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STRETCH INTENSITY AND THE INFLAMMATORY RESPONSE
Nikos Apostolopoulos BPHE (Sports Medicine)
A thesis submitted in fulfilment of the requirements of the University of
Wolverhampton for the degree of Doctor of Philosophy
University of Wolverhampton
2015
This work or any part thereof has not previously been presented in any form to the
University or to any other body whether for the purposes of assessment, publication
or for any other purpose (unless otherwise indicated). Save for any express
acknowledgements, references and/or bibliographies cited in the work. I confirm that
the intellectual content of the work is the result of my own effort and of no other
person. The right of Nikos Apostolopoulos to be identified as author of this work is
asserted in accordance with ss.77 and 78 of the Copyright, Designs and Patents Act
1988.
At this date, the author owns copyright.
Signature: …………………………………………………………..
Date:
………………………………………………………….
NATURE’S VEIL
For those who have gone before,
For those yet to come,
I dedicate this manuscript.
You have walked
Moreover, you will continue to walk,
Into and through the Abyss,
To gain a glimpse,
Of what lies beyond Nature’s Veil.
This cannot be defined and described by numbers alone,
It is a sensitive chaos,
Made all the more clearer,
By grasping for and comprehending,
The Divine
…Nikos Apostolopoulos (2015.12.03)
i
Abstract
ABSTRACT
Background: Stretching may be viewed as an external/internal force influencing the
range of motion of the connective tissue (muscles, tendons and the myotendon unit
(MTU)) The magnitude and rate of stretching may potentially induce mechanical
responses of the musculoskeletal system, such as increased range of motion
(ROM). The degree of the intensity of stretch (low, medium, or high) may be used to
optimize recovery from muscle damage via ameliorating inflammation; this is
however, a plausible hypothesis that needs to be appropriately investigated.
Aims: The present project aimed to investigate: 1) whether intense stretching (IS)
causes an acute inflammatory response (study 1), 2) the effects of stretching
intensity (low, medium, or high) in the onset of inflammation (study 2), and 3)
investigate whether stretching intensity is responsible for aiding in the recovery of the
muscle, post muscle damage (study 3).
Methods: Studies one and two were randomized crossover trials consisting of 12
and 11 recreational male athletes, respectively. The former investigated whether
high intensity stretching can cause an acute inflammatory response, with study two
examining the effects of different stretching intensities (30%, 60% and 90%) based
on a participant’s perceived maximum range of motion (mROM). Blood for both
studies was collected at pre-, post, and 24h post intervention, and analyzed for high
sensitivity C-reactive protein (hsCRP) (study 1 and 2), and for interleukin (IL)-1β, IL6, and tumour
necrosis factor (TNF)-α (study 1). In study three, a randomized
controlled trial investigated whether stretching intensity (low or high), can influence
the recovery from muscle damage. Thirty participants were randomized into three
groups, a) low intensity stretching (LiS) (30-40% ROM), b) high intensity stretching
(HiS) (70-80% ROM) and c) Control group. All participants performed both eccentric
ii
Abstract
(EPT) and isometric peak torque (IPT) tests prior to a muscle damage protocol
(MDP) (baseline). Participants were then assessed for EPT and IPT for three
consecutive days post MDP. Soreness levels were recorded immediately post
muscle damage and at 24, 48, and 72h, with blood samples collected at pre, 24, 48,
and 72h post muscle damage and analyzed for Creatine Kinase (CK) and hsCRP.
Results: Study one revealed a significant increase in hsCRP (P = 0.006) when
comparing IS to Control condition, also confirmed by the effect size analyses. In
study two, low (30% of mROM), and medium (60% of mROM) intensity stretching did
not elicit an inflammatory response while a pronounced inflammatory response was
observed when comparing 30 to 90 and 60 to 90% mROM. In study three, LiS
showed a significant increase in EPT compared to both HiS and Control, and these
findings were confirmed by magnitude based inferences analyses (i.e. LiS was
associated with a positive effect for both IPT and soreness levels compared to
Control and HiS). Blood biomarkers were associated with inconsistent effects
compared to Control and HiS for all three-time periods.
Conclusions: This thesis provides preliminary results suggesting that increased
stretching intensity may be responsible for causing an acute inflammatory response.
In addition, it was observed that LiS might be associated with faster recovery from
muscle damage with respect to muscle function (EPT and IPT) and soreness levels.
More research is needed to investigate these findings further.
iii
TABLE OF CONTENTS
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
Table of Contents
Nature’s Veil
i
Abstract
ii-iii
Table of Contents
iv-x
List of Figures
xi-xii
List of Tables
xiii-xiv
Glossary of
Terms
xv-xvi
Publications
xvii
CHAPTER 1
INTRODUCTION
1-4
CHAPTER 2
LITERATURE REVIEW
5-108
2.1 Introduction
6
2.2 The Definition of Stretching and it’s measurement
6-9
2.3 Types of Stretches
9-13
2.3.1
Static
9-10
2.3.2
Ballistic
10-11
2.3.3
Dynamic
11-12
2.3.4
Proprioceptive Neuromuscular Facilitation (PNF)
12-13
2.4 Macroscopic Level – Muscles, Tendons and the Myo-Tendon
Unit
14-20
2.4.1
Muscle
14-17
2.4.2
Tendon
17-18
2.4.3
Myo-Tendon Unit
18-20
2.5 Microscopic Level – Force, Cells, Tissue and the Extracellular
Matrix
20-27
2.5.1
Force, Cells, and Tissue
20-22
2.5.2
The Extracellular Matrix
23-24
2.6 The Strain Factors of Stretching: Intensity, Duration, and
Frequency
24-27
2.7 Inflammation
28-41
2.7.1
Introduction
28-32
2.7.2
Neutrophils, Macrophages, Cytokines and Acute Phase
Response
32
v
Table of Contents
2.7.2.1
Neutrophils, Macrophages
32-34
2.7.2.2
Cytokines
34-37
2.7.2.3
Acute Phase Response
37-41
2.8 Biomarkers
41-42
2.9 Inflammation and Exercise
43-108
2.9.1
2.9.2
Non-Damaging Exercise and the inflammatory response
43-47
2.9.1.1
Myokines
48-51
2.9.1.2
Muscle as an Endogenous Glucose Producing
Organ
52-53
2.9.1.3
Intensity, Duration, and Mode of Exercise
53-56
Damaging Exercise and the Inflammatory Response
2.9.2.1
Stages Associated with Muscle Damage Exercise 58-64
and Inflammation
2.9.2.1.1 Stage 1 – Muscle Damage
65-69
2.9.2.1.2 Stage 2 – Calpain
69-75
2.9.2.1.3 Stage 3 – Transmigration and
Adhesion Molecules
76-77
2.9.2.1.4 Stage 4 – Inflammatory Cells
77
1 Neutrophils
2&3
CHAPTER 3:
56-57
77-79
Pro- and Anti-inflammatory
Macrophages
80-81
Circulating Macrophages
(ED1+monocytes & pro-inflammatory)
81
Resident Macrophages (Ox6+,
ED2+Ox6-, ED2+Ox6+, and ED2+)
82-83
Spatial sequencing of inflammatory
cells
83-92
Concluding Remarks
93-95
2.9.2.2
Mechanotransduction
95-104
2.9.2.3
Stretching and Inflammatory Cells
104-108
STUDY ONE – Acute Inflammatory Response to Stretching
109-131
3.1 Introduction
110-111
3.2 Methods
112-119
vi
Table of Contents
3.2.1
Participants
112
3.2.2
Power Calculations
112
3.2.3
Procedures
113-114
3.2.4
Intense Stretch Intervention
115
3.2.5
Control Intervention
115
3.2.6
Biomarkers
116-117
3.2.7
Questionnaire
118
3.2.8
Statistical Analysis
118-119
3.3 Results
119-126
3.3.1
RMANOVA
120-123
3.3.2
Effect Sizes
124-126
3.3.2.1
Effect Sizes between Conditions
124
3.3.2.2
Effect Sizes within Conditions
124-125
3.4 Discussion
127-131
3.5 Summary
131
CHAPTER 4
STUDY TWO – Stretch intensity vs. inflammation – A dosedependent association?
132-156
4.1 Introduction
133-134
4.2 Methods
134-142
4.2.1
Participants
134
4.2.2
Power Calculations
134-135
4.2.3
Procedures
135-140
4.2.4
Blood Biomarkers
140-142
4.2.5
142
4.3 Results
143-152
4.3.1
RMANOVA
144
4.3.2
Effect Sizes
144-146
4.3.2.1
Pre-intervention hsCRP
144-145
4.3.2.2
Post-intervention hsCRP
145
4.3.2.3
24h post-intervention hsCRP
145
4.3.3
Secondary Analysis – Linear Regression Report 30 – 90%
mROM and 60 – 90% mROM
147-152
vii
Table of Contents
4.3.3.1 – hsCRP post
147-149
4.3.3.2 – hsCRP 24 post
150-152
4.4 Discussion
153-155
4.5 Summary
156
CHAPTER 5
STUDY THREE – The Effect of Different Stretch Intensities On
Recovery From Muscle Damage – A Randomized Trial
157-207
5.1 Introduction
158-161
5.2 Methods
161-173
5.2.1
Participants
161
5.2.2
Procedures
161-163
5.2.2.1
Pre Muscle Damage Protocol
164
5.2.2.2
Muscle Damage Protocol
164-165
5.2.3
Post Muscle Damage Protocol Stretching Exercises
165-169
5.2.4
Biomarkers
170-173
5.2.5
173
5.3 Results
5.3.1
5.3.2
5.3.3
174-197
Analysis – Strength and Soreness
179
5.3.1.1
Eccentric Peak Torque (EPT)
179-181
5.3.1.2
Isometric Peak Torque(IPT)
182-183
5.3.1.3
Soreness Level (SL)
184-187
Analysis of Blood Biomarkers
188
5.3.2.1
Creatine Kinase
188-190
5.3.2.2
hsCRP
191
Secondary Analysis – Relationship between Muscle
Function and Biomarkers
192-197
5.4 Discussion
198-207
5.5 Summary
207
CHAPTER 6
SUMMARY DISCUSSION
208-215
6.1 Introduction
209-210
6.2 Acute Inflammatory Response to Stretching
211
6.3 Stretch intensity vs. inflammation – a dose-dependent
212
viii
Table of Contents
association
6.4 The effect of different stretch intensities on Recovery From
Muscle Damage – A Randomized Trial
213-214
6.5 Summary
215
CHAPTER 7
LIMITATIONS
7.1 Limitations of Studies
217
7.1.1
Blood Collection
217-218
7.1.2
Methodology
218-220
7.1.3
Compliance
220
7.1.4
Nutrition
220-221
7.1.5
Mental Stress
221
7.16
Age
222
7.2 Summary
CHAPTER 8
216-223
FUTURE RESEARCH
222-223
224-236
8.1 Introduction
225-226
8.2 Further Research
226
CHAPTER 9
8.2.1
Further Research Based on Studies of the Thesis
226-228
8.2.2
Ultrasonography
229
8.2.3
Oxygen Free Radicals
230-231
8.2.4
Musculoskeletal Disorders and Pain
231-232
8.2.5
Recovery and Regeneration
233-235
8.2.6
Relaxation Response
235-236
CONCLUSIONS
CHAPTER 10 REFERENCES
237-238
239-278
ix
Table of Contents
APPENDIX
279-342
A FORMS
280-297
Questionnaire For Potential Participants In Projects Involving Blood
Analysis
Pre-Test Questionnaire
281
Consent Form Study One
283
Consent Form Study Two
284
Consent Form Study Three
285
Stretching Exercises for Study 3 – Low Intense Stretching (LiS)
286-290
Stretching Exercises for Study 3 – High Intense Stretching (HiS)
291-295
Image Release Form
296
Image Release Form
297
B SAMPLE OF RAW DATA
Table 1 - Study One - Age, Mass, Height, and hsCRP values
282
298-316
299
Table 2 – Study Two - Age, Mass, Height, and hsCRP values for 30, 300
60 and 90% maximum ROM
Table 3 - Study Three - Eccentric and Isometric Peak Torque and 301
Soreness Values
Graphs for Parallel Groups (Will Hopkins Material)
302-316
C SYSTEMATIC REVIEW
317-342
Systematic Review
318-342
x
Table of Contents
LIST OF FIGURES
CHAPTER
FIGURE
Chapter 2
2.1 The Sarcomere
15
2.2 The Cardinal Signs of Inflammation
29
2.3 Transmigration-Inflammatory cell migration into inflammation site
33
2.4 Summary of Cytokines
35
2.5 Acute Phase Response
38
2.6 Relationship of IL-6 Relative to Concentric and Eccentric NonDamaging Exercise
46
2.7 Comparison of Sepsis vs. Habitual Exercise Induced Cytokines
50
2.8 Cytokine Cascade Related to Muscle Damage
57
2.9 The Four Stages with Regard to Muscle Damaging Exercise and
Inflammatory Response
60
2.10 Neutrophils
61
2.11 Pro-Inflammatory Macrophages (Phagocytic)
62
2.12 Anti-Inflammatory Macrophages (non-phagocytic)
63
2.13 Measurement of Histological Changes
67
2.14 Subsequent Steps Associated with the Autolysis by Calpain
72-73
2.15 Expression of Anti-inflammatory Cytokines
93
2.16 Example of Tensegrity
98
2.17 Hierarchy of Systems
99-100
Chapter 3
3.1 Methodology Schematic of Interventions
114
3.2 CONSORT Flow Diagram
119
3.3 Change of hsCRP over Time
122
3.4 Perceived Soreness Levels Over Time
123
xi
Table of Contents
Chapter 4
4.1 Knee Immobilizer
136
4.2 Experimental Set-Up
137
143
4.4 Linear Regression between 30 and 90% mROM and hsCRP post
148
4.5 Linear Regression between 60 and 90% mROM and hsCRP post
149
4.6 Linear Regression between 30 and 90% mROM and hsCRP 24h
post
151
4.7 Linear Regression between 60 and 90% mROM and hsCRP 24h
post
152
5.1 Methodology Schematic of Intervention(s)
163
5.2 Hamstring Stretch
167
5.3 Hip Flexor Stretch
168
5.4 Quadriceps Stretch
169
174
Chapter 5
5.6a Eccentric Peak Torque
180
5.6b Eccentric Peak Torque (normalized)
181
5.7a Isometric Peak Torque
182
5.7b Isometric Peak Torque (normalized)
183
5.8a Soreness Levels
186
5.8b Soreness Levels (normalized)
187
5.9 CK in relation to Time
190
5.10 hsCRP in relation to Time
191
5.11 Relationship between EPT and hsCRP
194
5.12 Relationship between IPT and hsCRP
195
5.13 Relationship between EPT and CK
196
5.14 Relationship between IPT and CK
197
xii
Table of Contents
LIST OF TABLES
Chapter 2
2.1 Time Scale of the Inflammatory Cell Response in Relation to the
Phases of Muscle Regeneration Post Muscle Damage
64
2.2 Sequence of Inflammatory Cells
87
3.1 Mean (SD) of Raw and Ln Transformed Blood Biomarkers in
Relation to Time and Intervention(s)
121
3.2 Mean (SD) Soreness Levels of Muscle Post IS
123
3.3 Descriptive Statistics and Effect Sizes for all Between Conditions
and Within Conditions Comparisons
126
4.1 Mean Measurements and Effect Sizes for hsCRP
146
5.1 Contents of VITROS CK Slide
170
5.2 Mean and SD Measurements for EPT, IPT, and SL
175
Chapter 3
Chapter 4
Chapter 5
5.3 Changes in Creatine Kinase (CK), high sensitivity C-reactive 176
protein (hsCRP), perceived muscle soreness, eccentric peak
torque (EPT) and isometric peak torque (IPT) in low-intensity
stretching (LiS) compared to Control and qualitative inferences
about the intervention effects
5.4 Changes in Creatine Kinase (CK), high-sensitivity C-reactive 177
torque (EPT) and isometric peak torque (IPT) in high-intensity
stretching (HiS) compared to Control and qualitative inferences
about the intervention effects
5.5 Changes in Creatine Kinase (CK), high sensitivity C-reactive 178
torque (EPT) and isometric peak torque (IPT) Low-intensity
stretching (LiS) compared to High-intensity stretching (HiS) and
qualitative inferences about the intervention effects
5.6 Mean, SD, and Percent Change for Soreness Levels post muscle 185
damage
xiii
Table of Contents
5.7 Mean and SD Measurements for CK and hsCRP (Ln Transformed
& Raw Data)
189
5.8 Relationship between Muscle Function (EPT & IPT) and Blood
Biomarkers (CK & hsCRP). Results reported as means (95%CI)
193
6.1 Summary of Studies
210
7.1 Summary of Studies and Scheduled Time Periods for Blood
Collection
223
Chapter 6
Chapter 7
xiv
Glossary of Terms
GLOSSARY OF TERMS
AAM
ANOVA
APP
APR
BMNC
Ca2+
CAM
CI
CK
CRP
CR-PNF
CRAC-PNF
CONSORT
d
“d”
DOMS
ECM
ED1+
ED1+
ED2+
EDL
EMG
EPT
ε (epsilon)
FGF
h
HGT
HiS
HR max
hsCRP
IASP
ILIGF
IPT
IS
Kg
Km
LiS
Ln
LSD
M2n (a b c)
m
Alternatively Activated Macrophage
Analysis Of Variance
Acute Phase Protein
Acute Phase Response
Blood Mononuclear Cells
Calcium
Classically Activated Macrophage
Confidence Intervals
Creatine Kinase
C-Reactive Protein
Contract-Relax Proprioceptive Neuromuscular Facilitation
Contract-Relax-Antagonist-Contract Proprioceptive Neuromuscular
Facilitation
Consolidated Standards Of Reporting Trials
Days
Cohen’s d
Delayed Onset Muscle Soreness
Extracellular Matrix
Monocyte and Pro-Inflammatory Macrophage (phagocytic)
Macrophage (phagocytic)
Anti-inflammatory Macrophage (non-phagocytic) Resident Macrophage
Phenotype (ED2+OX6-, ED2+OX6+)
Extensor Digitorum Longus
Electromyography
Eccentric Peak Torque
Estimating Sphericity
Fibroblast Growth Factor
Hours
Height
High Intensity Stretching
Maximum Heart Rate
High Sensitivity C-Reactive Protein
International Association For The Study Of Pain
Interleukin (-6, -8, -10, -12, -15, -1β, -1βra (receptor antagonist), -1ra)
Insulin-like Growth Factor
Isometric Peak Torque
Intense Stretching
Kilograms
Kilometers
Low Intensity Stretching
Logarithm (Natural)
Least Significant Difference
Alternative Anti-inflammatory Macrophage Phenotype with subsets
Meters
xv
Glossary of Terms
MDP
mg/L
min
MiS
ml
Mm
μl
MPO
mRNA
mROM
MTU
n
Nm
NRS
PARQ
PBS
PGE2
PML
PNF
% mROM
r
rads/sec
RMANOVA
ROM
ROS
RPM
RT-PCR
s
SD
SPSS
sTNF-αR
TGF-β1
TNF-α
U/L
VO 2max
W
W max
Muscle Damage Protocol
Milligrams per Liter
Minutes
Medium Intense Stretching
Milliliters
Millimeter
Micro Liter
Myeloperoxidase
Messenger Ribonucleic Acid
Maximum Range of Motion
Myotendon Unit
Number of Participants
Newton Meters
Numerical Rating Scale
Physical Activity Readiness Questionnaire
Phosphate Buffered Solution
Prostaglandin E2
Polymorphonuclear
Proprioceptive Neuromuscular Facilitation
Percentage of Maximum Range Of Motion
Correlation Coefficient
Radians Per Second
Repeated Measures Analysis of Variance
Range Of Motion
Reactive Oxygen Species
Rotations Per Minute
Reverse Transcription Polymerase Chain Reaction
Seconds
Standard Deviation
Statistical Package for the Social Sciences
Soluble Tumor Necrosis Factor Alpha Receptors
Transforming Growth Factor – β1
Tumor Necrosis Factor – α
Units Per Liter
Maximum Amount of Oxygen
Watts
Maximal Exercise Capacity
xvi
Publications
PUBLICATIONS
1
Apostolopoulos N; Metsios GS; Flouris AD, Koutedakis Y and Wyon MA (2015).
The relevance of stretch intensity and position – a systematic review. Front.
Psychol. 6: 1128. doi: 10.3389/fpsyg.2015.01128
2
N. Apostolopoulos, G.S. Metsios, J. Taunton, Y. Koutedakis, and M. Wyon.
Acute Inflammation Response to Stretching: A Randomised Trial. Ita J Sports
Reh Po 2015; 2; 4; 368 – 381; doi: 10.17385/itaJSRP.015.3008 ISSN 23851988 [online] – IBSN 007-111-19-55
3
N. Apostolopoulos, G.S. Metsios, A. Nevill, Y. Koutedakis, and M. Wyon.
Stretch Intensity vs. Inflammation: A Dose-Dependent Association? Int J Kin
Sports Sci. 2015; 3; 1; 27 – 31; doi 10.7575/aiac.ijkss.v3n. 1p27; ISSN 2202946X; URL:http://dx.doi.org/10.7575/aiac.ijkss.v3n.1p.27
4
Abstract of Study: The Effect of Different Stretch Intensities on Recovery From
Muscle Damage: A Randomised Trial, presented at the PANEX2015
Conference in Toronto, Ontario, Canada, Saturday April 18th, 2015
5
In preparation for journal submission:
N. Apostolopoulos, I. Lahart, A. Nevill, M. Wyon, Y. Koutedakis, G.S. Metsios.
The Effect of Different Stretch Intensities on Recovery From Muscle Damage: A
Randomised Trial
xvii
1
INTRODUCTION
STRETCH INTENSITY and
THE INFLAMMATORY
RESPONSE
Introduction
Inflammation and the inflammatory process represents the body’s reaction to a
variety of conditions, including the presence of pathogens, chemical, thermal and
mechanical stressors (Butterfield et al., 2006). For instance, an acute muscle
damage, caused by crush injuries, overloading, and/or strain injuries, initiates a
sequence of cellular responses that define inflammation (Tidball, 1995). The
presence of necrotic and apoptotic cells could also trigger an inflammatory response
(Rock and Kono, 2008).
The inflammatory response can be both acute and chronic but in reality it represents
a continuum (Young et al., 2010), potentially dependent upon the intensity and/or
duration of the stimulus which may be responsible for initiating this response. The
acute inflammatory response – through the acute phase response (APR) induced in
the liver – refers to a generalized response of the body to tissue injury (i.e., chemical,
thermal or mechanical), with the sole purpose of healing (Smith, 1991). The major
characteristic of the acute inflammatory response is the mobilization of inflammatory
cells to the affected site, with the resolution being the elimination and subsequent
return of these inflammatory cells to homeostasis (Maskrey et al., 2011, Serhan et
al., 2007). In contrast, chronic inflammation is characterized by persistent
inflammatory stimuli (Serhan et al., 2007) and orchestrated by numerous chemical
mediators derived from the injured and damaged tissue (Young et al., 2010). Unlike
chronic inflammation, acute inflammation is self-limiting and does resolve itself
allowing the individual to return to somewhat normal function with the return of tissue
homeostasis. The current thesis focuses on the acute inflammatory response.
Stretching is an external and/or internal force that aims to increase muscle flexibility
and/or joint ROM. Along with duration and frequency, stretching intensity is part of
the parameters of stretching. Unlike duration and frequency which are easier to
2|Page
Introduction
quantify (i.e., how long to hold and how often to repeat the stretch), stretching
intensity is not. Its qualitative nature may explain why stretching intensity has
attracted little scientific interest thus far. However, the forces (intensity) applied to the
muscle architecture during stretching could potentially be a catalyst for tissue
damage and inflammation as shown by animal studies (Pizza et al., 2002).
If pain or discomfort is present during the act of stretching, it may potentially imply
tissue damage especially if one considers the definition of pain, as defined by the
International Association for the Study of Pain (IASP). According to IASP, pain is “an
unpleasant sensory and emotional experience associated with actual or potential
tissue damage, or described in terms of such damage” (Merskey and Bogduk, 1994).
An injured muscle typically undergoes stages of degeneration, inflammation,
regeneration and fibrosis, depending, partly, on the severity of the injury (Urso, 2013,
Li et al., 2001). The rupture and subsequent death of the myofibers results in the
infiltration of inflammatory cells due to the damage to the extracellular matrix (ECM)
and the surrounding vasculature (Urso, 2013). The acute inflammatory response is
orchestrated by numerous chemical mediators derived from the injured tissues, with
the most important being indicated at their site(s) of action (Wheater and Burkitt,
1996). In this thesis, reference will be made to such mediators, specifically to the
acute phase protein (APP) C-reactive protein (CRP), the pro-inflammatory cytokines
IL-1β, IL-6, and TNF-α, and to creatine kinase (CK) which is an enzyme associated
with muscle damage. By referring to these blood biomarkers, the current work will
examine how the magnitude and rate of force developed during stretching may
moderate and modulate inflammation and the inflammatory response. Much of the
research on inflammation has focused on inhibiting it with drugs. Though this
approach has some merit, since the degree and extent of the inflammatory response
3|Page
Introduction
may result in further injury to muscle, preventing it may hinder recovery (Mackey et
al., 2007, Ebbeling and Clarkson, 1989). Therefore, the purpose of this project is to
investigate the importance of stretching intensity with regard to aiding the recovery of
athletes from training, competition, and injury, as well as the rehabilitation of
individuals suffering from various musculoskeletal disorders.
4|Page
2
LITERATURE REVIEW
STRETCH INTENSITY AND
THE INFLAMMATORY
RESPONSE
Chapter Two – Literature Review
2.1 – INTRODUCTION
The practice of stretching is widespread in the arts (i.e., dancing), the general fitness
population (i.e., stretching programs), as well as in sports (Taylor et al., 1990).
