout,
followed by a two hour rest period. The two hour delay allows sufficient time for
the acute T2 signal to return to baseline (31). During the rest period, subjects
were instructed not to perform any lifting movements with the exercised arm

(e.g., lifting of book bags).

 

90
At the end of the two hour rest period, subjects were re-imaged to confirm

that the acute T2 signal had returned to baseline values. Immediately following
this scan, subjects performed the eccentric exercise. Since the muscle can
tolerate a larger load during the eccentric contraction, the eccentric exercise was
performed using a single dumbbell weighted to a higher normalized percentage
of each subject’s body weight (25% for women and 30% for men) (20,36).

Eccentric exercise began with the elbow in the fully flexed position. The
assistant placed the dumbbell in subject’s grip; the subject then fully extended
the elbow. Again, in an attempt to control the rate of elbow extension, subjects
followed the investigator’s verbal count of “one-one thousand, two-one thousand,
three—one thousand.” The weight was then removed from subject’s grip and the
subject retumed the unloaded arm to the starting position (i.e., elbow fully
flexed). Subjects repeated this movement for 3 sets of 10 repetitions at
approximately 1 repetition every 5 seconds, with a 1.5 min. rest period between
sets. Subjects were again encouraged to follow strict form and the palms were
supinated throughout the entire range of motion (9). Subjects were imaged
immediately following the eccentric exercise (PostEcc) and at 1, 2, 4, and 6 h
post-exercise.

At 24 h post-exercise, subjects wereimaged before and immediately after
performing the same light concentric exercise that was used at the start of the
experiment.

General muscle soreness was evaluated using a questionnaire (20).

Subjects were asked to rate their perception of overall soreness in the biceps

91
brachii muscle with the arm fully extended and relaxed on a scale of 1 (no

soreness) to 10 (extremely sore) before each exercise session, and at 12 h post

eccentric exercise and at 24 h intervals for 7 consecutive days (see Appendix C).

MR Imaging Parameters

Previous studies have shown that a standard two dimensional multiple
spin-echo pulse sequence with flow compensation maximizes the visibility of
exercise-induced contrast change (2,31,71,77,81). Pilot studies showed optimal
results with two echo times of 30 and 60 ms, with a repetition time of 2000 ms
and 0.75 NEX - number of excitations.

Images were acquired using a linear extremity coil and a 1.5T GE Signa
whole body imager. Subjects were positioned head first and prone, with the left
arm extended beyond the head. The coil housing was centered on a mark inked
on the brachial region 7.5 cm from the elbow joint along a line between the
olecranon and acromion processes. .

An initial coronal scan was used to locate the distal end of the biceps
muscle. From this point, ten axial slices 10 mm thick were acquired at 15 mm
intervals (5 mm gap) along the length of the brachium. With the TR/T E 2000/30
and 2000/60 and other parameters as previously noted, the total scan time for a

full series of 10 slices was 3 minutes and 32 seconds.

92
Images Analysis

Image sets were transferred to a Sun computer workstation to calculated
the muscle cross-sectional area and T2. Specifically, muscle cross-sectional
area and T2 were calculated using the analysis software package ‘x-vessel'
developed by Dr. Ron Meyer at Michigan State University (61). Images were
acquired over a 16 cm x 16 cm field of view using a 256x128 pixel matrix, giving
a resolution of 1.28 mm2 per pixel.

Cross-sectional area of the biceps brachii was calculated by the following
method: first the investigator selected an image that was near the belly or gaster
of the muscle, typically slice #6 out of the 10 slices acquired from the brachial
region. Then a region of interest was established by manually tracing around the
biceps brachii compartment, carefully excluding the subcutaneous fat and
skeletal tissue. Once the region of interest was established, the software
automatically calculated the number of pixels contained within this region.

Lastly, the biceps brachii cross-sectional area was calculated by multiplying the
total number of pixels within the region of interest by the per pixel area.

Past studies have used different methods of image analysis to calculate
the muscle T2, but none have directly compared the results of the different
methods within a single study (2,31,70,71 ,77,81). In the present study, three
different methods were used to calculate muscle T2, with the operator blinded to
subject identity in all cases.

In the first method, the investigator used a single image slice to outline a

small rectangular region of interest (‘box') within the biceps brachii muscle

93
compartment. The location of this region of interest or “box” was such that it did

not include any adipose tissue, blood vessels, subcutaneous fat, or bone. Image
slice #6 was selected out the 10 slices from the brachial region because pilot
data showed that this slice to have the largest change in the T2 immediately
following exercise and at 24 h post-eccentric exercise. Using x—vessel software,
a T2 value was calculated for each pixel in the region of interest and an overall
mean T2 value was reported.

Wrthin each subject, the same slice and rectangular region of interest was
used to calculate the mean T2 from the image sets acquired at the different time
points. Although the same slice was utilized in all subjects, the size and location
of the “box” varied slightly between subjects due to anatomical differences.