Stretching may be viewed as a physical strategy aimed at improving flexibility
(Weerapong et al., 2004), performance (Handel et al., 1997, Wilson et al., 1992),
pain relief and recovery from injury (Maffulli et al., 1994, Jackson et al., 1978,
Chandler et al., 1990). Besides, stretching has become the universal strategy for
injury prevention in sports (Thacker et al., 2004). Athletes, coaches, trainers, and
medical professionals (physicians, physiotherapists, etc) recommend stretching to
enhance performance and prevent injury and thus, stretching is regarded as an
essential component of numerous physical training programs (Caplan et al., 2009)
and rehabilitation protocols (Malliaropoulos et al., 2004, Feland et al., 2001b). A
review by Prentice has indicated that four methods are commonly used for sport
activities – static, ballistic, dynamic, and proprioceptive neuromuscular facilitation
(PNF) (Prentice, 1985).
2.2 – THE DEFINITION OF STRETCHING AND IT’S MEASUREMENT
Stretching is defined as movement applied by an external and/or internal force in
order to increase muscle flexibility and/or joint ROM (Weerapong et al., 2004).
Stretching uses an external/internal force that stresses connective and muscle tissue
mechanically (Martins et al., 2013). The force generated during stretching can be
viewed as a load, with load referring to the magnitude and the rate of force
developed (Elliot, 2006). An understanding of how force affects the soft and
connective tissue at macroscopic (muscle, tendons, and myo-tendinous unit (MTU))
and microscopic (proteins, enzymes, ECM, cells) level will provide a better insight as
to how the magnitude and rate of force generated during stretching may affect the
6|Page
body as a whole. This knowledge is pivotal in designing a proper stretching protocol
aiming at increasing athletic performance or the rehabilitation from musculoskeletal
pain and injury.
At the macroscopic level, the change due to stretching is primarily functional, with
changes at the microscopic level reflected in the cells and the ECM of the muscles,
tendons, and the MTU. A reduction in reflex sensitivity of the muscle with repeated
stretching, resulting in a change in the mechanical tension in the skeletal muscle is a
functional change (Avela et al., 1999, Proske et al., 1993). With tendons, stretching
may be responsible for changing their dynamic behavior; this is confirmed by
previous findings from animal studies demonstrating that the elasticity of tendons is
changeable through stretching (Witrouw et al., 2007, Viidik, 1969). Regardless of the
functional or structural change acquainted with stretching, an understanding of how
the body adapts and changes to this force, both macroscopically and microscopically
is essential. This understanding may help highlight the degree of force responsible
for the body’s tissues and cells to recover and repair themselves or be exposed to
greater damage.
The main application for stretching is to increase the ROM of a joint (Shellock and
Prentice, 1985). The increase in ROM is facilitated by the synchronized interaction
between many types of tissue (muscles, tendons) and their relation to bone (joints).
Therefore, stretching implies the use of mechanical stress (force) on these tissues,
using isolated movements, in order to improve the ROM of a specific joint. Range of
motion is an important factor in human athletic performance and in many traumatic
or orthopedic conditions (Maffulli et al., 1994). The degree and distribution of ROM in
individuals may be essential in maximizing physical performance as well as the
genesis and presentation of injuries (Jackson et al., 1978). Range of motion can be
7|Page
specific to each side of the body even in the same joint (Maffulli et al., 1994,
Chandler et al., 1990).
Referring to both the macroscopic (muscles, tendons, and MTU) and microscopic
(cells, tissues, ECM) levels in relation to stretching, an understanding of the degree
of force (intensity) developed during stretching may be responsible for structural and
mechanical changes to the tissue. These changes have been observed to serve a
number of functions such as mediating a load transfer as well as sustaining an
interaction between the cells and tissue critical for the interface of function and
homeostasis (Benjamin et al., 1986, Woo et al., 1988, Lu and Jiang, 2006).
Flexibility is typically measured in degrees of full range of movement to the point of
mild discomfort from a starting position (Luttgens and Hamilton, 1997). Both
goniometers and inclinometers have been used to measure ROM. The goniometer is
widely used in clinical settings in order to quantify any limitations and document the
effectiveness of intervention (Gajdosik and Bohannon, 1987). The two-arm
goniometer seems to be the most commonly used for the evaluation of ROM (Lea
and Gerhardt, 1995). Its usefulness, however, has been questioned since the
starting position, the long axis of the limb, the center of rotation, the vertical and
horizontal positions can only be visually estimated (Nussbaumer et al., 2010).
Nevertheless, the goniometer’s reliability is high confirming its continued use in
clinical settings (Rothstein et al., 1983, Mayerson and Milano, 1984).
Similar to the goniometer, an inclinometer is portable, lightweight and requires little
training to assess joint ROM (Kolber et al., 2012). Two types of inclinometers exist:
gravity-based and digital. The former makes use of a liquid compartment or magnet
that establishes zero degrees when positioned in the vertical. With constant gravity
as a reference point, this inclinometer can be used to assess joint mobility and ROM
8|Page
(deJong et al., 2007, Kolber et al., 2011). Digital inclinometers are costly, with a
major disadvantage being the requirement of the clinician or examiner to establish
the zero point accurately in order to minimize the risk of measurement error prior to
use (Kolber et al., 2012). Overall, the inclinometer possesses good reliability when
strict measurement protocols are adhered to (Kolber et al., 2011, Kolber et al.,
2012).
2.3 – TYPES OF STRETCHES
Stretches can be classified as static, dynamic, and pre-contraction. Within these
categories there are active (self- stretch) and passive (with or without a partner) for
static stretching, active and ballistic for dynamic stretching, and PNF for precontraction stretching (Page, 2012, Weerapong et al., 2004). This categorizing is not
universal with some authors suggesting only four types of stretches: static, ballistic,
dynamic, and PNF (Shellock and Prentice, 1985, Behm and Chaouachi, 2011).
However, what is prevalent is that each of these techniques place certain loads on
the muscles and the joint(s) being targeted.
2.3.1 – Static
Static stretching is commonly employed to improve joint mobility and prevent
contracture (Nakamura et al., 2012). It usually involves moving the limb to the end of
its ROM holding this position for 15 – 60s at a point of discomfort or pain (Behm and
Chaouachi, 2011, Smith, 1994, Sady et al., 1982, Bandy et al., 1997, Feland et al.,
2001b). It can be performed passively with use of a partner or therapist, or actively,
with the individual self-stretching. The primary use of static stretching has been for
increasing the ROM of a joint (Bandy et al., 1997, Feland et al., 2001b). This
increased ROM with an acute bout of stretching has been attributed to changes in
9|Page
the length and stiffness of the MTU of the limb being stretched (Behm and
Chaouachi, 2011, Power et al., 2004). However, a controversy exists whether this
change is due to a decrease in MTU stiffness (Wilson et al., 1991) or is it primarily an
increase in the tolerance to stretching (Magnusson et al., 1996a, Law et al., 2009).
Static stretching takes advantage of the inverse myotatic reflex promoting muscle
relaxation thereby causing a further stretch and an increase in ROM (Feland et al.,
2001a). Other benefits with regard to static stretching have been the reduction of
pain and injury (Smith, 1994, Safran et al., 1989), a decrease in soreness (High et
al., 1989) as well as improved performance (Young and Behm, 2002). However,
systematic reviews looking at the impact of static stretching for prevention of sports
injury risk, concluded that static stretching does not reduce injury rates (Bo Lauersen
et al., 2014, Small et al., 2008). In addition, it has been suggested that static
stretching may affect the development of explosive force, jumping, as well as sprint
performance (Young and Behm, 2003, Winchester et al., 2008). Nevertheless, a
study has observed that static stretching prior to competitions does not negatively
affect the vertical jump performance in trained women (Unick et al., 2005). These
disagreements as to whether static stretching improves performance or not, reduces
pain and soreness, or whether the increase in ROM is due to stretch tolerance or a
change in MTU stiffness, reflects the lack of our current understanding and thus, the
need for further research.
2.3.2 – Ballistic
Similar to static stretching, with “ballistic” stretching, the joint is passively moved to
its maximum ROM, whereupon a dynamic “ballistic” movement is applied on the
stretched structure at the end of the ROM (Konrad and Tilp, 2014). This movement is
in the form of a bouncing rhythmic motion using the momentum of a swinging body
10 | P a g e
segment to lengthen the muscle (Mahieu et al., 2007, Bandy et al., 1997). The
momentum causes the activation of the stretch reflex resulting in a contraction of the
muscle under stretch. This rapid change in tension which increases with the
magnitude and rate of the stretch can produce a strain or rupture of the tissue which
may make this form of stretching disadvantageous for improving ROM (Shrier and
Gossal, 2000, ACSM, 2006, Page, 2012). It is noteworthy that the articles referring to
ballistic stretching as being disadvantageous consisted of a clinical commentary
(Page, 2012), a non-systematic literature review (Shrier and Gossal, 2000), and
guidelines as suggested by the American College of Sports Medicine (ACSM, 2006).
The views expressed in these articles represent findings associated with the fact that
the rapid alternating “bouncing” at the end ROM may increase the risk of injury,
although relevant good quality studies in this field are currently lacking. In contrast,
the articles supporting ballistic stretching are all randomized clinical trials which
conclude that ballistic stretching is beneficial for increasing hamstring flexibility
(Kumar and Chakrabarty, 2010, Morcelli et al., 2013), or that ballistic stretching in
combination with other techniques presents a more appropriate approach with
regard to training and rehabilitation (Mahieu et al., 2007). The aforementioned
discrepancy illustrates the need for more robust research in order to provide better
and relevant information and create a better understanding of stretching techniques.
2.3.3 – Dynamic
Dynamic stretching involves moving a limb through its full ROM to the end ranges for
several times (Page, 2012). This method has been introduced as a better alternative
to static stretching for increasing ROM (Murphy, 1994a, Murphy, 1994b). With
dynamic stretching movement begins from the neutral position of the joint being
performed slowly and deliberately (Bandy et al., 1997). Murphy suggests that if the
11 | P a g e
limbs are moved too quickly, a tendency to swing the limb exists which may cause
the stretch reflex to be elicited at the endpoint of the movement in the lengthening
muscle (Murphy, 1994b). The slow deliberate movement of the limb back to the
neutral position is executed with the use of an eccentric contraction of the muscle.
This contraction by the antagonist muscle, results in the relaxation of the lengthening
muscle due to the principle of reciprocal inhibition. In otherwords, the muscle is
reflexively inhibited (Murphy, 1994a, Murphy, 1994b).
2.3.4 – Proprioceptive Neuromuscular Facilitation
This technique was developed to improve muscle elasticity in order to treat
neurological dysfunctions (Knott and Voss, 1968). and has been shown to have a
positive effect on active and passive ROMs (Hindle et al., 2012). Currently two
techniques, collectively referred to as PNF procedures (Etnyre and Lee, 1987) are
used, the contract-relax (CR) and the contract-relax-antagonist-contract (CRAC)
(Page, 2012, Hindle et al., 2012, Condon and Hutton, 1987). Each technique uses a
volitional contraction in order to increase ROM. The rationale behind the CR-PNF
technique is that the successive maximal excitations of the motor neurons during the
volitional contractions will reflexly promote their subsequent inhibition (Kabat, 1950).
The CRAC-PNF technique involves contraction and relaxation of the muscle to be
stretched, i.e. the stretching of the target muscle is followed by a concentric
contraction of the opposing muscle. The activation of the opposing muscle is
believed to reduce the activation of the target muscle through reciprocal innervation
(Kabat, 1950, Voss, 1967, Hindle et al., 2012, Knott and Voss, 1968). An example of
pairing target and opposing muscles would be the quadriceps and hamstring
muscles, respectively. It has been reported that PNF results in a greater increase in
ROM compared to static stretching (Magnusson et al., 1996a, Magnusson et al.,
12 | P a g e
1996b, Sady et al., 1982) with the effects lasting 90 minutes or more (Funk et al.,
2003). However, the mechanism which is thought to facilitate an increase in muscle
length following PNF has been questioned (Chalmers, 2004, Shrier and Gossal,
2000). In general, the PNF literature suggests that the activation of the opposing
muscle motoneurones, results in the simultaneous excitation of the Ia-inhibitory
interneurons which synapse target muscle motoneurones, which may lead in a
decrease in the neural activity of the target muscle (Sharman et al., 2006, Hultborn
et al., 1976). The inhibition of the proprioceptive structures in the target muscle (i.e.,
Golgi Tendon Organ), in response to the contraction, or shortening of the opposing
muscle, is responsible for the lengthening of the target muscle fibers (Sharman et al.,
2006, Hindle et al., 2012). However, instead of a decrease in the electromyographic
(EMG) activity occurring as suggested by reciprocal inhibition, inferring that the PNF
procedure has reduced the active force production which would resist the
lengthening of the muscle (Chalmers, 2004), the muscle(s) exhibit an active EMG as
well as an increase in muscle stiffness (Shrier and Gossal, 2000, Moore and Hutton,
1980). It has been observed that the increase in ROM of the quadriceps muscle
(target muscle) occurred while the hamstring muscle (opposing muscle) was still
under considerable tension suggesting that the common PNF techniques (CR- &
CRAC-PNF) do not evoke sufficient relaxation in muscles to overcome tension
generated by the stretch (Osternig et al., 1987). Regardless of this controversy, PNF
procedures have been consistently shown to enhance ROM compared to either
static or ballistic stretching (Wallin et al., 1985, Sady et al., 1982, O'Hara et al.,
2011b, Etnyre and Abraham, 1986, Feland et al., 2001a, Funk et al., 2003).
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2.4 – MACROSCOPIC LEVEL – MUSCLES, TENDONS, AND MYO-TENDON UNIT
In the following sections (2.4 and 2.5), reference will be made to both in-vivo
(humans and animals) and in-vitro studies (cells and tissues). Though in-vitro studies
possess a weakness by failing to replicate the precise cellular condition of an
organism, they - nevertheless - offer an understanding of how cells and tissues
respond to external forces. For instance, it was observed that the production of
tenascin-C and collagen XII, two ECM proteins typical of tendons and ligaments, is
high in fibroblasts attached to a stretched collagen matrix, but suppressed in cells in
a relaxed matrix (Chiquet, 1999). This study indicated that the response to a change
in stretch was rapid and reversible, reflected at the messenger ribonucleic acid
(mRNA) level (Chiquet, 1999). Though the cell may be removed from its native
environment, its response to force presents an understanding of what might happen
at the cellular level (in-vivo) relative to the force generated at the tissue level (i.e.,
muscles, tendons). Regarding in-vivo studies, it has been noted that “it is difficult to
separate the effects of one regulating factor from others and to determine the role
played by each in controlling muscle growth” (Vandenburgh, 1987).
2.4.1 – Muscle
All skeletal muscles have adaptive potential, capable of modifying their structure in
response to environmental changes, such as altered patterns of activity, pathological
processes, metabolic conditions, and ageing (Bruton, 2002). Muscle has also a
remarkable ability of repair or regeneration following damage via an effective cellular
repair system (Philippou et al., 2012). This is stimulated in order to recover normal
structure and function while preventing loss of mass since skeletal muscle is
considered an irreversibly postmitotic tissue, lacking an ongoing cell replacement
(Decary et al., 1997, Philippou et al., 2012).
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Isometric, concentric and eccentric are the three major muscular actions resulting
from both its active and passive components (Knudson, 2007). Isometric refers to a
condition where the muscle length does not change, with concentric and eccentric
describing the shortening and lengthening of muscle, respectively. Both active and
passive tension define the length-dependent properties of muscle which is strongly
related to stretching (Knudson, 2006). The interaction of each suggests that exercise
interventions, such as stretching, has a complex effect on skeletal muscle,
dependent on the interaction of the tissues and the nature of the training stimulus
(Knudson, 2006).
The primary functional units of muscle are the contractile proteins, actin (thin
myofilament) and myosin (thick myofilament); they are contained within the
sarcomere, the subunit of the muscle fiber (Huxley, 1985). Anchored at both ends of
the Z-line of the sarcomere, and projecting inwards are the thin filaments
surrounding the thick filaments found within the center of the sarcomere (Figure 2.1).
FIGURE 2.1 – The Sarcomere
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The production of mechanical force is the result of a combined dynamic interaction
between the actin and myosin proteins of these filaments (Geeves, 1991). According
to the cycling cross-bridge model of muscle contraction, the myosin heads of the
thick filament project from its surface forming cross-bridges with the actin of the thin
filament (Geeves, 1991, Huxley, 1985). The hydrolysis of adenosine triphosphate
(ATP) by the myosin drives a cycle of interaction between the actin and myosin
moving the two filaments past each other (Rayment et al., 1993, Huxley, 1985). This
results in a shortening of the muscle, which creates an increase in force. The tension
generated in skeletal muscle is a direct function of overlap between the thin and thick
filaments, with the force generated being a function of sarcomere length (Lieber and
Bodine-Fowler, 1993). With elongation of the connective tissue component of the
MTU, passive tension occurs (Knudson, 2007). In this case there is a decrease in
the overlap of the actin and myosin cross bridges. Therefore, the increase or
decrease in tension of skeletal muscle is a direct function of the magnitude of
overlap between the actin and myosin filaments (Lieber and Bodine-Fowler, 1993).
Though the microscopic structure (actin and myosin) is the functional unit of muscle,
with sarcomere length strongly influencing force generation or elongation,
macroscopically the best predictor of force generation function of muscle is
dependent on its architecture (Lieber and Friden, 2000). Relative to the axis of force
generation, the arrangement of muscle fibers within a muscle refers to the
architecture of skeletal muscle (Lieber, 1992). Skeletal muscle architecture
determines the force-velocity properties of the muscle (Moreau et al., 2010). In
otherwords, the orientation of the muscle fascicle partially determines the direction of
the muscle’s pull and its strength (Lieber and Friden, 2000). If the fibers extend
parallel to the muscle’s force generating axis, they possess a parallel or longitudinal
16 | P a g e
architecture (i.e., biceps brachii), with unipennate referring to muscle fibers running
at a fixed angle (0 – 30 degrees) (i.e., vastus lateralis), and multipennate to fibers
running at several angles relative to the muscle’s force generating axis (i.e. gluteus
medius). This structure-function relationship clarifies the basis of force production
and movement as well as explaining the mechanical basis of muscle injury during
movement (Lieber and Friden, 2000). According to Gans et al. the architecture of the
fiber(s) and how they are attached to each other influence force generation and
ultimately performance and function (Gans and Abbot, 1991).
2.4.2 – Tendon
The primary function of tendons is to transmit muscle force to the skeletal system
with limited elongation (Gelberman et al., 1988). It has also been suggested that the
tendons act as mechanical buffers during eccentric contractions protecting the
muscle from damage (Latash and Zatsiorsky, 1993). The tendon is structurally
attached to the muscle at the MTU and to the bone via the teno-osseous junction.
Tendons are highly organized tissues composed of dense parallel fibers with limited
elongation (Summers and Koob, 2002, Woo and Buckwalter, 1988). The fibers of the
tendon are predominantly type I collagen (strongest fibrous protein in the body) with
fibril associated proteoglycans (Summers and Koob, 2002), aligned along the
tendon’s long axis (Wren et al., 2000). The combination of type I collagen and the
parallel arrangement and alignment in the long axis, enables the tendon to withstand
very high tensile strengths, ranging between 50 to 150 megapascal (Woo and
Buckwalter, 1988). However, this structure is poorly suited to withstand compressive
loading (Wren et al., 2000). Chronic stress loads or overuse injuries on the tendon
result in multiple microtraumatic events that cause the disruption of its internal
structure and degeneration of the cells and matrix (Kraushaar and Nirschl, 1999).
17 | P a g e
An in-vitro study examining the effects of stretch on a rabbit tendon fibroblast cell,
revealed that the mechanical load (stretch) and the subsequent biochemical
response (pro-inflammatory cytokine IL-1β), was responsible for degenerative
changes (Archambault et al., 2002). The compromise in the fibroblast cell’s structure
in response to the stretch and the ensuing inflammatory response was responsible
for the changes. Tendons are viscoelastic material with their structure changing as
the load increases, a time-dependent behaviour, determined by the properties of the
tendon collagen fibers, and interfiber matrix (proteoglycans, glycoproteins, and
water) (Duenwald et al., 2009). The tendon deforms easily without much tensile force
since little realignment is required because of its innate parallel structure. As the
magnitude of the force is increased further, the tendon becomes very stiff. Once the
load is removed the majority of changes caused by the load are recoverable
(Gelberman et al., 1988). It has been observed that the time required for recovery,
after unloading, is greater than the time required during the loading period of the
tendon.
2.4.3 – Myo-tendinous Unit (MTU)
The MTU is the structural link between the tendon and the muscle. It is the location
where muscle cells adhere to tendon collagen fibers, and where the myofibrils
terminate at the cell membrane (Mair and Tome, 1972). The MTU is found at either
end of long cylindrical skeletal muscle fibers (Tidball, 1991). It is considered a
significant component of the tension transmitting mechanism (Garrett et al., 1988).
Both the muscle and the MTU is an integrated mechanical unit with the last
sarcomere of the muscle attaching to the tendinous enriched Collagen I molecules of
the tendon ECM matrix (Charvet et al., 2012). The MTU is dynamic in nature with
new sarcomeres added near the junction in response to stretch-induced hypertrophy
18 | P a g e
(Garrett et al., 1988, Williams and Goldspink, 1971). It has been shown that passive
stretch within a physiological range results in 2% strain on tendon, but an 8% strain
in the MTU demonstrates differences in the viscoelastic properties in various regions
of the MTU (Magnusson, 1998).
By referring to the function and architecture of muscle, tendon, and the MTU an
appreciation is gained of the role each structure plays in relation to the stress or
strain developed both extrinsically and intrinsically. With regard to intrinsic loading,
the transition of the force developed by the contractile filaments (Huxley, 1969,
McCall et al., 1996) of skeletal muscle progresses through the MTU to the tendon
transforming the force from the muscle to the bone as a mechanical force creating
joint movement. Throughout these transitions, the ECM, especially the connective
tissue with its collagen, is responsible for a functional link amongst the three tissues.
This continuum plays an important role in the force transmission and tissue structure
maintenance in the tendons, ligaments, bone, and muscle (Kjaer, 2004).
According to a comprehensive review, the ECM of both the tendons and intracellular
connective tissue are dynamic structures which adapt in a structural and functional
way to mechanical loading (Kjaer, 2004). Further support for the viewpoint of this
adaptation to the mechanical load, both structurally and functionally, can be found in
the articles referenced by Kjaer (Kjaer, 2004). For instance, a work was set to
identify potential genes specific to tendon surface epitenon cells and internal
fibroblasts that are regulated by mechanical load, growth factors, hormones or
combinations (Banes et al., 1999). Another one examined the expression of two
genes, Follistatin and Eph-A4 induced in the presence of transforming growth factor
(TGF)-β1, and expressed in the regions associated with early tendon formation in the
development of chick limbs (D'Souza and Patel, 1999). Follistatin was found around
19 | P a g e
the tendon and near the tips of the digits, while Eph-A4 was located within the body
of the tendon. It was observed that the development of cartilage and tendon are
related to one another in a coordinated manner, and that these genes are significant
in regulating the development of the tendon (D'Souza and Patel, 1999). TGF-B1 has
been found to have a synergistic relationship with mechanical loading, showing
significant increases in the synthesis of human tendinous tissue in response to
exercise (Heinemeier et al., 2003). More evidence comes from another interesting
set of data which demonstrated that acute exercise induced an increased formation
of type I collagen in the peritendinous tissue suggesting an adaptation to physical
load (Langberg et al., 1999). Finally, another review article was referenced by Kjaer
which examined the formation of the muscle and MTU, in particular the creation of
an adhesive joint between the muscle fiber and the tendon in order to maximize the
transmission of tension (Trotter, 1993). According to the latter author, the junctions
created between the muscle and tendons is a dynamic interaction between the
tissues and the muscle cells they adhere to providing not only a means to transmit
tension but as well as adapting to mechanical load.
2.5 – MICROSCOPIC LEVEL – FORCE, CELLS, TISSUE, AND THE
EXTRACELLULAR MATRIX
2.5.1 – Force, Cells, and Tissue
Many factors can regulate skeletal muscle performance and function such as
nutrition, hormones, tension development, as well as passive and mechanical forces
(Vandenburgh et al., 1991). Physical forces, including gravity, tension, compression,
pressure, and shear influence growth and remodelling of all living tissues with their
effects exerted at the cellular level (Ingber, 1997). Since force is both static and
dynamic in nature influencing the development of muscle and connective tissue
20 | P a g e
(Watson, 1991), its application over a given area results in stress to the tissue, with
the level of exposure defined by the magnitude, time, and the direction of the stress
application (Mueller and Maluf, 2002). In otherwords, if a cell protein undergoes a
structural change from an extended to a contracted state, the application of the
stretching force is responsible for changing its energy state (Khan and Sheetz,
1997).
Forces are increasingly recognized as major regulators of cell structure and function
(Jamney and McCulloch, 2007, Khan and Sheetz, 1997), modulating almost all
aspects of cell function, including growth, differentiation, migration, gene expression,
protein synthesis, and apoptosis (Alenghat and Ingber, 2002). It has been observed
that actively contracting and innervated muscle fibers subjected to active and
passive tensions, can regulate the growth of the myofiber both longitudinally
(Williams and Goldspink, 1971) and cross-sectionally (Goldberg et al., 1975).
Therefore, mechanical forces directly affect the form and function of tissues, with the
effects of compression on bone and cartilage and of tension on muscle being several
examples (Alenghat and Ingber, 2002).
Each organism is constantly subjected to external mechanical stress (e.g., gravity,
movements) as well as to internal (contractile and hemodynamic) forces generated
by both muscle and non-muscle cells (Chiquet, 1999). In processes ranging from the
contraction of muscles to the alignment of the chromosomes at the metaphase plate,
forces must be adjusted to the proper levels by cells in order for them to function
properly (Khan and Sheetz, 1997). Therefore, the ability for muscle tissue to respond
to mechanical force is central to and relevant to the restoration of its structure and
function (Orr et al., 2006).
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The impact of mechanical forces on tissue has been known for over a century, when
Julius Wolf postulated that bone tissue adapts its structure to the mechanical
environment (Ingber, 2004, Eyckmans et al., 2011). He observed that the trabeculae
of the bone matched the principal stress lines caused by physical loading (Eyckmans
et al., 2011). In other words, the coordinated growth of the tissue was guided by the
mechanical forces imparted on it (Orr et al., 2006). Mechanical force influences and
plays a role in the development and evolution of the tissues (Silver, 2006), since
tissues alter their structure to this physical stress to best meet the mechanical
demands placed on them (Mueller and Maluf, 2002). This ability to adapt suggests
that altering and manipulating the magnitude of the force(s) may influence the
outcome of the intervention.
The growth and development of both bone and muscle are in direct response to the
force, since tissue structures are dynamic and change in response to the physical
demands placed on them (Frost, 1990). Passive stretch in young animals has been
observed to cause an increase in muscle tension thus stimulating skeletal muscle
growth by inducing an increase in protein accumulation in the muscle tissue
(Goldspink, 1977, Vandenburgh et al., 1990). It has been observed that mechanical
tension in the form of a ‘passive’ stretch is associated with hypertrophy of the
skeletal muscle following a tenotomy of the gastrocnemius muscle (Schiaffino and
Hanzlikova, 1970). He further suggests that this mechanical ‘tension’ is one of the
basic ‘trophic’ factors acting on skeletal muscle, exerting a fundamental regulatory
function adapting the muscle to the variable physiological demands (Schiaffino and
Hanzlikova, 1970). Therefore, this ‘continuous integration’ of the internal responses
to a mechanical force is integral to the homeostasis of the tissue, a biological
adaptation and regulation of the ‘internal milieu’ (Schulkin, 2004).
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2.5.2 – The Extracellular Matrix
The ECM, specifically the connective tissue matrix, is a dynamic network composed
primarily of collagens, noncollagenous glycoproteins and proteoglycans, which are
regulated by diverse biochemical factors including growth factors, cytokines and
hormones (Bowen et al., 2013, Lee and Nelson, 2012). This matrix, with its collagen
content, links tissues of the body together playing an important role in the force
transmission and tissue structure maintenance for tendons, ligaments, bone, and
muscle (Kjaer, 2004). Altered expression of specific ECM proteins is part of the
adaptive response to various types of mechanical stress (Chiquet, 1999). This
“stress can regulate the production of ECM proteins indirectly, by stimulating the
release of a paracrine growth factor, or directly, by triggering an intracellular
signalling pathway that activates the gene” (Chiquet et al., 2003). The structural
integration between the individual muscle fibers and the ECM, as well as the
arrangement of the tendon fibers is pertinent for force transmission as well as the
absorption and loading of energy (Alexander, 1991, Kjaer, 2004, Alexander and
Bennett-Clark, 1977).
The tissues and cells can be exposed to diverse types of extrinsic mechanical forces
including mechanical stretch (tension), compression, and shear stress (Carver and
Goldsmith, 2013). An in-vitro study observed tension as a requirement to maintaining
the phenotype typical for tendon fibroblasts, for which collagen I amounts to more
than 20% of their total protein synthesis (Quinones et al., 1986, Chiquet, 1999).
Interestingly, the same cells (collagen I) at sites of compression, where the tendon is
bent over a bone pulley system, ‘transdifferentiate’ into fibrocartilage (Benjamin and
Ralphs, 1998, Chiquet, 1999), a basic requirement for proper function. In this thesis
our concern is mainly with tension and tensile strength (i.e. resistance to pull) of the
23 | P a g e
muscle and tendon, in the form of stretching. The tensile strength of the tissue,
specifically the matrix, is based on the intra- and inter-molecular crosslinks, as well
as the orientation and length of the collagen fibrils and fibers (Kjaer, 2004). The
connective tissue of skeletal muscle and tendon is a “plastic” structure with a
dynamic protein turnover possessing the capacity to adapt to changes in the external
environment such as mechanical loading or inactivity and disuse (Kjaer, 2004). The
realization that mechanical stimuli can contribute both to the health and
pathogenesis of the tissue illustrates the need to research both the magnitude, the
rate and type of force best contributing to the overall health and recovery of the
tissue(s) of the musculoskeletal system: the muscles, tendons, and the MTU.
2.6 – THE STRAIN FACTORS OF STRETCHING: INTENSITY, DURATION, AND
FREQUENCY
Regardless of the stretching methods used to facilitate an increase in ROM of a joint,
what is similar to all are the parameters of training or the strain factors of stretching
specifically the variables of intensity, duration, and frequency (Marschall, 1999,
Mujika et al., 1995). These variables can be further grouped in a manner defining
both stretching volume and intensity, with stretching volume referring to the total time
under stretch, and stretching intensity to the perception of how “hard” the stretch is
(Young et al., 2006). Volume may be manipulated by the parameters of duration and
frequency, with the perception of “hard” referring to the elongation of the muscle for a
given rate of stretch (Young et al., 2006). Various combinations of these variables
are believed to produce an adaptive response leading to improved performance
(Mujika et al., 1995, Seiler, 2010). In addition to these parameters, stretching initiates
a stress/strain response on the connective tissue dependent on the magnitude, rate,
and intensity at which the loading occurs (Ransone et al., 2012). It causes cellular
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changes as detected in an in-vitro study where tenascin C and collagen XII
concentration, two ECM components of fibroblasts, increased by 8-fold during
stretching (Chiquet, 1999). It has been found that the stretch of muscle cells within
the skeletal muscle tissue suggests an adaptation when subjected to this mechanical
force (Kjaer, 2004). This promotes sarcomeregenesis, the synthesis of a contractile
protein produced by specific muscle genes by mechanotransduction (Martins et al.,
2013). Mechanotransduction is the process by which mechanical energy is
converted into biochemical signals (Hamill, 1997). In other words, how the cells
sense mechanical forces and convert them into changes in intracellular biochemistry
and gene expression (Ingber, 2008b). The process of mechanotransduction is critical
for mediating adaptations to mechanical loading in connective tissues (Ko and
McCulloch, 2001). More on mechanotransduction will be presented below.
Ultimately, the cellular response to mechanical forces is inherently coupled to the
internal organization of the ECM (Chen et al., 2004). Stretching affects the ECM,
with the integrins, the transmembrane receptors bridging cell-ECM interactions,
detecting and transmitting this stimulus into the cell interior activating a series of
nuclear
proteins
modifying
gene
transcription
regulating
sarcomeregenesis
(DeDeyne, 2001). This cellular network may act as an integrated unit transducing
various stimuli (including mechanical loading) into coordinated tissue responses (Ko
and McCulloch, 2001).
The information above is based on in-vitro approaches, which have been used to
provide a plausible explanation as to what might occur in-vivo. As mentioned in
section 2.4, it is difficult to separate the effects of one regulating factor from others
and be able to determine the role that each plays with regard to controlling the
growth of muscle in-vivo (Vandenburgh, 1987). In-vitro studies such as organ, tissue,
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and cell-cultured models afford us the opportunity to isolate each structure in an
attempt to observe how it might respond to mechanical stress, or any other condition
of interest. For instance, with organ cultured methods, the stretching and relaxing of
the excised muscle, which mimics the actual in-vivo activity, has been observed to
stimulate several of the biochemical processes identified with muscle hypertrophy
(Vandenburgh, 1987). Use of collagen gels, either fixed or free floating, allowed
researchers to observe that more tenascin-C and pro-collagen I were secreted under
stressed vs. relaxed conditions (Chiquet-Ehrismann et al., 1994). Tenascin-C, an
ECM protein, is very important as it is expressed in many structures involved with
bearing high tensile forces (i.e., ligaments and tendons)(Chiquet and Farmbrough,
1984). The importance of looking at tenascin-C is that it may provide a better
understanding of how ECM is remodelled in response to a mechanical stress in-vivo.
Although methods and responses of in-vitro differ from in-vivo, they may be
complimentary representing the response of the organism to force as a whole.
However, care should be taken to not extrapolate primary in-vitro data to explain
phenomena in-vivo; the latter does provide a powerful tool for investigating the cells
ability to adapt to external conditions (Quinones et al., 1986).
Although duration and frequency are important in the design and implementation of a
stretching program, intensity, the magnitude of force being applied to the joint during
stretching (Jacobs and Sciacia, 2011) as well as the rate of force, may potentially be
of a greater significance. For instance, it has been observed that too much force
results in an inflammatory response and subsequent fibrosis (Brand, 1984, McClure
et al., 1994). However, unlike duration and frequency which are easier to quantify
with numerous articles referring to them in relation to stretching (Bradley et al., 2007,
Brandenburg, 2006, Kokkonen et al., 1998, Cornwell et al., 2002, Feland et al.,
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2001b), intensity is more difficult to quantify. Intensity, in relation to stretching may
be defined as a feeling, a perception associated with a certain amount of exertion.
According to psychophysical laws, this associated perception or feeling represents a
concept referred to as a “magnitude production” (Stevens, 1971). In otherwords, the
value associated with the intensity of the stretch, which is conveyed to the individual,
allows the individual to best adjust their response in order to produce a match. In
short, intensity is a qualitative response unique to individuals. This qualitative nature
may explain why intensity, with respect to stretching, remains under researched
accounting for the lack of relevant articles.
Therefore, the aim of the thesis is to look at stretch intensity and its influence on the
body. Particular attention will focus on the relationship of the magnitude of the force
generated during stretching and its potential relationship to inflammation and the
inflammatory response. Stretching imparts a mechanical energy on the body, with
stretching intensity (i.e., low vs. high) possibly influencing the mechanical equilibrium
between the tension generating muscle and tension-resisting tendons. In short,
manipulating this force on the body, both externally and internally, may determine
the recovery of the tissue and its function aiding in improving performance and in the
treatment of various musculoskeletal disorders.
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2.7 – INFLAMMATION
“Inflammation is an essential immune response that enables survival
during infection or injury and maintains tissue homeostasis under a variety
of noxious conditions. Inflammation comes at the cost of a transient
decline in tissue function, which can in turn contribute to the pathogenesis
of diseases of altered homeostasis”
…Medzhitov (Medzhitov, 2010)
2.7.1 – Introduction
The history of Inflammation, and its description can be traced back to both Ancient
Egypt and Greece (Granger and Senchenkova, 2010). Hippocrates, the father of
medicine, coined the term edema to describe inflammation, referring to it as a
necessary process of healing following tissue damage (Granger and Senchenkova,
2010). Based on a visual observation, the Roman medical writer, Celsus, is credited
with describing the four cardinal signs of acute inflammation: rubor et tumor cum calore
et dolore (redness and swelling with heat and pain) (Marmelzat, 1977, Medzhitov,
2010). The fifth sign, functio laesa (loss of function), was added a century and a half
later, by the Greco-Roman physician/physiologist, Galen (Rather, 1971, West, 2014,
Lawrence et al., 2002) (Figure 2.2 modified diagram (Lawrence et al., 2002)), and is a
characteristic sign for both acute and chronic inflammation. In the 18th century, the
Scottish Surgeon John Hunter wrote, “inflammation in itself is not to be considered as a
disease, but as a salutary operation consequent to some violence or some disease”
(p.296) (Palmer, 1835). His view further reinforced the importance of inflammation with
regard to body’s homeostasis.
Inflammation is a very important and useful host defense mechanism in response to a
foreign body challenge or tissue injury. It is a necessary process for our tissues and our
ability to function and survive, however, it may also be responsible for causing further
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damage (Slauson and Cooper, 2002). In 1972, Thomas Lewis wrote, “Our arsenal for
fighting off bacteria is so powerful, and involves so many different defense mechanisms,
FIGURE 2.2: The Five Cardinal Signs of Inflammation
that we are more in danger from them than the invaders. We live in the midst of
explosive devices; we are mined” (Lewis, 1972). Inflammation, a process by which the
body attempts to restore the damaged tissue to its preinjury state, consists of an innate
system of cellular and humoral responses (Rock and Kono, 2008, Ward, 2010). It is
either acute or chronic, with acute defining a series of tissue responses beginning within
hours and lasting for several days, characterized by the influx of neutrophils and
macrophages, cells of the immune response (Ward, 2010). The adaptive value of the
acute inflammatory response is the removal of cellular debris, necrotic tissue, with the
subsequent repair of the damaged blood vessels, myofibers, and the ECM (Cannon and
St. Pierre, 1998). However, if there is a persistent injury or a foreign material in the
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wound which cannot be eliminated, this progresses to a chronic phase, with a
preponderance of lymphocytes and macrophages (Lucke et al., 2015), suggesting that
chronic inflammation is not defined by the duration of the response (Ward, 2010). In
order for tissue(s) to return to a normal state, what is needed is a moderation of the four
components of a typical inflammatory response: the inducing stimulus, the sensors,
the inflammatory mediators, and the target tissues, with each component occurring
in various forms (Medzhitov, 2010). The inducing stimulus (i.e., muscle or tissue
damage, injury, infections etc) is responsible for initiating the inflammatory response,
with the outcome (i.e., apoptotic or damaged cells) being detected by the sensors of
the inflammatory pathway (i.e., resident macrophages, toll-like receptors, etc) at the site
of damage. These sensors are responsible for inducing the production of the
inflammatory mediators of the inflammatory process (i.e., cytokines (IL-1β, IL-6, and
TNF-α), which act both locally (site of damage), as well as a systemically (other parts of
the body) effecting various target tissues (i.e., blood vessels, endothelial cells, liver
etc) of the inflammatory response (Haslett, 1992, Medzhitov, 2010).
The disturbance of the microcirculation by an injurious stimulus (i.e., infection, tissue
injury) begins the inflammatory response, in which leukocytes and serum proteins move
from the blood to the extravascular tissue (Medzhitov, 2008, Lawrence et al., 2002).
This is controlled and regulated by the release of vasoactive and chemotactic mediators
(Lawrence et al., 2002). In turn, the nature of the inflammatory trigger (i.e., infection,
tissue damage) is responsible for the path that the inflammatory response will follow
(i.e., bacterial, viral infections, tissue damage etc) (Medzhitov, 2010). In this thesis, we
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are mainly concerned with the response to muscle damage caused by a mechanical
stimulus, in the form of stretching, since this occurs in the absence of an infection.
Several mechanisms are responsible for inflammation from, the injurious stimulation of
mast cells and nerves, bleeding caused by the damage, and cell death due to injury
(Rock and Kono, 2008). The mechanical injury to the myofiber, activates an immediate
necrosis of these fibers exposing the sarcoplasm, facilitating the perpetuation of the
necrosis along the entire length of the myofiber (Li et al., 2001). In turn, the myofibers
nearest the damage, undergo a hypercontraction, tearing apart into a series of irregular
dense masses of myofilaments in the form of contraction clots (Warren et al., 1993).
These clots activate an extracellular protein kinase which is responsible for the initiation
of degeneration of the muscle tissue and local inflammation (Aronson et al., 1998).
Eccentric exercise and acute stretch injuries, are examples of a mechanical stimulus,
given that the force generated in both causes an excessive overload of the contractile
elements of the skeletal muscle exceeding its habitual requirements (Toumi and Best,
2003). Structurally, there is a disarrangement of the myofilament in the sarcomeres,
damage to the sarcolemma, loss of fiber integrity, and the subsequent leakage of the
muscle proteins into the blood (Jones et al., 1986). This is a functional change that is
defined by a decrease or loss in muscle force (Clarkson and Hubal, 2002), changes in
mechanical (Brockett et al., 2001) as well as proprioceptive properties (Walsh et al.,
2006), and is responsible for triggering an acute response (Hellsten et al., 1997).
Muscle inflammation due to damage is defined by several phases: destruction, repair,
and remodelling (Jarvinen et al., 2005), with each characterized by the appearance of
predominant inflammatory cell types (Philippou et al., 2012). According to Tidball, the
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extent of the inflammatory response, and whether it will be beneficial or detrimental, is
determined and influenced by the degree of the muscle damage, the magnitude of the
inflammation, and the injury-specific interaction between the invading inflammatory cells
and the damaged muscle (Tidball, 2005).
2.7.2 – Neutrophils, Macrophages, Cytokines and Acute Phase Response
2.7.2.1 – Neutrophils, Macrophages
The release of reactive oxygen species (ROS), and F-actin, by dead cells caused by the
damage to the muscle tissue are a response of a relevant inducing stimulus, also
trigger the inflammatory response (Cheung et al., 2003). Once within the interstitium,
the inflammatory cells activate the resident satellite cells (the sensors) to release
chemotactic (movement of a cell in response to a chemical stimulus) agents and a
chemotactic signal, inducing the infiltration of the circulating inflammatory cells
(Clarkson and Hubal, 2002, Robertson et al., 1993). The inflammatory cells migrate
through the endothelial wall of the skeletal muscle cells, via the process of rolling,
adhesion, and transmigration, into the site of inflammation (Ley et al., 2007) (Figure
2.3).
The first responders, in the early stage of acute inflammation are the neutrophils,
detected within the first hour, peaking between 24 – 48h and remaining for up to 5d
(Smith et al., 2008, Tidball, 2005). They are phagocytic in nature, but also have the
capacity to release proteases that aid in the degradation and removal of the cellular
debris (Tidball, 2005). They are gradually replaced by the macrophages (Lawrence et
al., 2002, Philippou et al., 2012). Similar to the neutrophils, the macrophage population,
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which appear approximately 2d post damage, further contribute to the degradation of
the damaged tissue and the removal of necrotic cellular debris, through the process of
FIGURE 2.3 – Transmigration
inflammation site)
(Inflammatory
cell
migration
into
phagocytosis (Lawrence et al., 2002). In additions, macrophages play a key role in
repair (Cannon and St. Pierre, 1998, McLennan, 1993). Unlike neutrophils,
macrophages consist of two subtypes, the phagocytic pro-inflammatory phenotype,
responsible for the removal of cellular debris and necrotic tissue, preceding the nonphagocytic anti-inflammatory phenotype. The latter peaks at about 4d, remaining
33 | P a g e
elevated for many days afterwards (Tidball and Villalta, 2010, Tidball, 2005), and is
believed to be involved with muscle repair (Malm et al., 2000, Smith et al., 2008). Both
the neutrophils and macrophages are responsible for the release of cytokines.
2.7.2.2 – Cytokines
Cytokines are small proteins, released by activated immune cells, functioning as
intercellular messengers (Zhang and An, 2007, Kushner, 1993). They are responsible
for the movement of immune cells towards sites of inflammation in response to infection
as well as damage. These soluble mediators, which help regulate the immune
response, are multifunctional, such that more than one cytokine is capable of acting on
the same target cell, negotiating similar functions (Akira et al., 1990). The term cytokine
is a general term, with more specific terms describing their origin and creation. They are
subdivided into either pro- or anti-inflammatory, and act either locally in both an
autocrine (a hormone action in which a hormone binds to receptors on and affects the
function of the same cell that produced it) (Dictionary, 2016), and/or paracrine (a
communication between cells in which a cell produces a signal inducing changes to
nearby cells) (Wikipedia, 2016) manner. Cell to cell communication also occurs in an
endocrine (messaging over a long distance) (Walker, 2016) manner affecting a target
organ (i.e., liver). The types of molecules included under the cytokine umbrella are: the
tumor necrosis factors (TNF-x), the growth factors (x-GF), and the interleukins (IL-x)
(Figure 2.4).
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FIGURE 2.4 – Summary of Cytokines
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Muscle damage stimulates a local cytokine cascade, initiated by various cell types
recruited to the damaged site such as: fibroblasts, neutrophils, and macrophages
(Smith et al., 2008). In general, since cytokines can originate from a variety of cells,
and their biological activity may involve multiple coordinated and related steps (i.e.,
pro- or anti-inflammatory, degradation or repair of damaged muscle tissue), clear cut
and specific contributions of each cell type and the subsequent specific roles of each
cytokine is very difficult to determine (Smith et al., 2008).
In the early stages of the inflammatory response, neutrophils infiltrate the damaged
area. These polymorphonuclear (PML) cells are a source of chemokines, a group of
cytokines specifically released to recruit discrete leukocyte subpopulations (Scapini
et al., 2000). One such chemokine is the pro-inflammatory cytokine, IL-1β, a potent
chemoattractant for macrophages (Smith et al., 2008). In turn, both the neutrophils
and macrophages are responsible for the release of TNF-α. Both IL-1β and TNF-α
are responsible for initiating the degradation of the damaged muscle tissue (Peake et
al., 2005a). In turn, their production is responsible for the expression of a third proinflammatory cytokine, IL-6 (Frost and Lang, 2005). IL-6, a known pleiotropic
cytokine (cytokine that affects the activity of multiple cell types), is released into
circulation in response to multiple homeostatic perturbations including injury, trauma,
and acute infections (Streetz et al., 2001). In short, these three cytokines form a
network in which the expression of each influences and is influenced by the other. As
indicated above IL-1 and TNF-α are potent inducers of IL-6, with IL-6 in turn
regulating the expression of TNF-α (Akira et al., 1990, Akira et al., 1993, McGee et
al., 1995). This synergistic relationship between the three cytokines is an attempt to
regulate the immune response and the inflammatory reaction (Akira et al., 1990,
Akira et al., 1993, Streetz et al., 2001). In addition, IL-6 production in contrast to IL-
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1β and TNF-α, has also been linked to a contraction-induced response by skeletal
muscle (Smith et al., 2008). Finally, although the release of these three cytokines are
responsible for the inflammatory response occurring locally at the site of damage,
they are also responsible for causing a systemic response to the tissue damage, the
APR (Streetz et al., 2001).
2.7.2.3 – Acute Phase Response
The APR occurs due to a disturbance of the homeostasis of the organism (Kushner,
1982) (Figure 2.5). The main aim of the APR is to restore the disruption to the
homeostasis of the organism, suggesting that the response is both necessary and
beneficial to the organism. This response has been linked to playing both a major
adaptive and defense role with regard to tissue injury and infection. It is an intrinsic
response initiated after the first few days following the inducing stimulus, and is
associated with a vast number of changes, both systemic and metabolic (Kushner,
1982). Similar to acute inflammation, the objectives of the APR is concerned with the
containment and destruction of infectious agents, removal of damaged tissue, and
assisting in the repair of the affected organ (Kushner, 1982). Interestingly, the
cytokines involved during the early stages of acute inflammatory response, IL-1β,
TNF-α, and IL-6, are also important for activating the APR. However, according to
Akira et al., although IL-1, TNF are considered mediators with regard to initiating the
APR, they are not responsible for inducing a full APR (Akira et al., 1990). The
primary mediating factor is IL-6, initially referred to as a hepatocyte-stimulating factor
(Fuller and Grenett, 1989, Streetz et al., 2001, Gruys et al., 2005, Ramadori et al.,
1988) (Figure 2.5).
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FIGURE 2.5 – Acute Phase Response
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The APR refers to changes in the concentrations of numerous APP, representing a shift
in the gene expression pattern in the hepatocytes (Kushner, 1993). APP are produced
in the liver, helping to promote the immune response (Streetz et al., 2001). The APR, as
alluded to above, is induced mainly by IL-6, which acts as a messenger between the
hepatocytes synthesizing the APPs and the local site of damage (Baumann and
Gauldie, 1990, Whicher and Westacott, 1992). The APPs have the ability to influence
one or more stages of inflammation, healing or adaptation to a noxious stimulus (Gabay
and Kushner, 1999). Major APPs in humans are CRP, fibrinogen and serum amyloid
alpha with changes in their concentrations being largely due to their production by
hepatocytes, since these plasma proteins are mainly synthesized in the liver (Jain et al.,
2011). Normally present in trace amounts in the plasma, an averse stimulus can result
in a dramatic increase in their rate of synthesis as well as a subsequent increase in their
concentration (Kushner et al., 1989).
In this thesis, we are mainly interested in CRP as a measure of inflammation and its
response to various stretching intensities. In response to cytokines, predominantly IL-6,
CRP is synthesized in the liver (Heinrich et al., 1990, Castell et al., 1990). CRP has
been observed to be present at the very onset of any infection or tissue injury however,
its serum level decreases markedly with the resolution of the condition (Du Clos, 2000).
In otherwords, the increase or decrease in CRP levels is proportional to the
inflammatory stimulus (Libby et al., 2002). CRP, which is not specific to any disease, is
more accurate and the best reflection of the APR compared to any other
biomarker/APP, in demonstrating inflammation and the degree of tissue damage
(Kilicarslan et al., 2013, Libby et al., 2002). In line with this, CRP has a long biological
39 | P a g e
half-life of 19 – 20h (Marino and Giotta, 2008, Vigushin et al., 1993, Pepys and
Hirschfield, 2003), allowing for stability of levels with no circadian variation, as opposed
to IL-6 which is subject to a diurnal variation (Meier-Ewert et al., 2001). IL-6 has been
observed to be low in the morning with high concentrations measured both in-vivo or ex
vivo just before bedtime (Sothern et al., 1995, Vgontzas et al., 1999). CRP represents
and provides, a downstream integration of overall cytokine activation (Libby et al.,
2002).
C-reactive protein has been credited with playing a contradictory role, as either a pro- or
anti-inflammatory mediator. According to Tilg et al. it induces the synthesis of proinflammatory cytokines: IL-1α and β, TNF and IL-6, suggesting that one of its roles is
the magnification of the inflammatory response (Tilg et al., 1994). However, CRP has
more of a prominent role as an anti-inflammatory mediator, inducing anti-inflammatory
cytokines (i.e., interleukin 1 receptor antagonist (IL-1ra) a cytokine with known antiinflammatory properties) in circulating monocytes, with the subsequent suppression of
pro-inflammatory cytokines in tissue macrophages. This can partially be explained with
regard to its relationship with IL-6 (Samols et al., 2002). As previously mentioned, IL-6 is
the principle download mediator for APR, resulting in the synthesis of CRP (Streetz et
al., 2001, Ramadori et al., 1988, Heinrich et al., 1990, Castell et al., 1990). Interleukin-6
directly suppresses the synthesis of pro-inflammatory cytokines, IL-1β and TNFα, by
causing the synthesis or release of IL-1ra and the TNF-a antagonists (Tilg et al., 1994).