In the second method, the region of interest was defined by tracing
around the entire biceps brachii muscle compartment excluding subcutaneous
fat, bone, and residual blood flow artifacts within a single image slice. Therefore,
the mean T2 that was reported for each subject by this method provided an
overall average of the T2 values of the entire biceps muscle compartment within
a single image. Slice #6 was also used in this method to calculate the mean T2
of the biceps brachii muscle compartment from the image sets acquired at the
different time points.

The third method used the entire biceps brachii muscle compartment
excluding only the subcutaneous fat and bone from all ten slices to calculate an
overall average T2 at the different time points. Therefore, the mean T2 that was

reported for each subject using the third method provides an overall average of

94
the entire imaged region of muscle using all ten image slices at the different time

points.

Statistical Analysis

Initially, data were analyzed by using a repeated measures one way
analysis of variance (ANOVA) to assess difference with time in muscle soreness,
T2 relaxation times of the biceps muscle , change in T2 relaxation times from
baseline value, and muscle cross-sectional areas. If a significant difference was
noted (p<0.05) with the ANOVA, then post hoc tests were performed. These
tests make pairwise comparisons of the means. The Bonferroni's adjusted
paired t-test (p<0.05) was chosen for the post hoc analysis. Specifically, the
Bonferroni’s adjusted paired t-test was chosen over others because it allowed
the investigator to adjust the alpha values based on the number of comparisons.
Correlation analysis was conducted using the Speannan’s correlation test. The

results are presented as means i standard error (SE) of the means.

CHAPTER IV

RESULTS AND DISCUSSION

The content of this chapter is divided into five major sections. Subject
characteristics are presented first. Next, the acute T2 responses to a bout of
light concentric exercise performed before and after eccentric exercise are
described. Also included is a report on the time course of the delayed T2
increase following eccentric exercise. The third section presents the descriptive
analysis of the changes in muscle cross-sectional area 24 h following eccentric
exercise. This section is followed by a description of the self-reported muscle
soreness ratings following eccentric exercise. A discussion of the results as well
as comparisons with related literature are detailed in the final section. All results
are shown as mean :1: SE, with n = 8 for overall means or n = 4 for gender
subgroup means.

Subject Characteristics

Eight healthy non-weight-trained adults, four males and four females,

were recruited from the student population at Michigan State University. A

summary of subject characteristics is presented in Table 2.

95

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Subject characteristics. 96
Subject lD Gender Age (yr.) Height (cm) Weight (kg)
Subject 2 F 21 174 72.7
Subject 4 F 24 151.6 54.8
Subject 6 F 27 158 57.2
Subject 8 F 21 158.5 ' 54.1
Mean :1: SE (females) 23.3 :I: 1.4 161 :t 5 60 :I: 4
Subject 1 M 30 171.6 75.3
Subject 3 M 23 165.8 ' 67.8
Subject 5 M 27 178.4 91.1
Subject 7 M 26 182.9 76.6
Mean :1: SE (males) 26.5 :r: 1.4 175 a: 4 78 :1: 5
Overall Mean :1: SE 24.7 :r: 0.8 167 :1: 3 68 :1: 3

 

 

 

 

 

 

 

Acute and Delayed T2 Resmnses

In the present study, three different methods were used to calculate
muscle T2 values from the MR images acquired as described in Chapter 3. SE
as a percent of the mean T2 was calculated using all three methods for the
resting data set to determine which method minimized variance. SE as a
percent of the mean T2 calculated using the entire muscle compartment from
slice #6 and using all ten slices was 1%. On the other hand, SE as a percent of

the mean T2 calculated using the “box” method was 0.4%. Since the T2

 

97
calculated using the “box“ method reduced the variability considerably, all the T2

data presented in Table 3 and Figures 5, 6, and 7 were calculated using the
“box' method.

T2 relaxation times in milliseconds are given in Table 3. Baseline (resting
T2 of “normal" muscle) averaged 27.1 ms, typical of values reported in human
skeletal muscle (2,71). Concentric exercise resulted in a 30% rise in T2.
Eccentric exercise produced a smaller acute increase (16%), but resulted in a

delayed T2 elevation of about 1.5 ms at 24 hours post eccentric exercise.

Table 3. T2 data.

 

 

 

 

 

 

 

Time of Scan T2 (ms)
PreCon1 (baseline) 27.1 :t 0.1
PostCon1 35.2 i 1.0“
PreEcc 28.2 :I: 0.4
PostEcc 32.7 :I: 0.9”
PreCon2 (24h) 28.7 :I: 0.3‘
PostCon2 34.8 :r; 0.7“

 

 

 

 

Values are mean i SE, n=8. '
" significantly different from the pre-exercise T2 value (p<0.05).
' significantly different from baseline T2 value (P<0.05).