Indirectly, since APPs such as CRP are potent inducers of IL-1ra in-vitro, the
concentration of IL-1ra was further increased with the presence of IL-6 (Tilg et al.,
1994). Alternatively, it should be mentioned that as a pleiotropic protein, IL-6 is capable
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of producing more than one response, expressing a pro-inflammatory role with regard to
chronic inflammation (Srirangan and Choy, 2010). Unlike acute inflammation, in which it
exhibits a beneficial protective role aiding in the resolution of acute inflammation, with
chronic inflammation, it exhibits a detrimental role (Gabay, 2006). In this capacity it
favours the accumulation of mononuclear cells through the continuous secretion of
monocyte chemoattractant protein-1 and anti-apoptotic functions on T-cells at the site of
injury (Atreya et al., 2000). According to Gabay, this may be responsible for the
transition from the acute to chronic inflammation in response to the continued
expression and rise in the concentration of IL-6 (Gabay, 2006). Therefore, IL-6 is a
pivotal keystone cytokine, because if the damage to the tissue is not resolved during the
acute inflammatory response, its role shifts to one of perpetuating inflammation as seen
in some conditions such as rheumatoid arthritis (Srirangan and Choy, 2010, Yoshida
and Tanaka, 2014, Hashizume and Mihara, 2011, Metsios et al., 2015).
2.8 – BIOMARKERS
In this thesis, the main biomarker of interest investigating inflammation in response to
various stretching intensities is CRP. Serum levels of CRP are used to monitor the APR
in clinical practice (Vigushin et al., 1993, Jain et al., 2011, Roberts et al., 2001).
According to Kilicarslan et al., CRP is more accurate compared to any other parameter
for measurement of tissue damage (Kilicarslan et al., 2013). With a biological half-life of
approximately 19 to 20h (Pepys and Hirschfield, 2003, Marino and Giotta, 2008,
Vigushin et al., 1993), CRP has a rapid turnover, with elevated circulating levels
decreasing by up to 50% per day with the resolution of the acute phase stimulus (Cox et
al., 1986).
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With circulating CRP being only present in trace amounts in healthy individuals, and is
hardly detectable by standard clinical CRP tests possessing a detection limit of 3 –
8mg/L (Ridker, 2001, Nanri et al., 2007), hsCRP assay was used. The lower detection
limit for hsCRP is approximately 0.04mg/L, with a study of healthy donors indicating a
median of hsCRP levels being 0.8mg/L, ranging between 0.07 – 29mg/L (Nanri et al.,
2007). Use of hsCRP enabled the detection of CRP levels an order of magnitude lower
than traditional assays allowing us the ability to monitor inflammation within our
participants.
In addition, because of the synergistic relationship between IL-6, IL-1β, TNF-α to APR
and the subsequent synthesis of CRP (Akira et al., 1990), these biomarkers were also
measured (Study 1). Measurement for CK (Study 3) was conducted to determine if
muscle damage occurred to following a MDP (Paschalis et al., 2007). Since CK is found
exclusively in muscle (i.e., cardiac and skeletal), raised levels of CK are considered an
important indicator of muscle cell and damage (Apple et al., 1985, Siegel et al., 1981,
Brancaccio et al., 2007). The methods used to analyze IL-6, IL-1β, TNF-α, and CK are
described in studies one and three, respectively.
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2.9 – INFLAMMATION AND EXERCISE
In the literature, inflammation and the inflammatory response has been examined
relative to either non-damaging (e.g. concentric) or damaging (e.g. eccentric) exercise,
with the study by Canon et al. being the first to suggest that cytokines are released in
response to exercise (Cannon and Kluger, 1983). Since stretching is considered a form
of physical activity used by athletes and rehabilitation patients (Weerapong et al., 2004,
Page, 2012), by investigating the response of exercise in relation to the inflammatory
response, this provides a possible explanation of the relationship of stretching to
inflammation. Similar to exercise (non-damaging, and damaging), the efficacy of
stretching is determined by the parameters of training, intensity, duration, and frequency
(Marschall, 1999, Mujika et al., 1995, Seiler, 2010). In this section, both forms of
exercise relevant to the inflammatory response(s) will be discussed.
2.9.1 – NON-DAMAGING EXERCISE AND THE INFLAMMATORY RESPONSE
After the study by Canon et al. (Cannon and Kluger, 1983), exercise has been observed
to induce the cytokine cascade characterized by the release of the following specific
cytokines, the pro-inflammatory cytokines: TNF-α, IL-1β, and IL-6 (Sprenger et al.,
1992, Drenth et al., 1995), and the anti-inflammatory cytokines (cytokine inhibitors): IL1ra, TNF-α antagonists, and IL-10 (Drenth et al., 1995, Pedersen and Hoffman-Goetz,
2000, Ostrowski et al., 1999).
According to Pedersen et al., a marked increase in the concentration of plasma IL-6
compared to other cytokines occurs during habitual exercise (Pedersen and HoffmanGoetz, 2000), contradicting a study by Bruunsgaard et al. suggesting that this increase
is mainly due to muscle damage (Bruunsgaard et al., 1997). Support for Pedersen et al.
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was provided by Peake et al. who observed that IL-6 returned to resting levels post
exercise despite the continued expression of the two markers that associate with
muscle damage, namely CK and myoglobin, suggesting that this increase in IL-6 is not
related to inflammation (Peake et al., 2005b). Since the immune response depends on
the ability of leukocytes to transmigrate from circulation through the endothelium, to the
site of inflammation, an increase in the concentration of adhesion molecules (protein
molecules located on the cell surface involved in binding with other cells) (Alberts et al.,
2002) should be observed on the surface of leukocytes and endothelial cells during
muscle damage. This rise in the concentration of adhesion molecules helps to promote
the migration of the leukocytes to the site of inflammation (Reihmane and Dela, 2013,
Akimoto et al., 2002). This increase in the concentration of adhesion molecules is an
important early step in the induction of the inflammatory process (Springer, 1994, Carlos
and Harlan, 1994, Vestweber, 2007). According to Chen et al. and Kaplanski et al. this
rise of adhesion molecules on the surface of endothelial and leukocyte cells occurs
when these cells are exposed to pro-inflammatory cytokines, in particular IL-6 (Chen et
al., 2006, Kaplanski et al., 2003). However, during habitual exercise, instead of
observing an increase in the concentration of adhesion molecules post exercise or
recovery, adhesion molecules remain unchanged (Smith et al., 2000, Chaar et al.,
2011). Considering that numerous cells from a variety of tissues can produce proinflammatory cytokines (i.e., IL-6, TNF-α), alteration in the concentration levels of these
cytokines are not necessarily reflective of changes in their production by circulating
leukocytes (Reihmane and Dela, 2013, Starkie et al., 2001). Since circulating
leukocytes during habitual exercise are not solely responsible for the rise in
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concentration of plasma IL-6, it has been proposed that muscle contraction may be the
potential mechanism for this observed rise in IL-6 (Steensberg et al., 2002, Steensberg
et al., 2000, Steensberg et al., 2001).
Exercise, both eccentric and concentric, has been associated with the release of IL-6
(Hiscock et al., 2004, Steensberg et al., 2000, Ostrowski et al., 1998). Ostrowski et al.,
observed a rise in IL-6 mRNA in skeletal muscle in response to sustained marathon
running, a form of eccentric exercise (Ostrowski et al., 1998), while a study by
Steensberg et al. using a modified ergometer, also observed a rise in IL-6 from the
contracting knee extensor muscles of a single leg during a 5h concentric exercise
(Steensberg et al., 2000). The use of a concentric exercise provides the opportunity to
observe the expression of IL-6 during non-damaging exercise, since eccentric exercise
has been associated with micro-injuries to both muscle and connective tissue
(MacIntyre et al., 1995). Interestingly, the amount of plasma IL-6 released by the knee
extensor muscles of the exercised leg was ~100-fold greater compared to pre exercise,
with levels remaining relatively unchanged in the non-exercised leg (Steensberg et al.,
2000). This several fold rise in the concentration of IL-6 has also been detected in
clinical studies concerned with serious infection (Damas et al., 1992, Hack et al., 1989,
Bruunsgaard et al., 1999). Another study using concentric exercise in the form of
bicycling, also found an increase in plasma IL-6 levels with no increases in IL-1α, IL-1β,
and TNF-α (Ullum et al., 1994). More importantly no changes were observed for premRNA IL-6, IL-1α, IL-1β, and TNF-α in the blood mononuclear cells (BMNC) due to
exercise, suggesting that the increase in plasma IL-6 was less likely related to the
increased production of cytokines by BMNC (Ullum et al., 1994). This finding was also
45 | P a g e
confirmed in a study investigating prolonged running, (i.e., a marathon competition),
(Starkie et al., 2001). Unlike the previous two studies which had participants perform a
concentric exercise (i.e., bicycling, knee extension exercise) (Steensberg et al., 2000,
Ullum et al., 1994), the study by Starkie et al. involved the use of an eccentric exercise
in the form of running (Starkie et al., 2001). According to the aforementioned studies,
and as suggested by Pedersen et al., the rise in IL-6 observed in non-damaging
exercise was less likely due to circulating leukocytes (Pedersen and Febbraio, 2008).
The concentration of IL-6 associated with eccentric exercise was not much greater than
concentric exercise, demonstrating that damage is not a requisite for its release during
habitual exercise (Pedersen and Febbraio, 2008) ((Figure 2.6 diagram modified from
Pedersen et al. (Pedersen and Febbraio, 2008)).
FIGURE 2.6 – Relationship of IL-6 Relative to Concentric and Eccentric
Non-Damaging Exercise
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A study by Jonsdottir et al. on male wistar rats in which their calf muscles were
electrically stimulated concentrically or eccentrically, observed a local production of IL-6
in the exercising leg; this was not the case, for the non-stimulated leg in which levels of
IL-6 mRNA in the muscle remained unchanged (Jonsdottir et al., 2000). This further
supports the view that the expression of IL-6 is closely related to muscle contraction. In
addition, no-significant difference with regard to muscle fiber type was observed, with
similar levels of IL-6 mRNA measured in both the red fibers of the soleus and
gastrocnemius as well as the white fibers of the gastrocnemius (Jonsdottir et al., 2000).
Therefore, similar to the studies by Ullum et al. (Ullum et al., 1994) and Steensberg et
al. (Steensberg et al., 2000), the Jonsdottir et al. study confirmed that muscle damage
was not a requisite for the increase in plasma IL-6 during habitual exercise (Jonsdottir et
al., 2000). However, more importantly, it demonstrated that plasma IL-6 is produced
locally in the contracting muscle. Interestingly, an in-vitro study using human cultured
myoblasts (embryonic muscle cells with the potential of becoming a muscle fiber)
presented evidence that these cells were responsible for the release of IL-6 in response
to pro-inflammatory cytokines, IL-1β and TNF-α, (Gallucci et al., 1998), suggesting
indirectly that contracting muscle cells may be a source of IL-6. Production of IL-6 in
response to muscle activity has also been linked to muscle repair (Steensberg et al.,
2000), with Kurek et al. observing an increased expression of IL-6 by regenerating mice
myofibers exposed to a crush trauma (Kurek et al., 1996).
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2.9.1.1 – Myokines
Myokines are cytokines or other peptides produced, released, and expressed by muscle
fibers exhibiting either a paracrine or endocrine effect (Pedersen et al., 2007). Myokines
may account for exercise-associated immune changes, as well as playing a role in
resolving both exercise-associated metabolic changes and subsequent metabolic
changes following training adaptation (Pedersen et al., 2007). Habitual exercise is
responsible for the expression of several cytokines in circulation such as: IL-6, IL-1ra,
IL-8, IL-10, IL-15, and TNF-α; the latter cytokine appears in response to only very
intense long duration exercise (e.g. marathon running) (Febbraio and Pedersen, 2002,
Febbraio and Pedersen, 2005, Starkie et al., 2001, Ullum et al., 1994, Nielsen et al.,
2007). The condition for the release of TNF-α with regard to habitual exercise suggests
that it does not precede the expression of IL-6 as occurs with sepsis or muscle
damaging exercise (Pedersen and Febbraio, 2008) (Figures 2.6 Modified from
(Pedersen and Febbraio, 2008)). Interleukin-6, IL-8, and IL-15 have all been classified
as myokines since their expression is regulated by the type of muscular contraction
(Pedersen and Febbraio, 2008, Marino et al., 2011), with both IL-6 and IL-8 regulated
by concentric contraction at the protein and mRNA level (Febbraio and Pedersen, 2002,
Febbraio and Pedersen, 2005, Pedersen et al., 2007, Akerstrom et al., 2005, Chan et
al., 2004), and IL-15 regulated by resistance training (Nielsen et al., 2007). IL-8 is a
chemoattractant (a chemical agent inducing a cell to migrate toward it) cytokine with a
distinct specificity for neutrophils (Bickel, 1993). It is produced by a variety of blood and
tissue cells and is responsible for not only attracting and activating neutrophils at the
site of inflammation but as well as regulating neutrophil endothelial interaction (Bickel,
48 | P a g e
1993, Kirk et al., 1999). Interleukin-15 is a pleiotropic cytokine expressed at the mRNA
level of numerous normal human tissues (i.e., activated monocytes, dendritic cells,
fibroblasts, and osteoclasts) and has the property of inducing TNF-α synthesis (Liew
and McInnes, 2002). It is involved in both adaptive and innate immune responses
(Itsumi et al., 2009).
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FIGURE 2.7 – Comparison of Sepsis (a) vs. Habitual Exercise (b) induced cytokines [note during sepsis IL-6
release is preceded by TNF-α]
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In order to determine the source of IL-6 in contracting muscle a study using reverse
transcription-polymerase chain reaction (RT-PCR), a technique used to detect and
quantify gene expression (i.e., mRNA), was conducted (Keller et al., 2001). Levels of IL6 transcription and mRNA on the nuclei and total RNA were measured from muscle
biopsies obtained pre, during, and post non-damaging exercise from participants.
Participants performed a two-legged dynamic knee extensor exercise (50 – 60% of
maximal work load) for 180min, as well as being on a diet regimen eliciting either
normal (control trial) or low (~ 60% of normal; low glycogen trial) muscle glycogen
levels (Keller et al., 2001). Muscle biopsies prior to exercise indicated that the nuclear
transcription gene for IL-6 was nearly undetectable with a rapid and pronounced
increase observed post exercise (Keller et al., 2001). With IL-6 expressed in the muscle
fibers (Malm et al., 2000), the use of RT-PCR technique expressed that a factor at the
nuclear level was associated with the increase in the transcription rate of IL-6. Further,
in response to a reduction in muscle glycogen concentration, a greater rise was
observed in the transcription for the IL-6 gene ~40-fold after 90min, and ~60-fold after
180min of exercise (Keller et al., 2001). A significant increase in IL-6 mRNA (> 100-fold)
was also observed after 180min of exercise vs. the control trial (~30-fold). Therefore,
besides the type of muscular contraction (i.e., eccentric or concentric), the concentration
of muscle glycogen may be critical in the IL-6 response to non-damaging exercise.
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2.9.1.2 – Muscle as an Endogenous Glucose Producing Organ
It has been suggested that skeletal muscle is an endogenous glucose producing organ,
influencing the disposal of glucose, since a greater increase in glucose uptake was
evident during contraction of skeletal muscle compared to maximal stimulation by
insulin (Febbraio et al., 2004). Considering that IL-6 is produced by the contracting
skeletal muscles in the absence of inflammatory markers, and that the expression of
intramuscular IL-6 mRNA is exacerbated in response to compromised glycogen
concentration, the expression of IL-6 related to the glycogen content suggests skeletal
muscle may play a metabolic role as well (Pedersen and Febbraio, 2008). Studies by
Keller et al. (Keller et al., 2001) and Steensberg et al. (Steensberg et al., 2001)
demonstrated that the release of IL-6 from the contracting muscle is dependent upon
muscle glycogen concentrations. In addition, a study evaluating the metabolic effects of
IL-6 for the treatment of metastatic renal cell cancer, observed that IL-6 produced an
endocrine response in patients administered recombinant human IL-6 (Stouthard et al.,
1995). In otherwords, a greater increase in hepatic glucose occurred on the
recombinant human IL-6 infusion vs. the control day (Stouthard et al., 1995). In
agreement with the previous study, a study by Tsigos et al., which administered IL-6
sub-cutaneously to healthy volunteers noticed an increase in glucose (Tsigos et al.,
1997). IL-6 induced the release of glucagon accounting for an increase in hepatic
glycogenolysis contributing to the elevations of blood glucose (Tsigos et al., 1997). An
in-vitro study designed to investigate whether radioactive glucose was released from
cultured hepatocytes consisting of radioactive pre-labeled glycogen pools, observed
that IL-6 was a strong glucoregulatory cytokine stimulating the release of hepatic
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glucose (Ritchie, 1990). Interestingly, in the past, IL-6 was associated with the term
hepatocyte stimulating factor (Heinrich et al., 1998).
According to Gleeson, the release of IL-6 is related to both glucose and the contracting
muscle, suggesting that this myokine may act as a hormone mediating the hepatic
glucose output necessary for maintaining the homeostasis of glucose during nondamaging exercise (Gleeson, 2000). Therefore, since skeletal muscle has been
considered an endocrine organ, with its contraction stimulating the production, release,
and expression of several cytokines, an important link between skeletal muscle,
metabolic changes, and the modification of cytokine production in tissue and organs in
response to habitual exercise is established (Pedersen et al., 2001, Pedersen, 2006).
2.9.1.3 – Intensity, Duration, and Mode of Exercise
The extent to which muscle is activated depends on both the intensity, duration, as well
as the mode of exercise (i.e., the amount of muscle mass recruited) (Pedersen and
Febbraio, 2008). A study by Helge et al. investigated the importance of intensity and the
release of IL-6 (Helge et al., 2003). Seven untrained healthy male participants
performed a 45min knee extension exercise with both legs kicking at a frequency of 60
kicks per minute at 25% W max , on two independent parallel one-leg knee extension
ergometers, simultaneously. Blood was collected from both the femoral artery and veins
at 15, 30 and 40min. After 45min, the exercise was stopped and the load was adjusted.
When exercise resumed for another 35min, one leg performed a knee extension
exercise kicking at 65% W max (moderate intensity), with the other kicking at 85% W max
(high intensity). Blood was sampled at 15, 30, and 35min, with a muscle biopsy taken
pre and post 80min of exercise. The main finding of the study was that the release of IL53 | P a g e
6 by the working muscle was related to both the intensity of the exercise and the
glucose uptake (Helge et al., 2003), confirming Gleeson’s proposition that the release of
IL-6 by the contracting muscle is important for maintaining glucose homeostasis during
exercise (Gleeson, 2000). Interestingly, the author suggests that while the release of IL6 by the contracting muscle is prompted by the need to supply fuel, in response to
diminished muscular carbohydrate availability, IL-6 may also accommodate the need for
fuel by stimulating lipolysis in adipose tissue (Helge et al., 2003).
Besides intensity, duration of exercise has been associated with the release of IL-6. A
study by Steensberg et al. investigated the effect of a prolonged one-legged dynamic
knee extensor exercise with six healthy males for 5h at a power output of 25W,
representing 40% of their peak power output (W max ) (Steensberg et al., 2000).
Participants moved the ankle over a range of ~ 60 degrees (from 90 to 30 deg angle),
with blood collected before and after each hour of exercise from both the femoral artery
and vein of the exercising leg, and the femoral vein of the resting leg. An increase in the
production and a high turnover rate of IL-6 was observed, with the release of IL-6 from
the muscle over the last two hours of exercise being 17-fold higher than the elevation of
arterial IL-6 concentration (Steensberg et al., 2000). Ostrowski et al. examined the
effects of a prolonged exercise in the form of marathon running (Copenhagen
Marathon) (Ostrowski et al., 1998). Blood samples were drawn from the antecubital vein
of 16 male marathoners one week before, immediately after, and two hours post-race.
Similar to the time-periods for blood collection, muscle biopsies of the vastus lateralis
muscle of the quadriceps were taken from eight of the participants. The major finding of
the study was the detection of IL-6 mRNA in the skeletal muscle suggesting that IL-6
54 | P a g e
was produced locally. Since mRNA expression was not observed in the circulating
BMNC, this removed any contribution of mRNA to the muscle via blood contamination.
In addition, an immediate increase in plasma IL-6 was observed post exercise, with a
decline occurring thereafter, with the concentration of IL-1ra continuing to increase two
hours post exercise (Ostrowski et al., 1998). It is important to note that the later finding
indicates that acute exercise may create an acute IL-6 induced anti-inflammatory
environment within the human body, as opposed to the inflammatory environment
created by IL-6 during sepsis and inflammation.
In addition to intensity and duration, mode of exercise is considered as another source
for the release of IL-6. In otherwords, the mass of skeletal muscle recruited during
exercise may potentially influence the expression of the cytokines produced during
exercise. The study by Nieman et al., had 10 experienced triathletes performing two
cycling and two running sessions spread out over a four to six week period to determine
if exercise recruiting more muscle causes a greater cytokine response during exercise
on levels of IL-6 (Nieman et al., 1998). This study observed that since running involved
more muscle groups and was particularly eccentric in nature, this accounted for the
greatest release of IL-6 vs. cycling which uses mainly the lower body and is concentric
in nature. This observation was supported by Febbraio et al., similarly suggesting that
the pronounced increase in the systemic concentration of IL-6 was due to the greater
recruitment of muscle during running vs. cycling (Febbraio and Pedersen, 2002). In
conclusion, different studies indicated that circulating levels of IL-6 in response to
exercise can occur without muscle damage (Steensberg et al., 2000, Ullum et al., 1994,
Jonsdottir et al., 2000). The magnitude by which the levels of plasma IL-6 increased is
55 | P a g e
influenced by the intensity, duration, the amount of muscle mass recruited (Pedersen
and Fischer, 2007).
2.9.2 – DAMAGING EXERCISE AND THE INFLAMMATORY RESPONSE
Since the 1960s sports scientists have observed that small lesions expressing a small
foci of inflammation and degenerative changes, including fiber necrosis, were
characteristic of a single bout of exhaustive exercise – either short-lasting intensive or
long-lasting moderate (Vihko et al., 1978). This disruption of normal muscle structure
has been associated with crush and strain injuries, overloading, and eccentric exercise,
all initiating a sequence of cellular responses expressing an inflammatory response
(Tidball, 1995). Unlike the cytokine cascade observed for habitual exercise in which the
release of IL-6 is not preceded by TNF-α, muscle damage results in the release of the
pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6. It should be noted that IL-6 is
classified more as an “inflammation-responsive” cytokine since it does not directly
cause inflammation, even though its infusion has resulted in fever in humans
(Mastorakos et al., 1993). In addition, unlike IL-1 and TNF-α, capillary leakage or shock
are not observed with the overexpression IL-6 (Mastorakos et al., 1993). Moreover, the
increased expression of IL-6 is not associated with the upregulation of nitric oxide or
matrix metalloproteinases, which are major inflammatory mediators stimulated by both
IL-1 and TNF-a (Barton, 1997). Instead, as already mentioned in section 2.7.2.3, that IL6 is a primary inducer of CRP, as well as being responsible for the expression of
cytokine inhibitors, IL-1ra, soluble TNF-α receptors (sTNF-αR), and IL-10. Therefore,
with regard to muscle damage, the sequence/time response of the cytokines of the local
inflammatory response are initially pro-inflammatory in nature, with the release of IL-1β,
56 | P a g e
TNF-α, expressed in skeletal muscle (Cannon and St. Pierre, 1998). This is followed by
the release of and expression of the anti-inflammatory cytokines IL-6, IL-1ra, sTNF-αR,
and IL-10 (Figure 2.7, modified from Pedersen (Pedersen, 2006)). Although cytokines
are classified as either pro- or anti-inflammatory, this classification is quite simplistic
since a given cytokine may act as either a pro- or anti-inflammatory molecule (i.e. IL-6)
(Cavaillon, 2001). According to Cavaillon, the nature of the activating signal and the
produced cytokines, the sequence and timing of the action of the cytokine, and the
nature of the target cell greatly influence the properties of the cytokines, and therefore
their subsequent expression as either pro- or anti-inflammatory (Cavaillon, 2001).
FIGURE 2.8 – Cytokine Cascade Related to Muscle Damage
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2.9.2.1 – STAGES ASSOCIATED WITH MUSCLE DAMAGE EXERCISE AND
INFLAMMATION
The series of mind-maps and Table 1 that follow are intended to provide a road map
concerning the body’s response to acute inflammation in response to muscle damaging
exercise. The perceived divisions termed “stages” in the mind-maps were created to
provide a logical sequence of events, which define the acute inflammatory response.
These “stages” are not definitive in nature but rather suggestive (but evidenceinformed), since the complex processes affiliated with acute inflammation (proinflammatory myocytes in circulation, transmigration and adhesion molecules, cytokines
either pro- and/or anti-inflammatory etc.) overlap and are not isolated incidents. Based
on collective research findings, we have been able to identify and map four stages.
These stages are as follows: a) Stage 1 (Muscle Damage). This stage refers to the
causes of muscle damage responsible for the loss of the organization of the sarcomere,
b) Stage 2 (Autolytic Calpain). This refers to the disruption of the cellular Ca2+ resulting
in the expression and subsequent upregulation of calpain (calcium-activated proteases).
Calpain is known to be involved in the autolysis of the muscle components released in
response to mechanical disruption, Stage 3 (Transmigration of Leukocytes into
Inflammatory Site). This stage refers to the activation of the endothelium in order to
attract the leukocytes (neutrophils) to the site of inflammation, Stage 4 (Inflammatory
Cells). This final stage refers to the inflammatory cells that are attracted to (neutrophils
and
pro-inflammatory
macrophages)
as
well
as
activated
(anti-inflammatory
macrophages) at the site of inflammation. These “stages” were created to simplify and
provide an understanding of the complex processes associated with muscle damaging
58 | P a g e
exercises and acute inflammation (Figures 2.8 – 2.11). Table 2.1 provides a time scale
with regard to the inflammatory cells in relation to the phases of muscle regeneration.
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FIGURE 2.9 – The Four Stages with Regard to Muscle Damaging Exercise and Inflammatory Response
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FIGURE 2.10 – Neutrophils
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FIGURE 2.11 – Pro-Inflammatory Macrophages (Phagocytic)
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FIGURE 2.12 – Anti-Inflammatory Macrophages (non-phagocytic)
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Table 2.1 – Time Scale of the Inflammatory Cell Response in Relation to the Phases of Muscle Regeneration Post Muscle
Damage
Time Scale – Muscle Damage (Inflammatory Cell Response)
0h
Phases of Muscle Regeneration
Degeneration/necrosis
2h – 24h
24h – 2d
2d – 4d
4d – 10d
7-10d – 15d
15d – 20d 
Neutrophils Invasion
Phagocytic (M1) Macrophaes Invasion
Inflammation
Regeneration
Remodeling
Maturation/functional repair
Neutrophils Invasion
Phagocytic (M1) Macrophages Invasion
Non Phagocytic (M2) Macrophages Invasion
Satellite Cells Activation
Non-Phagocytic (M2) Macrophages
Invasion
Satellite Cells Activation
Regenerating Muscle Fibers
Regenerating Muscle Fibers
Remodeling Connective Tissue
Maturation of the Regenerated Myofibers
Rescue of the functional
performance of injured muscle
Innervation of regenerated
fibers
* h = hours; d = days
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2.9.2.1.1 – STAGE 1 – MUSCLE DAMAGE
The loss of normal sarcomeric organization, is the catalyst for inflammation at the site of
injury due to the retraction of the myofibrils in response to exercise-induced muscle
damage (Tidball, 1995). The first study to refer to morphological disruptions of muscle in
humans was by Friden et al. (Friden et al., 1981), in which five healthy males ran rapidly
down 10 flights of stairs for a total of 10 times, with the rest period consisting of taking
the elevator to the 10th floor. Muscle biopsies were obtained from the right and left
soleus muscle two weeks prior to and on the second and seventh day post exercise.
Each biopsy was divided into two halves, one-half prepared for electron microscopy,
with the other for enzyme histochemistry. At the cellular level the muscle fibers prior to
and post exercise appeared normal, tightly packed, in well-organized fascicles,
suggesting no signs of ischemic fiber necrosis or rupture in the sore muscles. However,
at the subcellular level focal disturbances of the characteristic cross-striated band
pattern of muscle were observed in biopsies obtained two days post exercise, with
disturbances estimated to be three times greater comparable to sections from both
control and seven days post exercise. The Z-band showed marked broadening,
“streaming” (structural disruption of the material of Z-band which appears ripped out
across a large portion of the sarcomere) (Luther, 2009), as well as total disruption, with
the myofilamentous material in the adjacent sarcomeres being either supercontracted or
disorganized. In addition, the normally regular and complex fine structure of the Z-bands
were observed to have gaps in their characteristic lattice pattern. The high myofibrillar
tension developed during the activation of the contractile material (interdigitating arrays
of the thick and thin myofilaments), may account for the mechanical disruption observed
65 | P a g e
in the Z-bands suggesting that this may be a potential weak link in the contractile chain
of the myofibers (Friden et al., 1981).
A study by Newham et al. suggested that the initial damage to muscle following
eccentric work is mechanically induced, with increases in the intensity of damage being
a result of both mechanical and biochemical factors (Newham et al., 1983b). In this
study, four healthy normal participants performed a 20min step test, with the height of
the step being 110% of the lower leg length (lateral knee joint line to ground) for each
participant (Newham et al., 1983b). The quadriceps of one leg performed a concentric
(stepping up) with the other an eccentric (stepping down) contraction, each lasting 1s
with a stepping frequency of 15 cycles/min. Muscle biopsies were taken bilaterally.
Three participants had a biopsy taken immediately prior to exercise, with biopsies of all
four taken immediately post, as well as at 24 and 48h post exercise. The 24 and 48h
times coincided with pain and tenderness of the eccentrically contracted quadriceps,
suggesting delayed onset muscle soreness (DOMS). The biopsies were prepared for
electron microscopy, with areas of myofibrillar disruption counted. These were classified
as either, “focal” (areas affecting one or two adjacent myofibrils and one or two adjacent
sarcomeres), “extensive” (areas affecting more than two adjacent myofibrils and
sarcomeres, or a fiber containing more than ten focal areas), and “very extensive”
(areas containing more than one extensive area of damage). No abnormalities were
observed in the internal architecture of the fiber in the concentrically activated
quadriceps, either pre or post exercise. However, abnormalities were observed in the
eccentric contracted quadriceps immediately post exercise and with marked changes
one to two days later. As time progressed, histological changes to the myofibers were
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observed, with 59% appearing “normal”, 16% expressing “focal” and “extensive”
changes, with 8% revealing “very extensive” changes for samples taken immediately
post. In samples taken on average 30h post exercise, 45% appeared “normal”, 6%
revealed “focal”, with 23% and 28% expressing “extensive” and “very extensive”
changes, respectively (Figure 2.12).
FIGURE 2.13 – Measurement of Histological Changes
Electron microscopy disclosed that the myofilaments of the sarcomeres in the eccentric
exercised quadriceps immediately and at 30h post exercise were disorganized with the
Z-line material “streaming” across the sarcomere (Newham et al., 1983b). The damage
varied based on the time-period (Figure 2.13), with more sarcomeres exhibiting greater
extensive damage at the 30h post damage. In addition, pain and tenderness was also
documented in the eccentrically vs. the concentrically contracted quadriceps. According
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to Newham et al. the time course of the morphological changes, with more damage
observed considerably later in the post exercise period vs. immediately post, seems to
reflect the delayed release of CK – an important biomarker used to measure muscle
damage – into circulation, peaking between four to five days post exercise (Newham et
al., 1983a). Interestingly, a study investigating the effects of passive stretching in
relation to the immobilized soleus muscle of male wistar rats, observed that stretching
was responsible for causing morphological changes to the muscle fiber (Gomes et al.,
2007). Ultrastructural examination observed a disruption of the sarcomere, with the
myofibrils appearing fragmented at the Z-line prompting the authors to conclude that
passive stretching needs to be applied carefully in order to prevent muscle damage
(Gomes et al., 2007).
Muscle damage initiates a rapid invasion of inflammatory cell populations lasting from
days to weeks and are instrumental in promoting further disruption in muscle
homeostasis or muscle repair (Tidball, 2005). This damage releases various
intercellular proteins, cytokines, as well as chemokines inducing chemotaxis (movement
of an organism or cell in response to a chemical stimulus) in nearby responsive cells
(Shek and Shephard, 1998). The magnitude of the response, the injury-specific
interactions between the invading inflammatory cells and muscle, as well as the
previous history of muscle use determines whether the overall inflammatory response
will be beneficial or detrimental (Tidball, 2005). In otherwords, the size and nature of the
disruption, as well as the previous history of the muscle relative to muscle damage (i.e.,
repetitive injuries vs. acute, eccentric vs. concentric exercise), may be responsible for
the continued expression of the pro-inflammatory cytokines that would be detrimental to
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overall recovery of the muscle from damage. Further, the sequence, timing, as well as
the concentration levels of either the pro- or anti-inflammatory cytokines relative to each
other may be potentially responsible for determining the overall recovery of the muscle.
2.9.2.1.2 – STAGE 2 - CALPAIN
According to Allen, studies referring to humans, intact animals, isolated muscle as well
as single muscle fibers have repeatedly shown that the primary site of damage is within
the muscle itself (Allen, 2001). These lesions are referred to as micro-injuries since they
are usually subcellular, occurring in small proportions to muscle fiber, in response to
either relatively intense, long duration or eccentric exercise (Armstrong et al., 1983). As
suggested above, damage to muscle initiates a sequence of cellular responses
regardless of the stimulus (i.e., crush injury, muscle strain, inflammation), caused by a
mechanical disruption preceding a biochemical response (Faulkner et al., 1993).
Although mechanical disruption is sufficient to explain a reduction in force production, it
is also believed that the increased expression of the inflammatory cells are responsible
for an additional decrease in function (Tidball, 2002, Faulkner et al., 1993, MacIntyre et
al., 1996). The extensive disruption of the structural components of muscle, expressed
as a loss of normal sarcomere organization associated with the retraction of the
myofibrils from the injured site, results in a structurally intact sheath surrounding the
damaged region consisting of a basement membrane and endomysial proteins (Tidball,
1995, Faulkner et al., 1993). This disruption may involve all or a few of the sarcomeres
of a muscle fiber, either in series or parallel, expressing a disturbance of the crossstriated band pattern affecting the myofibrillar Z-band, characterized by streaming and
broadening (Friden et al., 1983). Ultrastructural damage is associated with the
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crumpling of the interface between the thin and thick filaments, since their overlap is an
integral component of the myofilament structure (Brown and Hill, 1991, Higuchi et al.,
1988, Horowits, 1992). Sarcomeric disruption at the level of the Z-line, is associated
with both mitochondrial and sarcoplasmic reticulum vacuolization (Friden et al., 1983,
Belcastro et al., 1985). Vacuolization is an adaptive physiological response to numerous
environmental changes limiting damage, however, it has also been suggested that this
may a be a distinct form of cell death, a degeneration, leading to a lytic response
(Henics and Wheatley, 1999).
The catabolic events associated with muscle damage involve the autolysis of the
components due to the mechanical disruption (Armstrong, 1990, Huijbregts, 2001). The
key to the genesis of this autolytic stage is the disruption of the homeostasis of cellular
calcium (Ca2+) (Armstrong, 1990, Belcastro et al., 1998), released from either
intercellular stores or gaining entry from the extracellular space (Spencer et al., 1996).
The Ca2+ molecule is very important for triggering muscle contraction, relaxation, as well
as controlling the energetics of the muscle by regulating the provision of ATP (Berchtold
et al., 2000, Tate et al., 1991). Within the myofibrils a variety of Ca2+ binding proteins
(i.e., calmodulin, calpains) not involved directly in the primary process of contraction and
relaxation are found, however, these Ca2+ binding proteins are very important for muscle
plasticity and performance (Suzuki et al., 2004, Berchtold et al., 2000). The disruption of
the homeostasis of Ca2+ has been associated with myofibrillar damage such as;
sarcomeric derangement, fragmented or swollen sarcoplasmic reticulum elements and
mitochondria, and lesions in the plasma membrane (Belcastro et al., 1998). This
upregulation of Ca2+, and the muscle’s inability to buffer it, results in the expression and
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subsequent upregulation of calpain, an activated “calcium-dependent protease” (Tidball,
1995). According to Kumamoto et al., calpain is located exclusively inside skeletal
muscle cells existing in the cytosol as an inactive enzyme (Kumamoto et al., 1992).
Increases in cellular Ca2+ cause it to translocate to the membrane where it becomes
active in the presence of Ca2+ (Suzuki et al., 2004). This intercellular protease is known
to hydrolyze enzymes (phosphatases and kinases), as well as muscle, cytoskeletal or
membrane proteins (Kunimatsu et al., 1989). It is capable of cleaving prominent muscle
proteins, including myofibrillar and major Z-band proteins, and those involved with
myofibril linkage to cell membranes (i.e., talin and vinculin) (Takahasi, 1990). Calpain is
associated with both the I- and Z- band regions of the sarcomere, with higher
concentration observed at the Z-disk than anywhere else in the myofibril (Kumamoto et
al., 1992). When activated in response to a rise in the concentration of Ca2+, selective
proteolysis of various structural, metabolic and/or contractile elements occurs (Belcastro
et al., 1998). The location of the calpain at the Z-disk places them in the area of the
muscle that is most susceptible to degradation by calpain (Dayton et al., 1976). Its
protease activity has been observed in association with myofibrillar degradation,
particularly the absence of Z-lines or loss of Z-line proteins seen in 22% of the myofibrils
isolated from the skeletal muscle of exercising rats (Belcastro et al., 1988, Goll et al.,
1991). Calpain cleaves various cytoskeletal (i.e. spectrin, talin, α-actinin), membrane
(adhesion molecules (integrin, cadherin, N-CAM)), as well as myofibrillar (myosin light
chain kinase, troponin, tropomyosin) proteins (Belcastro et al., 1998). Its action is rather
disruptive destabilizing and altering the substrate proteins, making them more
susceptible to various cellular proteases (Saido et al., 1994) (Figure 2.14).
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Normal Sarcomere
Removal of Nebulin, and Titin by Calpain
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Removal of Tropomyosin by Calpain
FIGURE 2.14 – Subsequent Steps Associated with the Autolysis by Calpain
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Calpain has been associated with the proteolysis of skeletal muscle proteins (i.e. αactinin, tropomyosin, and desmin) following exercise (Belcastro et al., 1998), with
exercise induced protease activity linked to an increase in leukocyte populations
following strenuous bouts of exercise (Camus et al., 1992, Field et al., 1991, Gabriel et
al., 1992). In addition, Kunimatsu et al. revealed that the peptides released in response
to the activity of calpain were chemoattractive for neutrophils without producing any
cytotoxic effects (Kunimatsu et al., 1993, Kunimatsu et al., 1995, Kunimatsu et al.,
1989). A study designed to investigate the relationship between calpain-like protease
and neutrophil accumulation was conducted by Raj et al. (Raj et al., 1998). Neutrophil
accumulation was measured by the activity of myeloperoxidase (MPO), as also
discussed in a study by Belcastro et al. (Belcastro et al., 1996). In the study by Raj et
al., 15 male Wistar rats were randomly assigned to either a control (n = 5), or an
exercise (n = 5) group firstly to investigate the relationship between Ca2+-stimulated
proteolysis and neutrophil accumulation (MPO activity), with another exercise group (n =
5) set up to investigate the extent of how calpain may promote neutrophil accumulation.
This latter group was pre-injected with a cysteine protease inhibitor (to reduce the
activity of calpain-like protease), one hour prior to exercise. With speed of the motorized
treadmill set at 25m/min and at an 8% grade, the rats ran on the treadmill for 60min or
until voluntary termination. Post exercise, matched control and exercise rats were
euthanized, with blood and muscle tissue (plantaris and ventricles) collected.
Determination of calpain-like and MPO activity was performed on serial samples from
the same muscle. Plasma CK was also measured. A major finding of the study was the
positive relationship between the activity of the calpain-like protease and MPO activities
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for all the studied tissues (plantaris and ventricles). More importantly, this study
observed a link between the underlying processes with skeletal muscle calpain-like and
MPO activity showing a responsiveness to an exercise stimulus (Raj et al., 1998). The
rats injected with the cysteine protease inhibitor resulted in a lower MPO activity since
this inhibitor reduced the activity of the calpain-like protease. This finding suggests that
the neutrophil accumulation is dependent upon Ca2+ stimulated cysteine proteases (Raj
et al., 1998). The release of the peptides by the activity of calpain are chemoattractive
for neutrophils (Kunimatsu et al., 1989), signifying the shift from “stage 2” to “stage 3” as
outlined in the map above (Figure 2.9). With regard to the study by Raj et al., the
authors suggest that although the underlying mechanisms with regard to the enhanced
responsiveness to exercise stimulus of the plantaris muscle vs. the cardiac muscle of
calpain like activity and MPO are unknown, this enhancement may be related to the
greater force production generated by the plantaris muscle (Raj et al., 1998). Since
skeletal muscle contraction is associated with a metabolic demand, this demand may be
responsible for a breakdown in muscle protein, a mechanism dependent on the
disturbed Ca2+-homeostasis. Evans et al., investigating the metabolic effects of exercise
induced muscle damage, suggested that since eccentric exercise was affiliated with an
extensive delay vs. immediate damage, this may be linked to a continued degradation
of myofibrillar protein (Evans and Cannon, 1991). Interestingly, the elevation of muscle
Ca2+ associated with stretching was observed in female Sprague-Dawley rats,
suggesting that stretching may be a potential mechanism for the disruption of the
homeostasis of Ca2+ (Armstrong et al., 1993).
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2.9.2.1.3 – STAGE 3 – Transmigration and Adhesion Molecules
When skeletal muscle is damaged, leukocytes are attracted to the site of injury.
Robertson et al. investigated the chemotactic response of leukocytes (neutrophils and
macrophages) to skeletal muscle damage (i.e., crush injury) of the mid-region of the
tibialis anterior of female inbred Swiss mice at; zero (0), three (3), and at 24h post crush
injury vs. non-injured muscle (Robertson et al., 1993). All leukocytes were obtained from
the same breed of mice. A strong chemotactic response of the excised skeletal muscle
to leukocytes was observed for both the three and 24h muscle sample post damage,
with the migrated leukocytes seen to be accumulated in clumps near the region where
muscle fragments were pronounced. Even though an accumulation of PML was
observed within the muscle after three hours, a far stronger macrophage
chemoattractant agent was observed within the 24h crushed muscle, in particular for
exudate vs. resident macrophage populations (Robertson et al., 1993). No
chemoattractant was present for macrophages in the normal uninjured skeletal muscle
nor the muscle removed immediately after crush (zero hour) (Robertson et al., 1993).
Considering that PML and macrophages were expressed in the injured muscle several
hours post damage (three and 24h), and not immediately after, it has been suggested,
that the activation of resident fibroblast and macrophages may account for this.
Normally residing in a quiescent state in the endothelium, resident macrophages are
responsible for attracting and activating inflammatory cells to the site of injury during the
early stages of muscle injury (Tidball, 1995). In addition, activation of the endothelium
may be responsible for attracting inflammatory cells. Expression of IL-1β and TNF-α in
response to an active stretch during eccentric exercise (Malm et al., 2000), was
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responsible for the augmentation of leukocyte adhesion molecules has been observed
in studies which reported that IL-1 and TNF can act directly on cultured human
endothelial cells, increasing leukocyte adhesion (Bevilacqua et al., 1987). Use of
quantitative in-vitro endothelial-leukocyte adhesion assays, showed that IL-1 and TNF
were responsible for activating human endothelial cells increasing the adhesion of PML
leukocytes, and monocytes. Selectins are leukocyte adhesion molecules acting as
receptors for the recruitment of leukocytes into the site of damage and inflammation.
Interestingly, the expression of E-selectin has been observed to be induced by the
presence of TNF-α (Weller et al., 1992, Cannon and St. Pierre, 1998). In turn, activation
of the endothelium results in a further release of IL-1β, as well as other cytokines, such
as IL-6 and IL-8, both responsible for attracting neutrophils (Detmers et al., 1991, Liu
and Spolarics, 2003). Previous studies have shown that induction of IL-8 by IL-1β was
responsible for promoting endothelial adhesion and the chemotaxis for neutrophils
(Colditz et al., 1989, Willems et al., 1989). The blocking of IL-1β activity using IL-1βra
reduced production of IL-8 by 85% in isolated peripheral BMNC stimulated with IL-1β
(Porat et al., 1992).
2.9.2.1.4 – STAGE 4 – INFLAMMATORY CELLS
1 - Neutrophils
Neutrophils and macrophages dominate the inflammatory response. Within several
hours of damage, neutrophils invade skeletal muscle remaining for several days
following exercise (Tidball, 1995, Karalaki et al., 2009). Belcastro et al. reported a large
increase in neutrophil concentration in male Sprague-Dawley rat muscle immediately
after 1h of treadmill running at 0% grade at 25m/min until voluntary termination, by
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measuring the activity of the MPO enzyme (Belcastro et al., 1996). MPO is used to
measure and determine neutrophil migration into muscles and blood neutrophil
degranulation (Morozov et al., 2006). The presence of neutrophils was confirmed in a
study with nine healthy sedentary untrained men performing 3 sets of 15min bouts of
downhill treadmill running on a negative 16% incline, separated by 5min rest periods
(Fielding et al., 1993). The intensity of the exercise was set at 75% of each participants
HR max determined by a previous VO 2 max test. Percutaneous needle biopsies were taken
of the vastus lateralis before, 45min and 5d after exercise, with blood samples obtained
before, immediately after, as well as three and 6h, and 1, 2, 5 and 12d post exercise.
The study observed a relationship between neutrophil and immunohistochemical
staining for IL-1β with a significant positive correlation between neutrophil infiltration and
the ratio of ultrastructural damage to total Z-bands (r = 0.66; P< 0.05). A significant
accumulation of neutrophils was observed at 45min, while these remained elevated 5d
post downhill running protocol. Immunohistochemical staining of the muscle cross
sections revealed a 135% increase in IL-1β immediately after, increasing to 250% five
days post exercise. The association between neutrophils and IL-1 has been confirmed
by other studies as well (Smith et al., 2008, Pedersen et al., 1998, Figarella-Branger et
al., 2003, Philippou et al., 2012).
Neutrophils are the most abundant circulating leukocyte (Summers et al., 2010) playing
a prominent role in the early expression of the inflammatory response (Kanda et al.,
2013), with their increase occurring within one to six hours post injury (Orimo et al.,
1991, Papadimitriou et al., 1990). The elevation of neutrophils have also been
associated with passive stretching in four month old adult mice (Pizza et al., 2002). The
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importance of neutrophils post damage is twofold; firstly, they remove necrotic tissue by
the process of phagocytosis, and secondly, they continue the inflammatory response by
causing the release of pro-inflammatory cytokines (i.e., IL-1β, and TNF-α) (Smith et al.,
2008, Cannon and St. Pierre, 1998). In-vitro studies have demonstrated that neutrophils
are a potential source for both IL-1 (Tiku et al., 1986) and TNF-α (Dubravec et al., 1990)
expression. In addition, invading neutrophils are a source of ROS, cytotoxic molecules
(i.e., superoxide or hydrogen peroxide) which can lyze cell membranes causing further
muscle damage (Philippou et al., 2012). Reactive oxygen species are considered a
double edged sword: besides causing further oxidative damage to the differentiating
myoblasts and myotubes, under certain concentrations, and in conjunction with other
chemokines and growth factors, they participate in muscle repair (Barbieri and Sestili,
2012). Canon et al. suggests that a relationship exists between the concentrations of
these ROS and the expression of the pro-inflammatory cytokines IL-1 and TNF (Canon
et al., 1991). According to both Tidball (Tidball, 2005) and Nieman (Nieman, 1997),
expression of these cytotoxins is dependent on the type and the intensity of the exercise
causing the muscle damage. Therefore, muscle damage itself is believed to be a biphasic response characterized by the primary mechanical injury caused by the exercise
itself, followed by a secondary biochemical response linked to the inflammatory
response occurring within several hours to days post muscle injury (Howatson and van
Someren, 2008, Howatson et al., 2012). This secondary biochemical response has
been linked to further muscle damage or repair of the muscle (Tidball, 2005, Philippou
et al., 2012).
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2 & 3 – Pro-inflammatory and Anti-inflammatory Macrophages
After the initial invasion by neutrophils, macrophages begin to invade the damaged
skeletal muscle. Similar to the neutrophils, macrophages are active inflammatory cells
producing several pro-inflammatory cytokines, as well as promoting the removal of
cellular debris, and muscle tissue remodelling (Kanda et al., 2013). Macrophages
consist of several subtypes, with distinct functions within damaged skeletal muscle, with
their phenotype being determined by the molecular environment of the damage
(Philippou et al., 2012, McLennan, 1996). For instance, the continued presence and
accumulation of neutrophils, which are liable for the impaired tissue regeneration, are
also responsible for the continued presence of pro- rather than anti-inflammatory
macrophages at the inflammatory site (Pizza et al., 2005). Macrophages can adopt
either a pro- or anti-inflammatory phenotype with the pro-inflammatory phenotype
producing ROS as well as IL-1β and TNF-α (Bencze et al., 2012). The anti-inflammatory
macrophages, when activated, produce IL-10, which downregulates the production of
IL-12, characteristic of the inhibition of inflammation (Bencze et al., 2012).
With the use of a specific panel of antibodies based on the antigenicity of the various
macrophage subtypes (ED1+ monocytes, ED1+ macrophages, ED2+Ox6- and
ED2+Ox6+, and Ox6+), McLennan investigated the arrival and departure time, as well as
a specific location within the lesion of damaged muscle of the various macrophage
subtypes (McLennan, 1996). The tibialis anterior muscle of adult male Wistar rats (n =
55) was exposed to a freeze injury using the blunt end of a stainless steel surgical
probe (3 x 4mm) cooled in liquid nitrogen. The rats were sacrificed at various hours and
days ranging from 0.5, 1, 1.5, 2, 3, 5, 7, 9, 11 and 18h as well as 1, 2, 3, 3.5, 4, 4.5, 5,
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6, 7, 8, 11, 14, 19 and 21d after the lesioning of the muscle. Sections of the muscle
were prepared for analysis using antihaemopoietic cell antibodies. Within 1h of freezing,
damage/dead fibers had swollen differentiating them from healthy fibers. This initial
event occurred uniformly throughout the lesion with subsequent cellular events
occurring were the lesion bordered the undamaged fibers progressively moving from the
periphery to the center creating a gradient of maturity. Events within the central core
were usually delayed by two to three days. Cell infiltration on the periphery began by
three hours with some fibers being completely phagocytosed by one day. Myotube
formation was observed to occur by two days, with the boundary between the damaged
and undamaged portion becoming undiscernible by three weeks.
Circulating macrophages (ED1+ monocytes and ED1+ pro-inflammatory)
Within an hour of damage and in response to inflammatory signals (cytokines and
chemokines), the ED1+ monocyte are found to infiltrate the epimysium penetrating the
injury within three hours. The primary role of the monocyte is to replenish the population
of tissue-resident macrophages during homeostasis as well as during inflammation
(Murray and Wynn, 2011). Upon infiltration, the ED1+ monocyte is activated into the
ED1+ macrophages, which precedes the ED2+ macrophages and Ox6+ cells. According
to Butterfield et al. ED1+ monocytes are the predominant macrophage phenotype in
circulation transforming into the ED1+ macrophage upon activation (Butterfield et al.,
2006). The presence of ED1+ cells associates with mature lesions clearly indicating
where the damage had been. Near the end of the degenerative phase ED1+ are less
apparent.