 

98
Acute and delayed T2 changes (A T2) were calculated by subtracting the

baseline (PreCon1) T2 value from the T2s measured at the various post-exercise
time points (Figure 5). The acute AT2 following the first bout of concentric
exercise (Con1, Figure 5) was not significantly different from that following the
second bout of concentric exercise (Con2, Figure 5). It should be noted that the
results displayed in Figure 5 are consistent with the “non-additive effects“
scenario presented in the previous chapter (Figure 4, page 87). Therefore these
findings support the hypothesis that the mechanism underlying both acute and

delayed T2 is the same and that acute T2 has an upper limit.

 

AT2 (ms)

 

 

 

 

J i
I

Con1 Ecc24h Con2

Figure 5. The change in the acute and delayed T2 following exercise. Values
are means :I: SE, n=8.

99
Figure 6 presents the AT2 values in relative terms (percent increase

above baseline). This figure illustrates two additional points: first, the failure of
T2 to return completely to baseline before the eccentric exercise (PreEcc), and

secondly, the minimal magnitude of the delayed T2 increase (Ecc24h).

 

821.638

AT2 (percent change from baseline)
N
01

 

 

 

Con1 PreEcc PostEcc Ecc24h Con2

Figure 6. Percent change in T2 from baseline (PreCon1). Values are means i
SE, n=8.

To document the early time course of the delayed AT2 following eccentric
exercise, subjects were imaged immediately following eccentric exercise and at

1, 2, 4, 6, and 24 h post exercise. Figure 7 illustrates the finding that the delayed

100
T2, which peaks 24-48 h post eccentric exercise, begins to increase as early as

4 h post exercise. It should be noted that the elevation evident at 1 h is likely
due to the incomplete return of the acute T2 response to baseline, since the T2

value continues to fall between 1 h and 2 h.

 

 

 

 

 

PreCon PostEcc 1 h 2h 4 h 5 h // 24 h
1

Figure 7. Change from baseline (PreCon1) in T2 following eccentric exercise.
# significantly different from PreCon1 (p<0.05). Values are means :I: SE, n=8.

Figure 8 displays regional T2 changes along the imaged length of the
brachium. Relative T2 changes are given for acute responses to both concentric
and eccentric exercise as well as the delayed response to eccentric exercise.

These T2 numbers were derived using the entire biceps muscle compartment

1 01
rather than a small box as in the previous figures. Several investigators have

suggested that most of the damage caused by eccentric contractions occurs at
the belly of the muscle, whereas others report greater damage at the
myotendious junctions at the ends of the muscle. Using ge_l_a_ygd AT2 as an
indirect index of muscle damage, our findings show that the damage induced by
eccentric contractions (Ecc24h, open bars in Figure 8) occurred throughout the
length of the biceps muscle compartment. Using gcr__1t§ AT2 as a measure of
muscle recruitment, our results suggest that concentric contractions recruited
more motor units compared to eccentric contractions (Con1 vs. PostEcc, Figure
8). In addition, there was a trend towards increased motor activity towards the
belly of the muscle (slices 3 through 8) in both concentric and eccentric

contractions.

102

 

35
I Con1 ;
3° l PostEcc 3
U Ech4h

8

B

_A
(II

_A
0

0|

 

AT2 (Preoent change from baseline)

 

O

l
I I I
l' H I i I i I i I i'
12345678910
SliceNumber

Figure 8. Percent change in acute and delayed T2 in each slice along the arm.
Slices are numbered from distal (1, towards elbow) to proximal (10, towards
shoulder). Values are mean :t SE, n=8.

Muscle Cross-sectional Area

MR imaging allowed us to directly assess specific muscle compartment
swelling, vs. the total arm girth measurements utilized in previous studies. Since
girth data suggests maximal swelling occurs near the belly of the muscle
(20,50,74), we performed area analysis in image slice #6 at the middle of the
longitudinal axis of the biceps muscle. The percent change in the muscle
compartment cross-sectional area was calculated by subtracting the resting
muscle compartment cross-sectional area from the 24 h post eccentric muscle
compartment cross-sectional area and dividing it by the resting compartment
cross-sectional area. The overall mean percent increase in muscle

compartment cross-sectional area at 24 h post eccentric exercise was

1 03
11 :h 2% (n=8). Males showed a larger increase in muscle compartment cross-

sectional area at 24 h post eccentric exercise compared to females (13% vs. 9%).

However, this difference was not statistically significant.

Muscle Soreness

Muscle soreness was evaluated using a questionnaire. Subjects rated
their perception of soreness in the biceps muscle on a scale of 1 (no soreness)
to 10 (extremely sore) with the arm fully extended before and after each exercise
session and at 24 h intervals for 7 days following the eccentric exercise
(reference 20; also see Appendix B). All subjects reported no soreness prior to
the start of the experiment and immediately after performing the first bout of
concentric exercise. At 12 h post eccentric exercise all subjects reported mild
soreness (2 i 0). Males reported an increase in their mean soreness
immediately following the second bout of concentric exercise (5 i 0.3) compared
to before performing the second bout of concentric exercise (3 t 0.5). However,
this difference was not statistically different. On the other hand, females
reported similar mean soreness ratings before (3 :L- 0.9) and after (3 :1: 0.7) the
second bout of concentric exercise. Due to the large variance in the soreness
scores, these gender differences were not statistically significantly.