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Resident Macrophages (Ox6+. ED2+Ox6-, ED2+Ox6+, and ED2+)
Resident macrophages which represent up to 10 – 15% of the total cell number during
quiescent states in adult mammals, are versatile cells found in essentially all adult
mammal tissues, with this number increasing in response to inflammation (Italiani and
Boraschi, 2014). Concentrations of OX6+, a major subtype of resident macrophages,
are completely absent early in the lesion, possibly in response to the death of the
resident macrophages, as the damaged area contained some OX6+ cellular debris, with
their numerical density behind the lesion being normal. Their appearance in the core of
the lesion always occurred after the presence of ED1+ monocytes/macrophages with
their numbers exceeding ED1+ cells. Increased accumulation of Ox6+ occurring within
the necrotic fibers indicated early stages of degeneration, being less abundant in
extensively phagocytosed fibers. As the lesion regenerated, the Ox6+ cells became
more localized to the endomysium and perimysium, being initially more abundant than
ED2+ macrophages in the endomysium with the converse for the perimysium. They are
normally located in both the endo- and perimysium, with their concentrations being
greater during regeneration than in undamaged muscles. Three weeks post damage,
their numerical density declined, making it difficult to delineate between damaged and
undamaged tissue.
According to Honda et al. (Honda et al., 1990) and McLennan (McLennan, 1993)
ED2+Ox6- are a major type of resident macrophages in skeletal muscles. Within
lesioned areas, they appeared to die and rapidly regenerate. After one-hour post
damage, the ED2+ macrophages were no longer detectable; however, they were still
abundant in undamaged portions of muscle. At three hours a progressive increase in
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their numbers occur on the fringe, with a heightened accumulation forming on the
fringes of the lesions as well as the perimysia behind them by one to two days (Honda
et al., 1990, McLennan, 1993). It should be noted that the majority of ED2+ in the
connective tissue, both behind and overlaying the lesions, were of the ED2+Ox6phenotype. ED2+ macrophages are not associated with necrotic fibers during the early
stages of phagocytosis (Honda et al., 1990). Typically, these fibers are surrounded by
Ox6+ rather than ED2+ in response to the degeneration of the fiber since the density of
Ox6+ is initially higher in the muscle fiber prior to muscle damage ((McLennan, 1996).
Spatial sequencing of inflammatory cells
Evidence exists suggesting that overload, overuse, and compression can induce
structural damage and inflammation to both muscle and tendon tissue following an
injury (Messner et al., 1999, Soslowsky et al., 2000, Sharma and Maffulli, 2006,
Kuipers, 1994, Sorichter et al., 1999 ). A study specifically referring to the time course of
inflammatory cell accumulation in two animal models, with regard to structural damage
and inflammation following a tendon injury (acute tendinopathy), provides a summary of
the spatial sequencing of the various inflammatory cells, specifically the accumulation
and decrease in the concentration of the neutrophils, ED1+ (phagocytic) and ED2+
(non-phagocytic) macrophages (Marsolais et al., 2001). Although this study referred to a
tendon injury, regardless of the inciting factors, the events associated with inflammation
and tissue damage are considered to be the same with the kinetics and the magnitude
of the response dependent on the extent of the damage as well as the muscle damaged
(Butterfield et al., 2006, Tidball, 1995, Carlson and Faulkner, 1983, Charge and
Rudnicki, 2004, Lefaucheur and Sebille, 1995).
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The study by Marsolais et al., consisted of female Wistar rats weighing ~ 200g
(Marsolais et al., 2001). In the first model, a blunt dissection of the right isolated
exposed Achilles tendon was performed and subsequently injected with 30μl of crude
collagenase dissolved in sterile phosphate buffered solution (PBS) near the osteotendinous junction of 24 rats, inducing Achilles tendinitis based on a previous study by
Davidson et al (Davidson et al., 1997). This procedure was repeated with shamoperated animals with the same volume of PBS without collagenase. Ambulatory
controls not injected with either collagenase or PBS were allowed to roam freely in their
cage. Since the sham group in the first model expressed a high number of inflammatory
cells (neutrophils and ED1+ macrophages) following the exposed Achilles tendon
procedure, a second model was set up to determine whether the surgical intervention
and not the percutaneously injected collagenase and PBS was responsible for
inflammation. Rats in the second model were injected with the collagenase and PBS
percutaneously without any surgical intervention, with the same volume of PBS without
collagenase being injected in the sham animals, with the ambulatory control rats not
being injected with either collagenase or PBS. Rats in the first model (experimental,
sham, and ambulatory control) were sacrificed at 1, 3, 7, 14 or 28d post collagenase
and PBS injection, with the rats in the second model sacrificed after 1 and 3d. For both
models, the sham animals were sacrificed at 1 and 3d. A comparison of neutrophils,
ED1+ and ED2+ macrophages between the two groups (experimental and sham) for
both models was performed at 1 or 3d post collagenase or PBS injections, with this time
period being reflective of the time encompassing an extensive cell accumulation of the
inflammatory cells.
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The injured tendon expressed an accumulation of leukocytes with a rapid infiltration of
neutrophils followed by ED1+ macrophages, but to a lesser degree, with regard to post
collagenase injection. There was a 46-fold increase in neutrophil concentration at 1d
post injury in the exposed Achilles tendon animals, decreasing more than 70% after 3d,
with a return to control values at 7, 14, and 28d post injury. The time points used in this
study (1, 3, 7, 14, and 28d), are suggestive of the phases of inflammation, and the
resolution of the inflammatory response (Frenette et al., 2002) (Table 2.1). A
comparison of immunohistochemistry results revealed a high concentration of
neutrophils after one day of the injured tendon vs. being completely absent in the
tendon from both the ambulatory controls and those that had recovered at 28d.
Concerning ED1+, an 18-fold increase occurred at one day decreasing slightly at 3d post
injury, with the most significant decrease occurring by day seven matching ambulatory
control concentrations after 14d. Comparing ED1+ to neutrophils, the latter had a
twofold increase in numbers after one day, with ED2+ values augmented after 28d
(Table 2.2). Comparing the sham to the ambulatory control rats in the first model, a
significant increase in both neutrophils and ED1+ concentrations was observed in the
sham animals, which were exposed to the surgical procedure minus the collagenase
and PBS. This result is suggestive of the effect of the mechanical stimulus or stress as
a primary and major contributing factor for inducing muscle damage and inflammation
(Kuipers, 1994, MacIntyre et al., 1995, Frenette et al., 2002).
Interestingly, the appearance of leukocytes was similar in both the non-exposed and
exposed Achilles tendon groups at one and three days, confirming that regardless of the
insult causing the damage, the sequencing of the cells of the inflammatory response are
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the same (Tidball, 1995, Butterfield et al., 2006). The non-exposed Achilles tendon
procedure was associated with a decrease in the magnitude of the leukocyte cell
accumulation ~ 35% for neutrophils and ~ 39% for ED1+ macrophages. Following
collagenase injection, ED2+ macrophages increased in the non-exposed Achilles tendon
group after three days. With both the ambulatory controls and sham animals, which
underwent the non-exposed Achilles tendon procedure, no significant difference was
observed in the number of inflammatory cells. Based on the results of this study
(Marsolais et al., 2001), as well as several other studies, neutrophils and ED1+
macrophages express a phagocytic phenotype responsible for the removal of cellular
debris, and ED2+ macrophages are associated mainly with the regeneration of
damaged muscle (Al-Mokdad et al., 1997, McLennan, 1996, McLennan, 1993,
Massimino et al., 1997, St. Pierre and Tidball, 1994).
Referring to the table below (Table 2.2) based on the Marsolais et al. study; we notice
that in model one, which involved a mechanical insult to the Achilles tendon in the form
of a blunt dissection with a subsequent injection of collagenase and PBS, vs. model two
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Table 2.2: Sequence of Inflammatory Cells
Time scale – Achilles Tendon Damage
Inflammatory Cells
0h – 24h
Model One †
Neutrophils
ED1+
ED2+
3d
↑46-fold post trauma. Highly Significant ↓ (~70%)
concentrated after 1 day (note:
magnitude of ↑in cell number 2fold greater than ED1+).
↑ 18-fold increase
↓ Slightly
7d
14d
28d 
Return to control values post trauma.
Absent in ambulatory and animals
recovered for 28d
↓Significantly
Similar to control 
Note: Experimental (Exposed Achilles tendon & collagenase + PBS injection) > Sham (exposed Achilles tendon & PBS) > ambulatory control
(no collagenase & no PBS)
Model Two‡
Neutrophils
ED1+
ED2+
≈ 35% less than same time point
for neutrophils in model one
≈ 39% less than same time point
for macrophages in model one
Significant ↑ 3d
following injection of
collagenase
Note: no significant difference in the number of inflammatory cells between sham and ambulatory controls
†Model One (Exposed Achilles Tendon) = Experimental group (surgery + collagenase & PBS); Sham group (surgery + PBS), Ambulatory control
(no collagenase & no PBS)
‡Model Two (Non Exposed Achilles Tendon) = Experimental group (collagenase & PBS); Sham group (PBS), Ambulatory control (no collagenase
& PBS)
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in which the rats were not exposed to a mechanical stimulus (i.e., blunt dissection), the
ED2+ (non-phagocytic) macrophages associated with recovery accumulate earlier post
collagenase injection (Table 2.2, yellow box). As we observed in the studies which
referenced both the neutrophils as well as the pro- and anti-inflammatory macrophages
(P. 83 – 88), the neutrophils and the macrophages dominate the inflammatory response
to muscle damage, with the ED1+ and ED2+ macrophages possessing diverse roles
with regard to the inflammatory response and repair (Philippou et al., 2012, Tidball,
1995, Karalaki et al., 2009). The ED1+ macrophages feature prominently in the removal
of necrotic tissue with the ED2+ macrophages associated with muscle tissue repair
typically associated with the later stage of inflammation. The early appearance of the
ED2+ macrophage associated with the less traumatic insult on the tendon tissue (model
2 non-mechanical stimulus), suggests that the regeneration process may occur earlier
(Marsolais et al., 2001). Skeletal muscle has the ability to regenerate itself with the
repair process involving the activation of molecular and cellular responses (Charge and
Rudnicki, 2004, Mourkioti and Rosenthal, 2005, Doyonnas et al., 2004). Therefore, this
study by Marsolais et al. proposes that the degree of the muscle damage and the
interaction and coordination of the various infiltrating inflammatory cells are important
for the outcome of the repair process of muscle.
Neutrophils and macrophages play an important role in both the initial response to
tissue damage as well as the subsequent resolution and regeneration of muscle fiber.
At the site of tissue damage, as indicated by the several studies discussed above
(McLennan, 1996, Marsolais et al., 2001), these inflammatory cells coexist, with their
existence being dependent on their ability to perform distinct functions. According to
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Tidball et al. macrophages during the inflammatory process do not contribute to
membrane disruption even though the concentration of ED1+ (phagocytic) macrophages
are observed to peak with muscle damage (Tidball et al., 1999). However, the ability of
the macrophages, specifically ED2+, to repair the damage is quite limited in the absence
of neutrophils, since the primary role of the neutrophils is the removal of cellular debris
through phagocytosis, prompting the macrophages for cellular regeneration and repair
(Grounds, 1987). On the other hand, the continued presence and accumulation of
neutrophils may be responsible for impaired tissue regeneration, since they are
responsible for secondary muscle injury modifying skeletal muscle proteins oxidatively
(Pizza et al., 2005). Elevated levels of neutrophils have also been associated with
passive stretches without any signs of injury (Pizza et al., 2002).
With regard to the macrophages, although ED1+ and ED2+ phenotypes were described
in the studies by McLennan (McLennan, 1996) and Marsolais et al. (Marsolais et al.,
2001), other nomenclatures referring to the spectrum of macrophage activation are also
used. Type 1 pro-inflammatory (M1), and classically activated macrophages (CAM) are
similar to ED1+ (circulating macrophages) with type 2 anti-inflammatory (M2-a,-b,-c),
and alternatively activated macrophages (AAM) being similar to ED2+ (resident
macrophages) (Cote et al., 2013, Fernando et al., 2014). Regardless of the
nomenclature, the microenvironment in which the macrophage finds itself is essential
for its expressed phenotype (McLennan, 1996), with the polarization signals being
apoptotic cells (i.e. dead neutrophils) as well as cytokines released by other
inflammatory cells (Duque and Descoteaux, 2014, Butterfield et al., 2006). For instance,
a microenvironment populated predominately by necrotic fibers as early as one day post
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neutrophil invasion is responsible for the expressed ED1+ (M1) phenotype (McLennan,
1996, Marsolais et al., 2001). Similar to the neutrophils, ED1+ are activated by proinflammatory cytokines TNF-α and IL-1β (Hirani et al., 2001). Once ED1+ monocytes
are activated to the ED1+ macrophage phenotype, they contribute to the inflammatory
response by releasing pro-inflammatory cytokines such as PGE2 and IL-1β recruiting
more neutrophils heightening the inflammatory response (Scott et al., 2004). The
activated M1 macrophages produce nitrogen and oxygen intermediaries (nitric oxide
(NO) and super-oxide) which are highly toxic and have the capacity to cause damage to
neighbouring tissues (Nathan and Ding, 2010). They are believed to be involved in
various chronic inflammatory diseases (Sindrilaru et al., 2011) making it very important
to control their responses in order to prevent further collateral tissue damage (Murray
and Wynn, 2011).
As observed by the studies above, the ED2+ (M2 (a, b, c), AAM) macrophages appear
during the latter stages of inflammation (McLennan, 1996, Marsolais et al., 2001).
Similar to ED1+ macrophages, ED2+ macrophages originate in the bone marrow from
hematopoietic stem cells and are expressed in circulation as anti-inflammatory
monocytes becoming resident tissue macrophages (Yang et al., 2014), with these
resident macrophages representing 10 – 15% of the total adult mammal cell population
(Italiani and Boraschi, 2014). Unlike the preceding ED1+ macrophage, which are drawn
to the necrotic tissue to phagocytose the cellular debris and apoptotic neutrophils, the
primary role for ED2+ is that of tissue repair through cell signalling and cytokine
production (Philippou et al., 2012, Fernando et al., 2014). Interestingly, unlike the ED1+
macrophages that are derived from the circulating monocytes and are more involved in
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severe inflammatory injuries, resident macrophages are more involved in their
repopulation after a mild injury (Italiani and Boraschi, 2014). The resident macrophages
mediate tissue repair by releasing a number of growth-promoting factors and cytokines,
such as fibroblast growth factor (FGF), insulin-like growth factor (IGF), and TGF-β1
(Robertson et al., 1993, Philippou et al., 2012, Wahl et al., 1987), as well as IL-10, an
anti-inflammatory cytokine attenuating the ED1+ macrophage (Tidball and Villalta,
2010). The ED2+ macrophages produce matrix metalloproteinases and tissue inhibitors
of metalloproteinases controlling the turnover of ECM (Wynn, 2008), as well as
removing and digesting dead cells and debris that would otherwise promote the
responses of the ED1+ macrophage (Atabai et al., 2009, Baron and Wynn, 2011).
Interestingly, secretion of TGF-β1 by ED2+ signifies a shift from the ED1+ phenotype
(inflammatory macrophage) toward the anti-inflammatory ED2+ (non-inflammatory
macrophage) in response to the phagocytosis of apoptotic neutrophils and necrotic
muscle cells (Tidball and Villalta, 2010, Fadok et al., 1989, Arnold et al., 2007, Ashcroft,
1999). This shift serves to resolve muscle inflammation. Apoptosis (programmed cell
death) is an energy efficient means of removing the cellular debris since the cells that
phagocytose the debris (i.e., neutrophils and ED1+ macrophages) do not have to
migrate and do not cause further inflammation (Brown et al., 1992). More importantly it
is a necessary step in replacing cell populations in order to begin the next phase of
healing (Greenhalgh, 1998). However, if the natural apoptotic events are tampered with,
an exacerbation rather than a resolution of inflammation occurs, since the balance
between the cellular numbers is lost leading to non-normal tissue repair (Greenhalgh,
1998). Further, the cytokines FGF, IGF-1 and TGF-β1 are important for recruiting and
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activating fibroblasts which begin the repair process by secreting matrix molecules such
as collagen (Butterfield et al., 2006). In both animals and humans, a transient increase
in TGF-β1 and fibrosis is observed in acute limited injury (Border and Noble, 1994).
However, overexpression of TGF-β1 and the deregulation of its function is responsible
for the deposition of ECM and fibrotic tissue (Philippou et al., 2012, Border and Noble,
1994). It has been suggested that the stimulus of repeated injuries increases the
production of TGF-β1, sustaining its presence and levels leading to the further
deposition of ECM and the increased expression of tissue fibrosis (Chikenji et al., 2014,
Zimkowska et al., 2009).
Therefore, infiltration of the leukocytes to the site of injury and inflammation, sequencing
of the neutrophils and the macrophages (pro-inflammatory preceding anti-inflammatory),
their coexistence and communication in response to the early removal of cellular debris,
and the apoptosis of the inflammatory cells, and the release of growth factors and
cytokines, are essential for the resolution of tissue damage. Any delay in the
sequencing and proper function of these processes is responsible for slowing down the
recovery from tissue damage but as well for the disproportionate amount of tissue
destruction and increased fibrotic tissue (i.e. scar tissue), possibly affecting the function
of muscle. In addition, if the clearance of apoptotic cells is defective and there is a
continued accumulation and persistence of leukocytes (i.e., neutrophils), this may be
responsible for the progression from acute to chronic inflammation (Lawrence et al.,
2002). Since we are more concerned with the acute inflammatory response with regard
to stretching intensity, it is beyond the scope of this thesis to refer to chronic
inflammation and the subsequent mechanisms related to its expression.
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Concluding Remarks
In the sections above it was mentioned that neutrophils and macrophages were
responsible for the release of the pro-inflammatory cytokines IL-1β, TNF-α, and that
their production was responsible for the expression of IL-6 (Frost and Lang, 2005). In
turn, we mentioned that these three cytokines formed a complex network in which the
expression of each influences and is influenced by the other in an attempt to regulate
the inflammatory response (Akira et al., 1990). It was also pointed out that IL-6 is the
mediator of the acute inflammatory response (APR), resulting in the release of an acute
APPs, in particular CRP. According to Tilg et al. CRP plays a prominent role as an antiinflammatory mediator inducing the expression of IL-1ra, as well as sTNF-receptors,
potent anti-inflammatory cytokines (Figure 2.15) (Tilg et al., 1997, Tilg et al., 1994). The
importance of IL-1ra has also been discussed in the inflammation and non-damaging
exercise section.
FIGURE 2.15 – Expression of Anti-inflammatory Cytokines
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In conjunction with the previous sections, in this section specific reference was made to
the inflammatory cells, the neutrophils and macrophages, and the various macrophage
subtypes. It should be emphasized that the microenvironment of a given tissue is
responsible for the transformation of the macrophage monocyte into the macrophage
phagocytic phenotype (Duque and Descoteaux, 2014). As discussed above, many of
the cytokines that are capable of biasing the phenotype of the monocyte are provided
by the endothelium as well as the surrounding lymphocytes. These subtypes have been
defined as classically activated macrophages (CAMs, M1, or ED1 + ) or alternatively
activated macrophages (AAMs, M2, or ED2+) (Gordon and Taylor, 2005, Martinez et al.,
2008, Martinez et al., 2009). CAMs have been observed to produce pro-inflammatory
cytokines, IL-1β, TNF-α, IL-6, and IL-12, and are associated in mediating the
destruction of pathogens and the removal of cellular debris, while AAMs produce antiinflammatory cytokines IL-10, TGF-β1, and IL-6 and are associated with repair
(Fernando et al., 2014, Flynn et al., 2011). According to Fernando et al. IL-6 possesses
a Janus like nature with its ability to determine the phenotype of the macrophage based
on the micro-environment that surrounds it (Fernando et al., 2014), becoming either a
CAM or an AAM (McLennan, 1996). In turn, the absence of IL-6 prevents recovery
(McFarland-Mancini et al., 2010) since it is necessary for the resolution of inflammation
as observed in mice deficient in IL-6 (IL-6-/-) (Kopf et al., 1994). Although the IL-6-/- mice
did not express any developmental abnormalities they were unable to generate an
acute-phase response (Kopf et al., 1994). The induction of IL-6 due to injury or infection
is believed to be a very important in-vivo distress signal coordinating the activities of the
hepatocytes, macrophages and lymphocytes (Kopf et al., 1994), and therefore, the
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relationship between IL-6 and CRP represents the body’s mechanism for resolving the
acute inflammatory response.
2.9.2.2 – MECHANOTRANSDUCTION
The conditions under which muscle is mechanically loaded, whether in response to
eccentric and concentric exercise or passive stretching has been associated with the
expression of cytokines (Steensberg et al., 2002, Steensberg et al., 2000, Pedersen
and Fischer, 2007, Pedersen et al., 2001, Ostrowski et al., 1998, Ullum et al., 1994,
Helge et al., 2003), and with the elevation of neutrophils (Frenette et al., 2002, Koh et
al., 2003, McLoughlin et al., 2003, Pizza et al., 2002). In addition, the muscles response
to load and the ensuing inflammatory response may be influenced by the parameters of
intensity, duration, and frequency. These parameters have been associated with
training (Mujika et al., 1995) as well as stretching (Marschall, 1999). Of these
parameters, studies have indicated the importance of exercise intensity in determining
the response of cytokines to exercise. Ostrowski et al. noted a relationship between
exercise intensity and plasma IL-6 concentration, suggesting that the intensity of
marathon running was important in determining the increase in IL-6 (Ostrowski et al.,
2000). Peake et al. observed that with exercise lasting for approximately 1h, highintensity had a greater effect on the release of anti-inflammatory cytokines, IL-1ra and
IL-10, vs. moderate-intensity and downhill running, as well as compared to the
inflammatory mediators related to exercise-induced muscle damage (Peake et al.,
2005b). The study by Izquierdo et al. indicated that intensity with regard to resistance
training had an impact on cytokine levels after a relative or absolute load test post
seven weeks of resistance training, with high intensity being more conducive to
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increases of IL-6 and IL-10 (Izquierdo et al., 2009). Although an increase in IL-1β was
observed, this was related to a subsequent increase in IL-1ra potentially blunting its
activity after seven weeks of resistance training (Izquierdo et al., 2009). A study
investigating the effect of intense running with regard to the sequencing of cytokines, as
well as inflammatory and immunological responses, observed a significant increase in
IL-4, IL-6 and IL-10, with a simultaneous increase in leukocytes, neutrophils, and
monocytes, suggesting that inflammatory cells are a potential source of antiinflammatory cytokines as well (Ostapiuk-karolczuk et al., 2012). Therefore, although
we are not negating the influence of the other two parameters of stretching (duration
and frequency), we are hypothesizing, based on literature findings, that intensity of
exercise (either this is related to exercise per se or even potentially during stretching)
might play a far greater role in the inflammatory response.
Many physiological functions depend on the ability of cells and tissues to sense and
react to mechanical stimuli, whether these stimuli are internal or external, with their
response being essential for proper development and function (Hamill and Martinac,
2001, Kung, 2005). For instance, cells associated with baroreceptors, proprioceptors,
spindle receptors, and Golgi tendon organs, sense blood pressure, positions of limbs,
as well as muscle stretch and tension, respectively (Kung, 2005). Further, as mentioned
above, Wolff’s law suggests that bone remodelling occurs in response to loading.
According to Goldspink, skeletal muscle is and behaves similar to bone, possessing the
ability to adapt its structure, function, and metabolism in response to a mechanical
stimulus (i.e., stretching and overload ) and that to a large extent the mechanical signal
is responsible for regulating the expression of individual myosin genes (biochemical
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response)
(Goldspink,
1999).
Goldspink
is
alluding
to
the
concept
of
mechanotransduction, a process defining the response and relationship of the cells and
tissues to their environment, translating any physical input or mechanical perturbation
from the surrounding, into a biochemical or biological signal (Goldspink, 1999, Hamill
and Martinac, 2001, Kung, 2005, Previtera, 2004). Mechanotransduction has been
suggested as being a key regulator of many physiological process such as the
regulation of stem cell differentiation (Engler et al., 2006), fibroblast migration (Lo et al.,
2000), as well as being linked to the initiation of inflammation triggering cytokine
production (Previtera, 2004, Makino et al., 2007). Since stretching is defined as an
external and/or internal force (Weerapong et al., 2004) with the magnitude of the force
applied, as the intensity of the stretch (Jacobs and Sciacia, 2011), we believe that
potentially, stretching intensity may elicit a mechanotransduction response responsible
for stimulating an inflammatory response or not, aiding in the recovery of muscle from
muscle damage.
The concept of mechanotransduction looks at the behaviour of collective interactions
within complex networks adopting a “top-down” rather than a “bottom-up” approach
(Ingber, 2008a). In otherwords, rather than reverse engineer from the cellular level with
the relevant interactions, it tries to understand mechanical behaviour by referring
collectively to a higher order architecture and how it is affected by physical forces
(Huang et al., 2006, Ingber, 2008a). The main tenant of mechanotransduction is the
concept of tensegrity (Ingber, 2008a). It is concerned with how stability is created by
network structures through continuous tension (tensional tensegrity) and compression
with the system being in a state of pre-stress, essential for maintaining mechanical
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stability (Ingber, 2008a). Stability is achieved within the structure when the
compression-bearing rigid structures stretch or tense the flexible, tension bearing
members, which in turn impart a compression on the rigid structures, creating
counteracting forces achieving stability by equilibrating throughout the structure (Ingber,
1998) (Figure 2.16). According to Ingber, our bodies are constructed of a hierarchy of
systems within systems, each with its own tensional integrity, such that the tensegrity at
one level or size scale may itself be a tensegrity composed of smaller
tension/compression elements at another level or a reduced size scale (Ingber, 2008a)
(Figures 2.17)
FIGURE 2.16 – Example of tensegrity
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FIGURE 2.17 – Hierarchy of Systems (macro to micro)
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Chapter two – Literature Review
These prestressed hierarchies exhibit a mechanical responsiveness to a mechanical
stress or load by increasing their stiffness; this mechanical responsiveness is
influenced by intensity (McMahon, 1984). The relationship between the different
layers of the musculoskeletal system may serve as an example of tensegrity. At the
macroscopic layer, the stable vertical form of the body relative to the force of gravity
is maintained between the compression bearing bones of the skeleton and the
tensile pull of the muscles, tendons and ligaments (Ingber, 1998). The pre-stress
arises from the balance between the contractile forces generated by the cytoskeleton
of the muscle cells countered by the bone matrix’s ability to resist (Ingber, 2006).