Figure 9 shows the mean muscle soreness ratings by gender at 24 h
intervals following eccentric exercise. Males reported peak soreness earlier (48
h post eccentric exercise) compared to females (72 h), although the peak value

and variation were identical in both groups (5 :l: 2.0).

 

 

 

 

 

 

 

 

104
10
9 *- —D-Female
3 .. -I—Male
é”
a: 7 "
8
o 6 ~~
E
8 5 *- -
4 .,. I \
i , , \
E 3 ..
2 ..
1 t i % ‘fi .
0hr 24hr 48hr 72hr 96hr 120hr 144hr 168hr
Hours Following Eccentric Exercise

Figure 9. Mean muscle soreness ratings (1-no soreness to 10-extremely sore)
at 24 h intervals following eccentric exercise. Note that the males reported peak
soreness at 48 h and femalcs reported peak soreness at 72 h post eccentric.
Values are means :1: SE, n=4.

According to the acute inflammatory theory, the general muscle soreness
experienced during DMS is mostly due to the swelling. Figure 10 shows the plot
of mean muscle soreness ratings against the percent change in muscle cross-
sectional area (swelling) at 24 h post eccentric exercise. Spearman’s correlation

test revealed poor correlation between mean soreness ratings and the increase

in muscle cross-sectional area (r = .356). Mean muscle soreness ratings at 24 h

after eccentric exercise plotted against the 24 h T2 value are shown in Figure 11.

Spearrnan's correlation test did not show a significant correlation between
muscle soreness ratings and delayed T2 (r = -.074), as can be seen by the

nearly flat slope of the regression line in Figure 11.

 

105

10

 

 

 

 

 

B
3
8 7
‘I
8
8
(I)
{‘3’ 5 I
m
3
E 4 a 1:1 I
E
2 SI ____.__-——
0
0
2 u a
1<fi ; i 3 :
o 5 1o 15 20 25

Precent change in muscle cross-mctional area

Figure 10. General muscle soreness rating (1-no soreness to 10-extermely sore)
vs. percent change in muscle cross-sectional area at 24 h post eccentric
exercise (I females Elmales). Values are means, n=4.

 

 

 

 

 

10
9..
c»
5 8-~
to
a: 7,,
(I)
3
8 6"
'5 5-~ n
a)
_a_> 4«- II o
8
3 3«~ +
2
211' CI D
1 : 4 L c .
27 27.5 28 29.5 30 30.5 31

28.5 29
T2 (ms)

Figure 11. Mean muscle soreness ratings (1-no soreness to 10-extermely sore)
vs. T2 at 24 h post eccentric exercise (I females Elmales). Values are means, n
= 4.

1 06
Discussion

The main purpose of this study was to examine the acute T2 response to
a bout of light concentric exercise performed before (control) and after eccentric
exercise had been used to induce a delayed T2 increase. In addition, the early
time course of the delayed T2 contrast development was documented. As
reported in Table 3, the mean resting T2 of the biceps brachii muscle was 27.1 i
0.1 msec, in agreement with previously reported values for the biceps brachii
muscle (2,71). The concentric contractions induced a significant (p<0.05)
increase in the acute T2 (AT2 = 8.1 :1: 1.0 ms, 30% increase; see Figures 5 and
20). This elevation also falls within the range typically reported for acutely
exercised human muscle (2,71).

The acute T2 increase that occurs in response to concentric exercise has
been reported by Fisher et al. to return to resting levels by approximately 25 to
35 min post-exercise (31). However, in the present study, the T2 at the end of
the 2 h rest period following the first bout of concentric exercise remained slightly
elevated by about 4% compared to the resting or baseline value (Figure 6).
Although this increase was not statistically significant, it suggests that the muscle
had not completely recovered from the first bout of concentric exercise. Since
the T2 response is sensitive to small increases in motor activity, it is possible that
the elevation could be due to the casual use of the exercised arm during the rest
period. Thus, the fact that the subjects in the Fisher et al. study spent most of
the rest period laying supine with the exercised extremity fixed in position inside

the magnet may account for the difference in the acute T2 recovery.

107
Alternatively, the small but statistically nonsignificant difference could be due to

random statistical variation in this small sample size.

As shown in Table 3 and Figures 5 and 6, the acute T2 increase following
eccentric contractions was significantly less than that induced by the concentric
exercise. Several investigators have also reported similar results following
eccentric contractions (1,2,10,57,70). This result is not surprising given that T2
increases have been shown to scale with exercise intensity (31) and because of
the well-known fact that eccentric contractions consume less energy than
concentric contractions at the same load (1 ,10,57). Although the concentric load
was actually 10% less than the eccentric load, concentric contractions likely
recruited more motor units compared to eccentric contractions in each slice
along the upper arm. These results are due to the fact that muscles have an
inherent ability to generate greater force during eccentric contractions with a
given number of active motor units (10,51).

MR images were also acquired at 24 h post eccentric exercise to confirm
that the new resting T2 was elevated, a condition crucial for testing the research
hypothesis. There was in fact a significant increase in the biceps brachii muscle
T2 at 24 h post eccentric exercise (PreCon2) compared to baseline (PreCon,
Table 2). Shellock et al. reported similar results at 24 h post eccentric exercise
(70). However, the very small magnitude of the delayed T2 elevation (AT2 = 1.6
i 0.4 ms, 6% increase) reduced the capacity to discriminate between the

additive and nonadditive conditions.

1 08
There are two possible explanations as to why the eccentric exercise

protocol employed in the present study did not cause more notable muscle
trauma at 24 h post eccentric exercise. One is that the subjects did not actively
resist lengthening during eccentric contractions. In another words, they simply
allowed the weight to drop. As a result, the individual biceps brachii muscle
fibers would not be exposed to high enough mechaniml strain to cause structural
damage. The second possibility is that the eccentric exercise intensity in the
present study was simply not high enough to induce structural damage even
when performed correctly.

After the 24 h post eccentric images were acquired, each subject
performed another bout of the original concentric exercise and was then re-
imaged (PostCon2, Table 2). Acute AT2 following the first bout of concentric
exercise (8.1 :I: 1.0 ms, 30% increase) was not significantly different from the
acute AT2 after the second bout of concentric exercise (7.7 :I: 0.5 ms, 28%
increase). Therefore, these findings support the hypothesis that the mechanism
underlying both acute and delayed T2 is the same and that acute T2 has an
upper limit.

The secondary or descriptive purpose of this study was to document the
early time course of the delayed T2 contrast development. Takahashi et al.
observed that the T2 signal following eccentric exercise has a bimodal response
with the first peak occurring immediately after the exercise (acute T2), and the
second peak sometime after 12 h post-exercise (delayed T2) (81). These

investigators acquired MR images before, immediately after, and at 7,15,20,30,

109
and 60 min and 12, 24, 36,48, 72, and 168 h post-exercise. In the present

study, MR images were acquired before, immediately after, and at 1, 2, 4, 6 h
following eccentric exercise (Figure 7).

In our results the characteristic first peak immediately following eccentric
exercise was evident (Figure 7). At 1 h post eccentric exercise, T2 remained
elevated compared to baseline (p<0.05). Takahashi et al. reported similar
findings at 1 h post eccentric exercise. At 2 h post eccentric exercise, T2
remained slightly elevated compared to baseline, although the T2 values had
decreased from the 1 h time point. It is our belief that the increase in T2 values
measured at 4 and 6 h post eccentric exercise is the early development of the
second peak in the bimodal response of T2 following eccentric exercise found by
Takahashi et al. at 12 h post-exercise. No previous report has documented T2
values in the interval between 1 and 12 h after eccentric exercise. However,
other investigators have reported evidence of damage as early as 1.5 h post
eccentric exercise based on elevated levels of serum CK activity, circulating
leukocytes and neutrophils (64).

In the present study, the time course of the development of soreness was
consistent with past reports (6,11,20,61). Subjects reported mild soreness at 12
h post eccentric exercise, reached peak levels 2-3 d post-exercise, and by day
seven the soreness had completely dissipated. Females in our study tended to
report peak soreness a day later than the males (2-3 d vs. 1-2 d). However, this

is still within the time frame reported in the literature of 2-3 d for peak soreness

 

 

 

1 10
development. In addition, there is no evidence in the literature to suggest that

the time to reach peak soreness is longer in females.

In the present study, at 24 h post eccentric exercise, males reported a
mean soreness rating of 3 :I: 0.9 (Figure 9). Immediately following the second
bout of concentric exercise they reported an increase in soreness, 5 i 0.3. On
the other hand, females reported similar soreness ratings before (3 i 0.5) and
after (3 d: 0.7) the second bout of concentric exercise. Due to the large variance
in the soreness scores neither the increase in soreness reported by males
following the second bout of concentric exercise nor the gender difference was
statistically different. In Hough’s 1902 report on delayed muscle soreness, he
noted that a second bout of exercise performed while the muscles were already
sore actually decreased the sensation of soreness (45). In addition, two studies
conducted by De Vries found stretching exercises performed after eccentric
exercise reduced muscle soreness (23,24). However, in a more recent study,
High and Howely reported that static stretching and warm-up exercises did not
reduce the development of DMS (44). The discrepancy in our results, as well as
between studies, on the ameliorating effect of exercise on DMS can likely be
attributed to the subjective nature of the muscle soreness scores as well as to
the relatively low level of soreness induced in our study. A

In an attempt to better explain the delay between fl1e immediate tissue
injury and later soreness development, several investigators have proposed that
the mechanism underlying soreness development is acute inflammation, based

on the similar time courses of the two conditions. The time course data on the

1 1 1
development of swelling and sensation of pain in DMS correlated very well,

lending support to the notion that the pain reported during DMS might be the
result of inflammatory swelling. Unfortunately, the studies that have examined
the causal role of swelling in initiating the sensation of DMS by increasing the
intramuscular pressure are not conclusive (74).