Below this level, mechanical force is distributed through the myofascial and ECM
connections between adjacent muscle bundles, blood vessels and nerve tracts
(Ingber, 2006). Further down, at the cellular level, balance between compression and
tension of the individual muscle bundles and blood vessels is achieved between
tractional forces at the parenchymal cells resisting the forces exerted by the stiffened
ECMs surrounding the connective tissue cells, ensuring stability (Ingber, 2006). This
rearranging at many size scales of the musculoskeletal system tries to protect it
against injury since the molecular components that comprise the tensed ECMs and
interconnected cytoskeletal elements within adherent cells, adjust accordingly to the
mechanical load imparted on the system as a whole (Ingber, 2006, Ralphs et al.,
202, Komulainen et al., 1998). The importance of this architectural hierarchy, in
interpreting the biochemical response to a mechanical perturbation, has also been
expressed in terms of the importance of muscle and its ability to adapt to its
immediate environment (Gans, 1991). In particular, muscle’s effectiveness
(efficiency) in terms of energy consumption and the generation of force, which is
facilitated by the placement of sarcomeres in fibers, fibers into muscle, and the role
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muscle plays in relation of the organism to its environment (Gans and Abbot, 1991).
As mentioned in section 2.4.1 (Muscle) above, the relationship of the fibers and their
attachment to one another, and to the tendons and aponeuroses, and skeletal
elements (bones, joints, etc) will determine the displacement of force and its related
responses in terms of tissue damage and the response to stretching. The ensuing
responses to the magnitude and rate of force imparted on the tissue will be a
determinant as to whether the tissue will recover with the abatement of inflammation,
the inflammatory response with the various stages as discussed above.
Since the process of mechanotransduction is concerned with the conversion of a
mechanical perturbation into a biological signal and stretching is a physical activity
related to force development (Weerapong et al., 2004), the intensity of the stretch
(i.e., magnitude of force), may be linked to either the recovery from muscle damage
or not. In section 2.9.2 (Damaging Exercise and the Inflammatory Response),
muscle damage caused by exercise results in the loss of the sarcomeric organization
of the myofibrils (mechanical response) (Tidball, 1995). This is subsequently
followed by the autolysis of the damaged components as a result from the loss of
and upregulation of Ca2+. Severity of stretching, defined as muscle fibers stretched
by 50% of optimum force generating length (L o ), was observed to cause a reduction
in the maximum Ca2+-activated force (contraction), with 25% (L o ) in adult mice
skeletal muscle not associated with a force deficit (Balnave et al., 1997). Sarcomere
inhomogeneity was observed with severe stretching (Balnave et al., 1997). In
addition, an elevation of muscle Ca2+ via an influx of Ca2+ from the extracellular
space was observed during static stretching of rat soleus muscle, suggesting a
disruption of the homeostasis of Ca2+ (Armstrong et al., 1993). This loss of and
upregulation of Ca2+ is responsible for the activation of calpain (Belcastro et al.,
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1998, Armstrong, 1990). As discussed in Section 2.9.2.1.2, calpain is responsible for
cleaving a wide variety of myofibrillar and cytoskeletal proteins found in muscle, such
as α-actinin (Takahasi, 1990), vinculin (Evans et al., 1984), and talin (Fox et al.,
1985), with these cleaved proteins acting as chemoattractants for neutrophils
(Kunimatsu et al., 1989). According to a study, passive stretching was associated
with elevated levels of neutrophils in three to four month adult male mice (Pizza et
al., 2002).
Concerning stretching, the parameters of intensity, duration, and frequency are
potentially important in terms of influencing the recovery of the tissue from muscle
damage mitigating the inflammatory response, however intense or severe stretching
may be damaging. The adjustments of the musculoskeletal system related to
stretching are dependent on these parameters, in a similar manner to exercise. Their
manipulation may affect the elastic and plastic responses of the MTU, observed to
influence the flexibility and the generation of muscle strength (Gajdosik, 2001). In
this thesis, we are interested in investigating stretching intensity (but not ROM) and
its
influence
on
the
acute
inflammatory
response.
The
concept
of
mechanotransduction may be a potential mechanism by which stretching may
ameliorate the acute inflammatory response. Muscle stretching generating a
mechanotransduction response is believed to be responsible for the expression of
specific genes, promoting sarcomeregenesis and the remodelling of the ECM in
shortened and atrophied muscles (Martins et al., 2013). As well, fibroblasts of
ligaments quickly modified their morphology, cellular orientation, and shape in
response to cyclic stretching, before upregulating genes encoding for cellular and
extracellular components (β1 integrins, β-actin, and type I and III collagen) involved
mainly in mechanotransduction (Kaneko et al., 2009). Muscle is considered to be a
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highly plastic tissue adaptable to various situations such as physical activity, injury
and regeneration, as well as stretching (Salvini et al., 2012). Stretching is defined as
movement applied by an external and/or internal force in order to increase muscle
flexibility and/or joint ROM (Weerapong et al., 2004). It uses an external/internal
force (that can be of various different intensities) to stress connective and muscle
tissue mechanically (Martins et al., 2013), with intensity being defined as the
magnitude of force or torque applied to the joint during a stretching exercise (Jacobs
and Sciacia, 2011).
2.9.2.3 – STRETCHING AND INFLAMMATORY CELLS
A study specifically looking at the expression of neutrophil concentrations relative to
lengthening, isometric contractions, or passive stretching observed a significant rise
in neutrophil concentrations to either passive stretching or isometric contractions
without any overt histological or functional signs of injury (Pizza et al., 2002).
Seventy-one adult male Harlan Sprague Dawley mice were divided into groups
performing lengthening or isometric contractions, or passive stretching. With the
animals under anesthesia, measurement of the force generated by the extensor
digitorum longus (EDL) muscle and control of its length was accomplished by
exposing and tying its distal tendon to the lever arm of a servomotor, with stimulation
being done with use of needle electrodes in the peroneal nerve. During lengthening
and isometric contractions, the EDL was stimulated at 150Hz, with the muscle being
lengthened 20% relative to its optimal fiber length during lengthening contractions, or
held at its optimal muscle length, during isometric contraction. Passive stretching
was similar to the lengthening contraction protocol without stimulation. Each exercise
lasted 5min consisting of 75 repetitions performed at 0.25Hz. Controls were allowed
to roam freely in their cage or underwent similar surgical procedures without
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exercise. All mice were sacrificed at either 6h or 3d after initial in-situ procedure.
With 10μm cross sections excised from the midbelly of EDL, immunohistochemistry
for both neutrophils and macrophages was conducted. The inflammatory cells were
counted manually, and expressed as a number per cubic millimeter, with the number
of fibers invaded by neutrophils or macrophages counted and expressed as a
percentage of the total number of fibers. At 3d, passive stretching and isometric
contractions accounted for a 5.5 and 3.7-fold increase in neutrophils, respectively
compared to controls. With regard to lengthening contractions, there was a 51.2- and
7.9-fold increase in macrophages and neutrophils respectively. The rise in
neutrophils suggests that this increase in concentration can occur whether the
muscle activity results in an injury or not. Further, the authors postulated that a minor
injury, one defined without any overt impairments in function or histological
disruptions, may be a stimulus for one or more chemoattractants for neutrophils
(Pizza et al., 2002). This minor injury needs to be further investigated for this may be
related to the release of Ca2+ with the subsequent expression of calpain. In addition,
it was observed that training with passive stretching induced protection of adult mice
muscle following lengthening contractions of the EDL, by reducing the force deficit,
the number of overtly injured fibers, as well as being responsible for a reduction in
the accumulation of inflammatory cells (Koh and Brooks, 2001, Koh et al., 2003).
A study by Smith et al. was the first study interested in documenting whether
stretching (i.e., static or ballistic stretching) may be responsible for causing DOMS
(Smith et al., 1993). Most studies concerned with stretching and DOMS are
interested in investigating whether stretching (i.e., static, passive, active, ballistic,
dynamic and PNF) can influence DOMS (Lund et al., 1998, Wessel and Wan, 1994)
rather than cause it. This point of view is reflected further in a Cochrane
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Collaboration Review with regard to stretching research (Herbert et al., 2011). This
review was interested as to whether stretching before, after, or before and after
exercise is beneficial in preventing or treating DOMS. Based on the eligible
randomized clinical trials, it was concluded that muscle stretching regardless of
whether it is performed before or after exercise, does not produce any clinically
important reductions in DOMS (Herbert et al., 2011). However, stretching intensity
was not taken into account.
In the Smith et al. study, in order to determine if stretching (i.e., static or ballistic) was
responsible for muscle damage, CK was measured as the primary outcome relative
to either static or ballistic stretching of a similar intensity and duration (Smith et al.,
1993). Twenty active college males were randomly assigned to either a static or
ballistic stretching group (Smith et al., 1993). Participants were asked not to partake
in regular stretching activities and were required to remain sedentary during the
entire study. After marching on the spot for 5min as a warm-up, both groups
performed three identical sets of 17 stretching exercise, with the static group
remaining stationary during each 60s stretch, while the ballistic group performed
bouncing movements in time to a metronome (60 bounces/min). Each stretching
session lasted 90min being synchronized to a pre-recorded video in order to
standardize procedures. Approximately two stretches per muscle group were
performed with the chosen stretches being both safe and easy for beginners, with no
mention as to the intensity of the stretching. Blood samples (3ml) to assess CK were
drawn from the antecubital vein pre- as well as at 24, 48, 72, 96, and 120h post
exercise. Rate perceived exertion scores were recorded following each stretch using
the Borg 6 – 20 scale. Since each stretch was repeated three times, the mean of the
three scores for each of the 17 stretches was computed. Similar to the times for
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blood collection, a 10 point scale was used to assess daily soreness (1 = no
soreness, 10 = unbearable soreness) with participants rating DOMS in nine separate
muscular sites. The major finding of this study was that although both stretching
exercises resulted in a significant increase in DOMS, static stretching was
responsible for causing more DOMS than ballistic. Interestingly, the time course and
extent of DOMS observed, was similar to that reported with regard to eccentric
exercise mainly that discomfort develops during the first 24 – 48h peaking between
24 and 72h, and subsiding within 5 to 7d (Ebbeling and Clarkson, 1989). In addition,
the significant rise in CK, albeit it small, according to Smith et al. was consistent with
a previous report observing a rise in CK levels at 24h relative to an eccentric
exercise (Smith et al., 1993).
With passive stretching associated with morphological changes to muscle fiber (i.e.,
disruption of sarcomere) (Gomes et al., 2007), the disruption of the homeostasis of
Ca2+ (Armstrong et al., 1993), an increase in neutrophils (Koh et al., 2003, Pizza et
al., 2002), and static stretching suggested as a primary cause of DOMS (Smith et al.,
1993), we were interested in investigating whether stretching intensity may be
responsible for the magnitude of the acute inflammatory response. Stretching has
been associated with force (intensity), with this force being potentially responsible for
stressing connective and muscle tissue (Martins et al., 2013). The stimulation and
subsequent response of all the interconnected layers of this pathway, from the
macro level (muscles and tendons) to the micro level (sarcomeres) may be
influenced and defined by the magnitude of force generated relative to stretching
intensity. In otherwords, tissues and cells may respond differently to a low intense
gentle stretch associated with no pain vs. a high intense maximum ROM stretch
associated with pain and discomfort. To investigate our hypothesis, three studies
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were designed. The first study investigated whether acute IS (i.e., discomfort with
some pain), which is used in many sports, and in particular in aesthetic (i.e.,
gymnastics) and martial arts (i.e., taekwondo, judo, etc) may be responsible for an
acute inflammatory response. The blood biomarkers measured were the APP, CRP,
specifically a more sensitive assay hsCRP, and the pro-inflammatory cytokines IL1β, TNF-α, the cytokine IL-6. Study two, looked at comparing various stretching
intensities: low, medium, and high to determine if a relationship exists between these
different intensities and the expression of the acute inflammatory response. The
blood biomarker measured was hsCRP. Finally, based on the results of the two
previous studies, study three was designed to determine the applicability of
stretching intensity with regard to recovery of the musculoskeletal tissue from muscle
damage and DOMS. The blood biomarkers hsCRP and CK were assessed, with CK
to evaluate muscle damage.
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3
STUDY ONE
ACUTE
INFLAMMATORY
RESPONSE TO
STRETCHING
Chapter Three – Acute Inflammatory
Response to Stretching
Stretching is a complex time-dependent activity relying on intensity, duration and
frequency (Marschall, 1999). Of these three factors, intensity, defined as the magnitude
and rate of force applied to a joint during stretching (McClure et al., 1994, Jacobs and
Sciacia, 2011), may directly influence ROM levels (Marschall, 1999). According to
available data, intensity may play a primary role in determining stretching outcomes; if
the applied force is too high it may injure the tissue and trigger an inflammatory
response (Jacobs and Sciacia, 2011). In this article, a contradiction is presented
between the benefits attributed to either low-intensity or high-intensity stretching, by
referencing two different studies (Light et al., 1984, Stephenson et al., 2010). The study
by Light et al. revealed that the use of a low load prolonged stretch was superior to a
high-load brief stretch with regard to an increase in passive ROM in treating knee
contractions in elderly patients. In contrast, Stephenson et al. demonstrated that a highintensity stretch vs. a low-intensity stretch with use of a mechanical therapy devise was
superior with regard to reducing the risk of knee-attributable re-hospitalization.
However, it should be pointed out that the Stephenson et al. study was an observational
cohort study compared to the Light et al. study that was a randomized study with the
participants acting as their own control. These disparate methodological designs
contribute differently to our current understanding on the mechanisms around
inflammation and its associations/effects with stretching. This discrepancy illustrates the
need to design and implement better quality research studies in an attempt to determine
if the intensity of the applied force, whether low or high may injure the tissue potentially
triggering an inflammatory response.
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A previous study comparing different stretching types, concluded that static stretching
produced higher levels of DOMS than ballistic stretching (Smith et al., 1993). It revealed
that stretching was associated with the release of CK, a marker of muscle damage
(Smith et al., 1993). In turn, muscle damage has been associated with the release of
pro-inflammatory cytokines, namely IL-1, IL-6, and TNF-a, which have overlapping
functions in the immune response (Cunniffe et al., 2010, Cunniffe et al., 2011, Lund et
al., 2011, Chiu et al., 2013, Chatzinikolaou et al., 2010). Of the pro-inflammatory
cytokines released, IL-6 is directly responsible for the release of CRP, a principal
downstream mediator of the APR (Pradhan et al., 2001, Gabay, 2006, Akira et al., 1990,
Ramadori et al., 1988) stimulated in response to trauma, bacterial infection as well as
collagen tissue disorders (Kilicarslan et al., 2013). However, it is not entirely clear
whether IS is responsible for an inflammatory response.
The aim of this present study is to investigate whether acute IS causes an inflammatory
response. It is assumed that changes in the concentrations of the APP, CRP, as well as
the pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α should be observed between the
IS and Control interventions, and that an increase in muscle soreness would occur.
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3.2 – METHODS
3.2.1 – Participants
Twelve recreationally active male athletes (age: 29 ± 4.33yrs, mass: 79.3 ± 8.78kg,
height: 1.76 ± 0.06m) were recruited for the study. A recreational athlete is referred to
as an individual who does not participate in an organized team or individual sport
requiring systematic training and regular competitions against others. Each participant
read and signed an informed consent form and answered a health and safety
questionnaire, with the rights of each participant being protected. The University of
Wolverhampton Ethics Committee granted ethical approval. Participants were
requested and informed to abstain from any form of exercise post stretching intervention
as well as during the pre 24h blood collections. In addition, they were instructed to get
adequate sleep as well as to avoid alcohol prior to the assessments. We have also
provided a Physical Activity Readiness Questionnaire (PARQ, Appendix P. 282) that
screened our participant sample for confounding factors that may influence the
outcomes (e.g. smoking, injury, chronic disease).
3.2.2 – Power Calculations
C-reactive protein was selected as the primary end-point for its importance as an
inflammatory biomarker as it has been previously assessed pre- and post-exercise in
stretching (Frey et al., 1994). Assuming a detectable difference of 2mg/L with 2mg/L
standard deviation, an 80% power with an alpha level of 5%, a sample of 11 participants
was required; to allow for potential drop-outs we recruited 12 participants (nQuery
Advisor v 6.0, Statistical Solutions, MA, USA).
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3.2.3 – Procedures
During the first session, participants were familiarized with their respective activity
(stretch intervention and/or Control) and measurements for height and body mass were
taken. Each participant was randomized into an IS or Control group using a computerrandomized program (www.sealedenvelope.com) (Figure 3.1). They were all tested in a
controlled setting at the University of Wolverhampton’s physiology laboratory.
Participants acted as their own controls by taking part in both the IS and Control
conditions, with testing set a week apart.
In this study, measurement of hsCRP was utilized as a method for quantifying CRP in
serum. Studies conducted with apparently healthy individuals require hsCRP methods
(Roberts et al., 2001), allowing for the detection of CRP levels an order of magnitude
lower than traditional assays, enabling measurement of low-level elevations in CRP
where there was local, low grade inflammatory component (Pearle et al., 2007). Low
grade inflammatory component refers to a term describing conditions (i.e., type 2
diabetes, and atherosclerosis) typically exhibiting a two- to threefold increase in
systemic concentrations of CRP, IL-1, IL-6, and TNF-α (Petersen and Pedersen, 2005).
With the clearance of CRP from the plasma having a biological half-life of 19 – 20h,
falling by up to 50% per day afterwards, the week between conditions ensured a full
clear out of any potential effects, with this clearance being independent of physiological
and pathophysiological circumstance(s) (Vigushin et al., 1993, Marino and Giotta, 2008,
Pepys and Hirschfield, 2003).
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FIGURE 3.1 – Methodology Schematic of Intervention(s)
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3.2.4 – Intense Stretch Intervention
Participants were asked to rest in the supine position on a floor mat for 10min with
support provided under the knees. After the rest period, a trained therapist started
stretching the muscles of the lower extremity (hamstrings, gluteals, and quadriceps) for
each participant. The therapist was instructed to maintain a constant pressure on the
muscle groups throughout the duration of each stretch (60s). With the perception of
discomfort or pain varying amongst the participants, a numerical rating scale (NRS)
adopted from McCaffery et al. (McCaffery and Beebe, 1989), was utilized in order to
standardize the intensity of the stretch. This scale was anchored at zero (0) ‘no pain’ to
ten (10) ‘worst pain possible’. Since participants were required to experience pain and
discomfort during each stretch during the stretch intervention, they were verbally
encouraged to maintain a level equivalent to an eight out of 10 on this scale. All
participants were stretched for three sets. Each set consisted of stretching the right
hamstring and gluteal muscles in the supine position followed by both the right and left
quadriceps muscles in the prone, proceeding with the left gluteal and hamstring
muscles. The stretches were completed with no rest between sets. The total time for the
completion of the three sets was approximately 18min. Blood samples were collected
immediately post and at 24h post.
3.2.5 – Control Intervention
Similar to the stretching intervention, participants rested on a floor mat in the supine
position with support provided under the knees for 10min. This position was maintained
for a further 18min, mimicking the time allotted for the stretch intervention. Blood
samples were collected post rest ending the Control session, as well as 24h post.
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3.2.6 – Biomarkers
Approximately 5ml of the participant’s blood was drawn from the cephalic vein of the
arm of choice by a certified phlebotomist pre-, post and 24h following both the IS and
Control sessions. Serum hsCRP (eBioscience – BMS8288FF), IL-6 (eBioscience –
BMS8213FF), IL-1β (eBioscience – BMS8224FF) and TNF-α (eBioscience –
BMS8223FF) were measured by means of a FlowCytomix Simplex Kit (San Diego, CA,
USA) with a Beckman FC500 flow cytometer. The flow cytometer measures the light
scatter and fluorescence emission properties of individual cells in each sample
(Aghaeepour et al., 2013). It is used routinely in both research and clinical labs to study
normal and abnormal cell function and structure, as well as diagnose and monitor
human disease and responses to therapy, respectively (Aghaeepour et al., 2013). Flow
cytometry is well adapted to measure soluble analytes (a compound of interest in an
analytical procedure) such as variations in cytokines, and CRP in the sera and plasma
samples of both healthy and non-healthy individuals (O'Hara et al., 2011a, Prunet et al.,
2006). The advantage of using flow cytometry procedures is that it allows for the
detection of several functional characteristic of a cell simultaneously (O'Hara et al.,
2011a, Green et al., 2011).
The Beckman cytometer measures beads coated with antibodies specifically reacting
with each of the analytes to be detected. After determining, the number of microwell
strips required to test the desired number of samples plus appropriate number of wells
needed for running blanks and standards; 50µl assay buffer was added to the filter
plates to pre-wet the wells. Rows 1 and 2 of the microplate were designated for
standards with 25µl of standard mixture dilutions added from rows 1 to 7 of the plate,
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with 25µl of assay buffer added to the blank wells below row 7. A 25µl standard mixture
dilution was added to the first well of column 3 for cytometer setup. Afterwards 25µl of
each analyte sample for that particular microplate was added to the designated wells.
Biotin-conjugate mixture was added to all the designated samples including the
remaining blank wells. The wells were covered with adhesive film and an aluminium foil
to protect from light and the microplate was incubated at room temperature (18° to 25°)
for 2h on a microplate shaker at 500rpm. After incubation, the adhesive film was
removed and the wells were emptied using the vacuum filtration manifold whereupon
100µl assay buffer was added to the microwell strips and emptied again. A 100µl assay
buffer with 50µl of streptavidin-PE solution was added to all the wells including the blank
wells. The microplate was again covered with adhesive film, protected from light with an
aluminum foil, and incubated for a further 1h on a microplate shaker at 500rpm. Using a
vacuum filtration manifold the wells were emptied after the adhesive film was removed
with 100µl assay buffer added to each microwell strip and emptied with the vacuum
filtration manifold followed by the addition of 200µl assay buffer to each well. The
contents of each well were mixed with repeated aspiration and ejection with each 200µl
sample transferred to a separate acquisition tube and filled up to 500µl with an addition
of 300µl of assay buffer. The samples were then measured using a flow cytometer. The
coefficient of variation for each analyte were as follows: 6.4% for hsCRP, 6.2% for IL-6,
9.3% for IL-1β, and 6.3% for TNF-α.
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3.2.7 – Questionnaire
All participants were asked to provide information about their pain and or soreness
levels for all muscle groups stretched during the stretch intervention, immediately post
as well as at 24, 48 and 72h post intervention with use of the same 10-point numerical
rating scale used during the stretching intervention. The high validity and reliability of
these scales to rate a subjective sensation such as soreness have been previously
established (McCaffery and Beebe, 1989, Downie et al., 1978, Horsley et al., 2007,
Cunha et al., 2008, Trampas et al., 2010).
3.2.8 – Statistical Analysis
Pre analysis screening using the Kolmogorov-Smirnov test was employed to establish
distribution of all the variables. Accordingly, Ln transformation was used to overcome
skewness and kurtosis in the dependent variables of interest (hsCRP, IL-6, IL-1β, and
TNF-α) in relation to the independent variables (IS and Control). A repeated measures
analysis of variance (RMANOVA) evaluated the changes in the dependent variables
over time, intervention and time for all the dependent variables including soreness
levels, with the LSD post hoc test evaluating any differences based on the observed
main effect. With the level of significance set at p < 0.05 all statistical analyses were
performed via SPSS (version 20.0, SPSS Inc., USA). It should be noted, that although
twelve participants were recruited for study 1, there were missing data for hsCRP blood
biomarkers. With regard to the Control condition, one value was missing for pre-hsCRP,
two for post hsCRP, and one for 24h post hsCRP. Complete set of data points for both
pre- and post IS exists with one missing value recorded for 24h post. Complete data for
both conditions and all time-points (pre-, post, and 24h post) existed for nine
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participants. In addition, effect sizes (Cohen’s “d”) were calculated comparing inbetween (pre-, post, and 24h post) and within groups (pre- vs. post, pre- vs. 24h post,
and post vs. 24h post) for both the IS and Control conditions.
3.3 – RESULTS
All participants were involved in both interventions (IS & Control). There were no
withdrawals from the study. Figure 3.2 highlights the involvement of the participants in
the study and analysis.
FIGURE 3.2 – CONSORT Flow Diagram
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3.3.1 – RMANOVA
A RMANOVA revealed that Mauchly’s test for sphericity was violated for time, and time
x intervention for hsCRP (p = 0.002), X2 (2) = 12.48, p < 0.05. With the degrees of
freedom corrected using the Greenhouse-Geisser estimates of sphericity (ε = 0.658),
significant differences were detected for time (p = 0.005) and time x intervention (p =
0.006). No significance was observed for IL-6 (time, p = 0.274; time x intervention, p =
0.537), IL-1β (time, p = 0.277; time x intervention, p = 0.815), and TNFα (time, p =
0.462; time x intervention, p = 0.435), however it should be emphasized that there were
insufficient data of detectable values for these pro-inflammatory cytokines (Table 3.1).
With respect to soreness, no significant difference was observed between participants
in any of the muscle groups studied up to 72h post IS (Table 3.2, Figure 3.4).
The LSD post hoc test revealed a significant difference between pre and 24h post
measurements for hsCRP (p = 0.012). The mean and standard deviation Ln
transformed values for pre Control and IS (0.604 ± 0.155mg/L and 0.727 ± 0.378mg/L)
compared with 24h post (0.604 ± 0.155mg/L and 1.343 ± 0.599mg/L) support this
increase (Table 3.2, Figure 3.3).