In the present study, muscle cross sectional area was calculated at 24 h
post eccentric to evaluate the relationship between swelling and soreness in
DMS. Overall there was a significant increase in muscle cross sectional area or
swelling (11 i 2%, n = 8). Although males showed a larger increase in muscle
cross sectional area at 24 h post eccentric exercise compared to females, this
difference was not statistically different. If this trend towards greater swelling in
males is indeed real, it could possibly stem from the larger loads lifted rather
than from a gender difference per se.

Speannan’s correlation test revealed only a modest realtionship between
mean soreness ratings and swelling (r = 0.356) at 24 h post eccentric exercise
(Figure 10). Bar et al. also observed that the peak soreness was reported at 48
h post eccentric exercise, when signal intensity and the muscle surface area had
only started to increase (9). Therefore, at least at 24 h post eccentric exercise,
swelling is not likely to be the primary cause of soreness.

The relationship between soreness ratings and T2 signal intensity was
also evaluated at 24 h post eccentric exercise in the current study (Figure 11).
The nearly flat slope of the regression line in Figure 10 suggests that there is no

correlation between T2 signal intensity and soreness ratings at 24 h post

1 12
eccentric exercise. Based on our results and Nurenberg et al.’s findings that

there is a high correlation between areas with increased signal intensity and
ultrastructural injury, it can be concluded that the soreness experienced at 24 h
post eccentric exercise is not solely dependent on the extent of the initial
structural damage.

In summary, the primary findings of our study were that (1) acute T2
elevation is not additive to chronic T2 elevation, and (2) the delayed T2 response
begins as early as 4 h after eccentric exercise. Secondary results included (1)
the observed trend towards a later soreness peak in females vs. males, (2) a
confirmation of increased T2 activation in the belly vs. ends of the muscle and
(3) lack of correlation of soreness with either swelling or T2 response. The final
chapter of this thesis will discuss the significance of these findings as well as
present recommendations for future experiments addressing the limitations of

the current study.

CHAPTER V
CONCLUSIONS, SIGNIFICANCE, AND RECOMMENDATIONS

The purpose of this study was to examine the acute T2 response to a
bout of light concentric exercise performed before (control) and after eccentric
exercise had been used to induce a delayed T2 increase. The secondary
purpose of this study was to document the early time course of the delayed T2
contrast development. As described in Chapter 4 and summarized briefly in the
following paragraphs, the results of this study demonstrated partial success in
achieving both objectives. In addition to the direct outcomes of the current study,
this work has led to numerous suggestions for future refinements and additional
studies.

Regarding the main study objective, the results indicated that the acute T2
change was my; significantly different whether concentric exercise was
performed using “normal” muscle or with muscle having an elevated resting T2.
The immediate interpretation of these results would be that the mechanisms of
both the acute and delayed T2 response are identical, hence non-additive.
However, the very small magnitude of the eccentric-induced delayed T2 change

resulted in a reduction in the power of the statistical test of the mechanism

113

1 14
hypothesis. Suggestions for remedying this loss of conclusiveness are

presented near the end of this chapter.

Documentation of the early time course of the delayed T2 contrast
development was somewhat more successful, although this objective would also
have benefited from an increase in the eccentric stimulus. A statistically
significant elevation in T2 values were observed as early as 4-6 h post eccentric
exercise, vs. the 12—24 h initial rise reported by other researchers.

Additional secondary findings included (1) the observed trend towards a
later soreness peak in females versus males, (2) a larger non-significant
increase in muscle compartment cross-sectional area in males, and (3) no
significant correlation between soreness and either swelling or T2 response at 24
h post eccentric exercise.

In addition to these findings, a pair of unexpected observations arose from
the current study. The first of these was the failure of acute concentric T2 to
return to baseline values at the end of the 2 h rest period. It is conjectured that
the elevated T2 at the end of the rest period was due to casual use of the
exercised arm during the rest period. The other unexpected finding was that the
trend towards increased T2 activation in the belly of the muscle versus the ends
of the muscle following both concentric and eccentric exercise. The increased
T2 activation in the belly of the muscle suggests that the metabolic demand was
greater in the belly of the muscle compared to the distal and proximal ends.

Further studies directly measuring the regional energy utilization within a muscle

1 15
will be useful in confirming the trend towards increased metabolic activity in the

belly of the muscle as indicated by T2 values.

Significance

In “normal” muscle the increase in acute T2 has been reported to be
roughly linear at submaximal intensities. The findings of this study suggest that
the acute T2 elevation following exercise in damaged muscle does not exhibit a
similar dependence on exercise intensity as in normal muscle, as evidence by
the smaller acute T2 response to the same bout of concentric exercise (PreCon2
(at 24h) minus PostCon2).

Clinically, the findings of the current study suggest that acute T2 elevation
is not a good index of motor recruitment in muscles that exhibit an elevated T2
as a result of exercise-induced damage and/or neuromuscular disease.
Therefore, the acute contrast change can only be used as a measure of the
extent of muscle recruitment in “nonnal' muscles following various activities.

The findings of relatively early initiation of the second or delayed T2
response to eccentric exercise may shed some light on the mechanism of the
damage induced by lengthening contractions. This early time course supports
the inflammatory theory, since initial events of the inflammatory response also
begin within 4-6 hours as documented in Chapter II. The fact that this T2
response becomes visible before CK normally begins to appear in the
bloodstream is not surprising, since the relatively large kinase protein must

transverse the slow-moving lymph system before finding its way into the

 

1 16
systemic circulation. Studies measuring myoglobin, a much smaller muscle

protein, have in fact documented its appearance in the bloodstream within the 4—
6 hour time flame of the initial T2 response.

In addition to these two potential appliations of the results of this study,
the other main contribution of the current results is to the development of more
refined and powerful methods of utilizing the basic method of the current study to
more effectively examine the question of mechanisms of the acute versus
chronic T2 responSes. These suggested refinements are described in the

following section.

Recommendations

The results of this investigation support the research hypothesis of
identical mechanisms for the acute and delayed T2 responses to exercise as
described in Chapter I. However, the small magnitude of the delayed T2
elevation (AT2 = 1.6 i 0.4 ms, 6% increase) reduced the power of the statistical
test of the research hypothesis. The following recommendations are offered as
points to be considered in future studies, both to remedy the low statistical power
problem and to address other shortcomings of the present study.
1. Since a larger increase in the delayed T2 elevation has been reported at 48 h
post eccentric exercise compared to 24 h, performing the second bout of
concentric exercise at 48 h post exercise will increase the capacity to

discriminate between the additive and nonadditive conditions.

1 17
2. The relatively small elevation in the delayed T2 at 24 h post eccentric

exercise is partly due to the fact that the eccentric exercise intensity was not high
enough. Instructing the subjects to actively resist the lengthening during
eccentric contractions will increase the exercise intensity. In addition, the
eccentric exercise intensity can be increased by raising the resistance and the
number of repetitions, for example, five sets of 10 repetitiOns at 110% one
repetition maximum (1 RM), the maximum amount of weight lifted one time with
proper form. Furthermore, setting the load relative to the 1RM value rather than
body weight normalizes the exercise intensity between subjects. On the other
hand, the concentric exercise intensity can be relatively low, for example, three
sets of 10 repetitions at 15% of the 1RM, since the acute T2 is sensitive to small
increases in concentric motor activity.

3. No experimental studies have documented the acute T2 recovery following
the second bout of concentric exercise performed with an elevated resting or
baseline T2. Likewise, a high time resolution study of the T2 recovery in the
hour after eccentric exercise has never been reported. Such a study would help
clarify the magnitude and time course of the chronic or delayed T2 response, as
discriminated from the acute response.

4. It has been reported that the T2 signal following eccentric exercise follows a
bimodal response with the first peak occurring immediately after the exercise
(acute T2), and the second peak sometime after 12 h post-exercise (delayed
T2). In the current study, as expected, the first peak was observed immediately

following the eccentric exercise and by 2 h post exercise the T2 had nearly

 

1 18
returned to baseline. It is our belief that the elevations in the T2 at 4 and 6 h

post eccentric exercise is the early development of the delayed T2, which peaks
24-48 h post exercise and is an index of damage. In order to confirm that the
elevation in T2 at 4 and 6 h post eccentric exercise is due to muscle damage, it
might be useful to document the development of the damage using blood borne
inflammatory markers within the same time frame. Several investigators have
used a combination of inflammatory markers to document damage, for example,
serum CK activity, serum myoglobin, and circulating leukocyte and neutrophil
counts.

5. According to the acute inflammatory theory, the soreness experienced
during DMS maybe the result of swelling. In the current study, the correlation
between soreness scores and swelling was not significant. Although it has been
claimed by some investigators that swelling is not likely to be the primary cause
of soreness at 24 h post eccentric exercise, this could be due to lack of precision
in the methods applied to measure swelling. In the current study, swelling was
calculated by measuring the change in the biceps brachii muscle compartment
cross-sectional area at 24 h post eccentric exercise, excluding the subcutaneous
fat compartment around the muscle. However, most of the swelling in DMS
occurs in the interstitial space. Therefore, in future swelling should be calculated
by measuring the cross-sectional area of the muscle compartment plus the
subcutaneous fat compartment around the muscle. It would also be useful to
compare swelling of the non-exercised (triceps) muscle compartment to that of

the exercised compartment. It seems likely that the increased eccentric exercise

 

1 19
intensity recommended in item 2 above might in fact result in detectable swelling

in the biceps compartment alone, which could then de discriminated from
changes in the triceps or subcutaneous fat compartment.

6. In the present study, females reported peak soreness a day later than the
males (72 h vs. 48 h). However, this difference was not statistically significant
due to the large variance in the soreness scores. In general, soreness scores
collected using a questionnaire are subjective in nature. Noneflieless, these
soreness scores may be useful in documenting soreness development across
different time points of a single subject On the other hand, comparing soreness
scores of different subjects is problematic since different individuals perceive
pain differently. In an attempt to develop a more objective measure of soreness,
several investigators have elected to measure soreness in the following fashion.
First, the investigator applies a small amount of pressure directly on the muscle
experiencing DMS. Next, the subject is asked to verbally rate the perception of
pain. Since the soreness experienced in DMS has been reported to worsen
during movement or palpation, researchers suggest that the pressure induced
soreness rating is more valid because it measures soreness in a manner similar
to that experienced in DMS. Therefore, in future studies, investigators should
consider collecting pressure induced soreness scores in addition to general
soreness ratings. Finally, an increase in the size of the gender subgroups would

also provide an obvious boost to the power to discriminate gender differences.

 

120
Summagy

The current study has provided preliminary evidence supporting the
hypothesis of identical mechanisms underlying the acute and delayed T2
responses to exercise, as well as novel information on the early time course of
the delayed T2 response. The major value of this work, however, has been to
provide a platform from which to launch future studies of the T2 response to
exercise. This project has this investigator about the theory and methods of
magnetic resonance imaging. It has also brought into focus for the numerous
potential pitfalls in empirical research. The research design and execution
recommendations resulting from the present study are already being
incorporated into an expanded project which will constitute the first phase of my
doctoral dissertation research. It is this investigator’s expectation that the skills
and insights gained from the current study will pay off in more conclusive results
and more extensive potential signifiwnce of these ongoing and planned future

studies.

 

APPENDICES

 

APPENDIX A

APPENDIX A

APPLICATION FOR PARTICIPATION IN EXERCISE-INDUCED DELAYED
MUSCLE SORENESS RESEARCH PROJECT

 

 

 

 

Name:
first middle last
Address: -
street city state zip
Local Phone:( )- - Age: Date of birth: / l
Height : Weight :

Have you lifted weights or done any strength or resistance training in the past
two years? Yes No

If yes, please describe (how long ago, duration of sessions, sessions per week,
over how many months/years):

Please describe your typical weekly exercise/sportslphysical activity:
(If none, please indicate when you last participated in such activity, if ever.)

Do you have any surgically implanted devices (e.g. metal plates/pinslscrews
Irods. Pacemaker, etc.) or other metal objects such as shrapnel or braces?
Yes No

Do you suffer from any type of acute or chronic disease?
Yes No
If yes, please describe in detail:

 

121

 

 

APPENDIX B

 

APPENDIX B

GENERAL MUSCLE SORENESS QUESTIONNAIRE.

Instructions: The following is a muscle soreness scale which will allow us to
measure each individual’s perception of pain/soreness in the exercised arm as a
result of the eccentric exercise. Rate the level of soreness in the exercise arm
(left) in the following position: standing with the arm hanging completely relaxed
to the side. Write the number that corresponds to the perception of soreness in

the space provided next to the appropriate date and time.

1 - No soreness

2

3 - Mild soreness

4

5 - Moderately sore
6

7 - Very sore

8

9

10 - Extremely sore

122

 

APPENDIX C

APPENDIX C

INFORMED CONSENT
MRI analysis of muscle function. Departments of Physiology and Radiology,
Michigan State University. .
1. l have freely consented to participate in a study using Magnetic Resonance
Imaging. I understand that there are no known hazards or risks associated with
Magnetic Resonance Imaging in patients who do not have a pacemaker.

2. I understand that the study involves the following procedures: I will perform
repeated bouts of maximal arm curls (biceps curls) on free weights. In some
cases this exercise may resuIt in the development of muscle soreness one to
three days later. MR images of my muscles will be acquired immediately before
and after the exercise bouts, with a follow-up MR imaging session 2 days later.
Each MR exam will require about 20 minutes, and the exercise session will last
about 15 minutes. My participation may also involve taking a standard
therapeutic dose of the over-the—counter anti-inflammatory drug ibuprofen (three
doses of 400mg each over 24 hours, with initial dose given four hours prior to the
exercise bout; each dose to be taken with milk or food). I understand that if I am
not asthmatic or allergic to aspirin, and if I take the drug with milk or food, the risk
of side effects is very small.

3. I understand that any information I provide, and all data collected will be
treated in strict confidence. No information or data will be traceable to my name.

4. I understand that I am free to discontinue my participation in this study at any
time without penalty.

5. I understand that the results of this study will be made available to me at my
request. Furthermore, on my request I can receive an additional explanation of
this study after my participation is completed.

6. I understand that my participation in this study does not guarantee any
beneficial results to me.

7. I understand that if I am injured as a result of my participation in this research
project, Michigan State University will provide emergency medical care if
necessary. I further understand that if the injury is not caused by the negligence
of MSU I am personally responsible for the expense of this emergency care and
any other medical expenses incurred as a result of this injury.

 

 

Participant’s Signature Date
1 23

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