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TABLE 3.1 – Mean (SD) of Raw and Ln Transformed Blood Biomarkers in Relation to Time and Intervention(s)
Blood Biomarkers
Time
hsCRP(mg/L)
IL-6(mg/L)
IL-1β(mg/L)
TNF-α(mg/L)
Control
IS
Control
IS
Control
IS
Control
IS
0.85
(0.27)
0.60
(0.16)
1.24
(1.14)
0.73
(0.38)
Immediately post
(Raw)
0.79
(0.35)
1.81
(1.87)
5.0 e-3
(0.012)
5.0 e-3
(0.012)
7.0 e-3
(0.02)
0.61
(2.01)
0.19
(0.61)
0.61
(2.01)
0.21
(0.63)
0.12
(0.35)
0.29
(0.66)
1.02
(2.83)
0.31
(0.74)
0.88
(2.16)
0.04
(0.11)
0.04
(0.09)
0.05
(0.13)
6.52
(21.53)
0.42
(1.29)
3.41
(11.17)
Immediately post
(Ln)
0.57
(0.22)
0.91
(0.45)
7.0 e-3
(0.02)
0.19
(0.61)
0.17
(0.37)
0.33
(0.68)
0.04
(0.11)
0.36
(1.09)
24h post (Raw)
0.85
(0.27)
3.55
(2.96)*
24h post (Ln)
0.60
(0.16)
1.34
(0.59)*
5.0 e-3
(0.01)
5.0 e-3
(0.01)
0.61
(2.02)
0.19
(0.61)
0.21
(0.63)
0.12
(0.35)
0.87
(2.31)
0.29
(0.69)
0.04
(0.11)
0.04
(0.09)
2.99
(9.77)
0.35
(1.05)
Pre (Raw)
Pre (Ln)
Key Ln = Natural Logarithm; IS = Intense Stretching; hsCRP = high sensitivity C-reactive protein; IL-6 = interleukin 6; IL-1β = interleukin 1 beta;
TNF = tumor necrosis factor alpha
* repeated measures ANOVA significantly different between conditions over time
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p=0.012
FIGURE 3.3 – Change of hsCRP over Time (h).
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TABLE 3.2 – Mean (SD) of Soreness Levels of Muscles Post IS
Muscle
Time
Hamstrings
Glutes
Quadriceps
0h
1.25 (0.62)
1.00 (0.60)
0.92 (0.52)
24h
4.83 (2.79)
4.92 (2.75)
2.58 (2.78)
48h
5.33 (2.06)
5.75 (2.93)
5.00 (2.52)
72h
1.58 (1.56)
1.92 (2.39)
1.08 (0.793)
FIGURE 3.4 – Perceived Soreness Levels over Time
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3.3.2 - Effect Sizes
3.3.2.1 - Effect Sizes between Conditions
See Table 3.3 for descriptive statistics and effects sizes for all between group and within
group comparisons. The effect size for the comparison between IS and Control at
baseline suggests a small elevated concentration of hsCRP in the IS intervention (“d” =
0.49, 95% CI, 0.13 to 0.86). The effect sizes for the comparison between IS and Control
immediately post-conditions suggests a small difference in hsCRP concentrations (“d” =
0.41, 95% CI, -0.26 to 1.07), with higher concentrations in the IS group. However, the
upper 95% CI for Cohen’s “d” crossed zero (0) (i.e. the line of no effect). Comparisons
of effect sizes for between groups at 24h post-condition suggests a large difference (“d”
= 1.32, 95% CI, 0.40 to 2.24), with higher concentrations again in the IS group.
3.3.2.2 – Effect Sizes within Conditions
The effect sizes for comparisons between pre- and post-conditions measures revealed
a small increase in hsCRP in both the IS and Control conditions (“d” = 0.39, 95% CI, 0.00 to 0.78 and d = 0.37, CI, -0.28 to 1.02, respectively). However, the 95% CI for
Cohen’s “d” contained 0 for both comparisons, which means that there may be no
effect. When comparing pre-condition values to post-24h, we found a large increase for
hsCRP in the IS condition (“d” = 1.06, 95% CI, 0.08 to 2.03), but no effect in the Control
condition (“d” = -0.00, 95% CI, -0.17 to 0.16), suggesting a higher concentration is
associated within the 24h post vs. pre-conditions for IS. The effect sizes for
comparisons of between immediately post-conditions and 24h post-conditions revealed
a moderate increasing effect on hsCRP in the IS condition (“d” = 0.75, 95% CI, -0.32 to
1.81), and a small lowering effect in the Control condition (“d” = -0.39, 95% CI, -0.78 to
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0.002). However, the 95% CI for Cohen’s “d” contained zero (0) for both comparisons,
which again may mean that no effect exists.
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TABLE 3.3 - Descriptive Statistics and Effects Sizes for all Between Conditions and Within Conditions Comparisons
Comparisons Between
Conditions
Pre IS – pre Control
Mean difference (SD)
Cohen’s “d”
95% CI (lower)
95% CI (upper)
0.42 (0.94)
0.49
0.13
0.86
Post IS – post Control
0.08 (1.44)
0.41
-0.26
1.07
24h post IS – 24h post Control
2.84 (2.74)
1.32
0.40
2.24
Post – pre IS
0.52 (0.79)
0.37
-0.28
1.02
24h post – pre IS
2.31 (2.40)
1.06
0.08
2.03
24h post – post IS
1.75 (2.13)
0.74
-0.32
1.81
post – pre Control
-0.05 (0.34)
0.39
-0.00
0.78
24h post – pre Control
-0.00 (0.00)
-0.00
-0.17
0.16
24h post – post Control
0.05 (0.34)
-0.39
-0.78
0.002
Comparisons within IS
Comparisons within Control
Key: Cohen’s “d” cut off points for effect size interpretation: small effect ≥ 0.20, medium effect ≥ 0.50, and large effect ≥ 0.80
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3.4 – DISCUSSION
The aim of the study was to assess whether IS results in acute inflammation by
specifically referring to selected inflammatory biomarkers (hsCRP and pro-inflammatory
cytokines, IL-1β, IL-6, and TNF-α). We found a significant increase in hsCRP at 24h
post IS intervention compared to Control. However, with regard to the pro-inflammatory
cytokines IL-1β, IL-6, and TNF-α, there were insufficient data of detectable values for IL6, IL-1 and TNF-a., thus not allowing us to conduct the relevant statistical analyses
intended. Based on the effect sizes, although there was slightly higher baseline hsCRP
concentrations in the IS condition, there appeared to be a large increase in hsCRP at
24h post in the IS compared to Control. The effect sizes comparing each measurement
time within each condition revealed an at least a small elevating effect on hsCRP (i.e.
lower 95% CI = 0.08) when comparing baseline values to 24h post, but no consistent
effects for any of the other within group comparisons (Table 3.3).
The reaction of muscle to the IS may have been responsible for causing inflammation,
since it has already been established that too much force in the form of stretching may
result in inflammation (Jacobs and Sciacia, 2011). In addition, although an increase in
soreness levels was observed for all the muscle groups, this was not statistically
significant. According to Gullick et al. (Gullick and Kimura, 1996), the degree of
discomfort causing soreness depends largely upon the intensity and duration of effort as
well as the type of activity. We postulate that since participants were stretched passively
while lying down, and were not involved in active stretching, this may account for the
observed results.
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To our knowledge, this is the first study to investigate the relationship between IS and
acute inflammation with reference to an APP (CRP, specifically hsCRP) and the proinflammatory cytokines, IL-1β, IL-6, and TNF-α. These aforementioned biomarkers are
capable of cross regulating one another. Although we did not observe changes in the
pro-inflammatory cytokines, mainly because we missed the opportunity to measure their
half-lives, numerous studies in the literature exist which have observed the relationship
between these three pro-inflammatory cytokines (Akira et al., 1990, McGee et al., 1995,
Akira et al., 1993). The release of IL-1β and TNF-α can induce the production of IL-6,
with IL-6 inversely regulating the TNF-α expression (Akira et al., 1990, Akira et al.,
1993). This synergistic relationship is in an attempt to regulate the immune response
and the inflammatory reaction (Akira et al., 1990, Streetz et al., 2001). The increase in
IL-6 has been related to an acute phase inflammatory response (Cunniffe et al., 2010,
Cunniffe et al., 2011, Chatzinikolaou et al., 2010, Kim et al., 2007, Streetz et al., 2001,
Ramadori et al., 1988), subsequently associated with a delayed increase in CRP as well
as to the growth and repair process following intense exercise (Cunniffe et al., 2010,
Cunniffe et al., 2011, Streetz et al., 2001).
In line with the aforementioned studies (Cunniffe et al., 2010, Cunniffe et al., 2011, Kim
et al., 2007, Chatzinikolaou et al., 2010), a delayed sharp increase in hsCRP was also
observed in the present study the day after the IS session. However, unlike these
studies, we did not observe an increase in IL-6 as expected since the collection of blood
missed the half-life for IL-6 (Limitations, Section 7.1.1 – Blood Collection, P. 217–218).
According to Pedersen (Pedersen, 2000) IL-6 is produced in larger amounts than any
other cytokine during exercise with a peak in concentration levels (half-life) ranging
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between one to two hours post prolonged activity.(i.e. marathon running). In contrast,
with an eccentric exercise model IL-6 levels did not peak until 1 - 1.5h post activity
(Rohde et al., 1997, Bruunsgaard et al., 1997). Therefore, we postulate that an increase
in IL-6 may have occurred explaining the sharp increase in hsCRP seen approximately
24h later, since the half-life for hsCRP is approximately 19 – 20h (Vigushin et al., 1993,
Marino and Giotta, 2008, Pepys and Hirschfield, 2003). Support for this view has been
demonstrated in studies observing a relationship between IL-6 and CRP, since the
primary mediator of the APR and CRP is IL-6 (Pradhan et al., 2001, Gabay, 2006, Akira
et al., 1990, Ramadori et al., 1988). This association has been investigated and
observed in animal studies. In mice deficient of IL-6- (IL-6-/-), proper recovery from
inflammation was prevented since this cytokine is necessary for the resolution of the
inflammatory response (McFarland-Mancini et al., 2010). Further, although mice did not
express any developmental abnormalities they were unable to generate an APR in the
absence of IL-6 (Kopf et al., 1994). The induction of IL-6 due to injury or infection is
believed to be a very important in-vivo distress signal coordinating the activities of the
hepatocytes, macrophages and lymphocytes and therefore, the relationship between IL6 and CRP represents the body’s mechanism for resolving the acute inflammatory
response (Kopf et al., 1994, McFarland-Mancini et al., 2010).
This increase in hsCRP may indirectly reflect muscle damage as well, because of its
relationship to IL-6, as mentioned above. A study by Bruunsgaard et al. (Bruunsgaard et
al., 1997), showed a significant correlation between IL-6 and CK (r = 0.722, p = 0.028)
two hours post strenuous activity, suggesting that this increase in serum IL-6 may
reflect a pronounced inflammation in muscle. However, our data suggest that this may
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not be accompanied by muscle soreness. With respect to TNF-α and IL-1β, both
increase post strenuous exercise with TNF-α exhibiting a small increase in
concentration (Ostrowski et al., 1999). Interleukin-1β concentrations also increase post
exercise however since they are produced locally they are rapidly cleared from the
circulation (Ostrowski et al., 1999). Considering blood was extracted at 24h post
intervention, this may account for the inability to observe any rise in these inflammatory
biomarkers, representing a potential limitation of the study (see Chapter 7 Section
7.1.1).
In the current study participants were not subjected to aggressive forms of activities
such as rugby (Cunniffe et al., 2010, Cunniffe et al., 2011), plyometric exercise
(Chatzinikolaou et al., 2010), or a long duration run (Mooren et al., 2006, Kim et al.,
2007), but to an intense passive stretching exercise of a relatively short duration
(18min). Nevertheless, similar to these studies we observed a significant increase in the
concentration levels of hsCRP post treatment, with the rise increasing 14-fold. It is
interesting to note that the study by Kim et al., showed a 23-fold increase in hsCRP in
the latter half of a 200km ultra-marathon race (Kim et al., 2007). Although we are not
claiming that IS is similar to intense physical activities of high duration or eccentric
exercise, this rise in the concentration of hsCRP following IS is very interesting.
Increases in the concentration of this APP has been shown to increase a 1000-fold with
acute inflammatory events such as infection, surgery, and trauma (Roberts et al., 2000).
The increase in hsCRP may potentially be in response to local inflammation occurring at
the muscles during IS possibly triggering the release of IL-6, which in turn may stimulate
the delayed systemic release of hsCRP as measured. In addition, according to Febbraio
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et al. (Febbraio and Pedersen, 2002), the appearance of IL-6 into circulation may also
depend on the mass of muscle recruited. This was observed in both a 160km triathlon
race (Taylor et al., 1987) and a 6d ultramarathon (Fallon, 2001), where the sharp
increase in IL-6 resulted in a massive increase in CRP, 50.8 and 37.5mg/L,
respectively. In relation to this study, although the value for hsCRP for 24h post was not
as high (1.34mg/L), this increase in hsCRP relative to pre- and immediately post values
(Table 3.1 RMANOVA & Table 3.3 Effect Sizes) may be a reflection of the IS being
applied to more than one muscle group (hamstrings, gluteal, and quadriceps), resulting
in acute inflammation. Therefore, in order to show the importance of the relationship
between IL-6 and hsCRP further studies are needed with blood samples taken at least
two hours post IS, as well as comparing stretches that may involve just one muscle
group. In turn, the measurement of CK will help to determine if muscle damage does
occur since this relationship has been established by Bruunsgaard et al.(Bruunsgaard et
al., 1997), concerning IL-6.
3.5 – SUMMARY
In summary and within the study’s limitations, the present data demonstrated that IS
may be associated with an increase in systemic hsCRP. This rise in hsCRP levels may
possibly be attributed to inflammation occurring at muscle groups subjected to IS.
Further research into the probable causes of inflammation with regard to IS will
elucidate if this form of activity is detrimental to the health of the muscle tissue and its
proper function.
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4
STUDY TWO
STRETCH INTENSITY VS.
INFLAMMATION: A DOSEDEPENDENT
ASSOCIATION?
Chapter 4 – Stretch intensity vs. Inflammation:
A Dose Dependent Association?
The training parameters of intensity, duration and frequency, feature prominently in
stretching and training programs (Marschall, 1999, Mujika et al., 1995), with appropriate
combinations of these parameters believed to produce an adaptive response (Mujika et
al., 1995, Seiler, 2010). The magnitude of force being applied to the joint during a
stretching exercise is defined as the intensity of the stretch (Jacobs and Sciacia, 2011).
Adopting the view that stretching is a mechanical force or load applied to the tissue, this
may be viewed as a physical stress. Over a given area of tissue, application of force
results in stress to the tissue (stress = force/area), where force may be applied in any
direction (tension, shear, compression) (Mueller and Maluf, 2002). Intensity is very
important for the application of too much force may injure the tissue resulting in
inflammation (Jacobs and Sciacia, 2011).
In response to a protocol of passive stretching using three to four month old adult male
mice, it was demonstrated that passive stretching induced an elevation of neutrophils
within the skeletal muscle (Pizza et al., 2002). Activated neutrophils have been shown to
secrete different pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α (Gresnigt et
al., 2012). IL-6 features prominently in the release of the APP, CRP, a principal
downstream mediator of the APR primarily derived via an IL-6 dependent hepatic
biosynthesis (Pradhan et al., 2001, Gabay, 2006, Kilicarslan et al., 2013). This response
occurs in response to trauma, bacterial infection as well as collagen tissue disorders
(Kilicarslan et al., 2013). Both IL-6 and CRP are markers of inflammation (Pradhan et
al., 2001). The release of IL-6 initiates an inflammatory response, with the acute phase
changes reflecting the presence and intensity of inflammation, with the subsequent
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expression of CRP levels being proportional to the inflammatory stimulus.
Given the importance of stretching on performance, the aim of the present study was to
investigate the effect of various stretching intensities (30%, 60%, and 90% of mROM)
with regard to eliciting an inflammatory response. We hypothesize that low (30%
mROM) and medium (60% mROM) intensity stretching does not cause inflammation via
measuring the expression of circulating hsCRP (primary outcome).
4.2 – METHODS
Eleven recreational male athletes (n = 11) were recruited for this study (age: 26 ±
6.20yrs, mass: 85.4 ± 8.55kg, height: 1.79 ± 0.57m). A recreational athlete is referred to
as an individual who does not participate in an organized team or individual sport
requiring systematic training and regular competitions against others. Each participant
read and signed an informed consent form and answered a PARQ (Appendix P. 282),
prior to participation in the study, with the rights of each participant being protected. The
University of Wolverhampton ethics committee granted ethical approval. Participants
were advised to maintain normal physical activity, refraining from the introduction of new
forms of activity that may cause DOMS for the period of the study.
4.2.2 – Power Calculations
C-reactive protein was selected as the primary end-point for its importance as an
inflammatory biomarker as it has been previously assessed pre- and post-exercise in
stretching (Frey et al., 1994). Assuming a detectable difference of 2mg/L with 2mg/L
standard deviation, an 80% power with an alpha level of 5%, a sample of 11 participants
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was required (nQuery Advisor v 6.0, Statistical Solutions, MA, USA).
As part of the familiarization process, during their first visit in the laboratory, the mROM
for the right hamstring muscle for each participant, using an isokinetic dynamometer (Kin
Com, East Ridge, Tennessee, USA), was measured. All measurements and the relevant
stretching interventions were conducted in the biomechanics laboratory of the University
of Wolverhampton. Lying in the supine position, the right knee was fitted with a knee
immobilizer brace (Őssur Exoform® knee immobilizer 2220 style, Grjothals, Iceland)
(Figure 4.1) which straightened and locked the knee preventing knee flexion. With the
pelvis, left leg and the upper body immobilized, the fulcrum of the lever arm of the Kin
Com was aligned with the greater trochanter of the right leg.
With use of the Kin Com dynamometer, the straight leg was raised, increasing hip
flexion until mROM was achieved, indicated by the sensation of maximal physical
pain/effort by the participant (Figure 4.2). This procedure was repeated three times with
the best value, often from the last attempt, recorded as their mROM (Mean =
109.50± 20.19). The ROM for hip flexion in males varies with mean values ranging from
121.3 (Boone and Azen, 1979) to 130.4 (Soucie et al., 2011) degrees. Following the
identification of the mROM (independent variable), the three stretch angle interventions
were calculated as 30%, 60%, and 90% of the individual’s mROM. All participants
completed the three stretch interventions within a three-week period, being randomized
into each percentage of mROM intervention using a computer randomization program
(www.graphpad.com).
In this study measurement of hsCRP was conducted as a method of quantifying CRP in
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serum, since hsCRP allows for the detection of CRP levels an order of magnitude lower
than traditional CRP assays, (Roberts et al., 2001, Pearle et al., 2007). This allowed for
the measurement of low-level elevations in CRP where there was a local, low grade
inflammatory component (Pearle et al., 2007). Low grade inflammatory component
refers to a term describing conditions (i.e., type 2 diabetes, and atherosclerosis) typically
exhibiting a two- to threefold increase in systemic concentrations of TNF-α, IL-1, IL-6
and CRP (Petersen and Pedersen, 2005).
FIGURE 4.1 – Knee Immobilizer
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FIGURE 4.2 – Experimental Set-Up
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During their second visit to the laboratory, participants were informed about the
percentage of ROM that they would be tested on that day (i.e., 30%, 60% or 90% of
their mROM), with the other percentages of mROM also being randomized by graphpad,
and performed during the following consecutive two weeks (Figure 4.3). Prior to each
intervention, a 5ml blood sample was withdrawn from the cephalic vein of the arm by a
certified phlebotomist. With the participant in the supine position, and their right knee
placed and strapped in the immobilizer, their hips, shoulders, and left lower limb were
also strapped into the Kin Com aiding in preventing any internal/external hip rotation of
the whole right lower limb. To prevent any rotation of the lower leg in relation to the
femur of the upper leg, the ankle attachment of the Kin Com was used. With the right
lower leg, resting on the ankle attachment just above the lateral and medial malleoli, and
the foot placed in the neutral position (i.e., no supination or pronation) the lower leg was
strapped into the ankle rest. During the intervention (both rest and stretching), as well as
for the duration of the study, the same designated assistant was assigned. The straight
right leg was then moved to the selected angle by the dynamometer and held in that
position for 60s. After a minute, the leg was lowered to the starting position to rest for
10s. The sequence was repeated another four times (total 5 x 60s with 4 x 10s rest in
between).
The use of 5 sets of 60s represents a methodology change from the previous study (3
sets of 60s) and therefore, does not represent a continuity in methodologies with regard
to the number of sets used per stretching intervention. However, there is evidence that 5
sets elicits similar responses to 3 sets, and that no statistical difference exists between
the two (Taylor et al., 1990). These authors have observed that for the first four
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stretches of the MTU to 10% beyond resting length and to a set tension demonstrated
the greatest changes in the MTU. They concluded that with repetitive stretching there
was little alteration of the MTU after four stretches implying that this is the optimum
number needed for the greatest elongation of the MTU during stretching (Taylor et al.,
1990). In an attempt to reduce the total assessment time from the participants point of
view, a rest interval of 10s was chosen as previously suggested (de Weijer et al., 2003).
In this study, participants performed 3 sets of 30s stretches on the right hamstring with a
10s rest interval between each stretch length and ROM, in order to examine the effects
of a static stretch and a warm-up exercise on hamstring length over 24h. However, we
realize that both the increase in the number of sets and a change in the rest interval
represent a potential limitation of the methodology for the current work with regard to
continuity and as such this has been noted in the limitations chapter (Section 7.1.2 –
Methodology, P. 218 – 220).
Post intervention blood was immediately taken, with a third sample taken at 24h post. All
stretch interventions were performed with the right leg, over three consecutive weeks.
Each stretch intervention was conducted on one day during the week and was followed
by a full one-week rest period. This rest period ensured a full clear out of any potential
residual effects. This was decided since the clearance of CRP from the plasma has a
biological half-life of approximately 19 – 20h, falling by up to 50% per day when the
acute stimulus is resolved (Marino and Giotta, 2008, Vigushin et al., 1993, Pepys and
Hirschfield, 2003), since a significant determinant of plasma CRP levels is the rate of
synthesis justifying its use in monitoring the activity of inflammation (Ablij and Meinders,
2002).
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The day prior to the intervention as well as the day during the assessments, participants
were requested to refrain from any form of exercise or activity that might cause any
physical or mental stress. In addition, they were instructed to get adequate sleep and
avoid alcohol consumption. We have also provided a PARQ (Appendix P. 282)
questionnaire that screened our participants for factors that may influence the outcomes
(e.g. smoking, injury, chronic disease). However, we could not control whether the
aforementioned instructions were actually followed, and therefore realize that this is a
potential limitation of the study (Limitations, Section 7.13 – Compliance, P. 220).
4.2.4 – Blood Biomarkers
Blood was collected in a vacutainer (BD Vacutainer, K2E EDTA). After the whole blood
was left to rest in the vacutainer for 10min, it was spun at 3000rpms in a centrifuge
(SciQuip Sigma 2-6E) for another 10min. Serum hsCRP (eBioscience – BMS8288FF),
was measured by means of a FlowCytomix Simplex Kit (San Diego, CA, USA) with a
Beckman FC500 flow cytometer. The flow cytometer measures the light scatter and
fluorescence emission properties of individual cells in each sample (Aghaeepour et al.,
2013). It is used routinely in both research and clinical labs to study normal and
abnormal cell function and structure, as well as diagnose and monitor human disease
and responses to therapy, respectively (Aghaeepour et al., 2013). Flow cytometry is well
adapted to measure soluble analytes (a compound of interest in an analytical procedure)
such as variations in cytokines, and CRP in the sera and plasma samples of both health
and non-healthy individuals (O'Hara et al., 2011a, Prunet et al., 2006). The advantage of
using flow cytometry procedures is that it allows for the detection of several functional
characteristic of a cell simultaneously (O'Hara et al., 2011a, Green et al., 2011).
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The cytometer measures beads coated with antibodies specifically reacting with each of
the analytes to be detected. After determining, the number of microwell strips required to
test the desired number of samples plus appropriate number of wells needed for running
blanks and standards 50µl assay buffer was added to the filter plates to pre-wet the
wells. Rows 1 and 2 of the microplate were designated for standards with 25µl of
standard mixture dilutions added from rows 1 to 7 of the plate, with 25µl of assay buffer
added to the blank wells below row 7. A 25µl standard mixture dilution was added to the
first well of column 3 for cytometer setup. Afterwards 25µl of each analyte sample for
that particular microplate was added to the designated wells. Biotin-conjugate mixture
was added to all the designated samples including the remaining blank wells. The wells
were covered with adhesive film and an aluminium foil to protect from light and the
microplate was incubated at room temperature (18° to 25°) for 2h on a microplate
shaker at 500rpm. After incubation, the adhesive film was removed and the wells were
emptied using the vacuum filtration manifold whereupon 100µl assay buffer was added
to the microwell strips and emptied again. A 100µl assay buffer with 50µl of streptavidinPE solution was added to all the wells including the blank wells. The microplate was
again covered with adhesive film, and protected from light with an aluminium foil and
incubated for a further 1h on a microplate shaker at 500rpm. Using a vacuum filtration
manifold the wells were emptied after the adhesive film was removed with 100µl assay
buffer added to each microwell strip and emptied with the vacuum filtration manifold
followed by the addition of 200µl assay buffer to each well. The contents of each well
were mixed with repeated aspiration and ejection and each 200µl sample was
transferred to a separate acquisition tube and filled up to 500µl, with an addition of 300µl
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of assay buffer. The samples were then measured using a flow cytometer. The
coefficient of variation for hsCRP was 6.4%.
With circulating CRP being only present in trace amounts in healthy individuals, and is
hardly detectable by standard clinical test which possess a detection limit of 3 – 8mg/L
(Ridker, 2001, Nanri et al., 2007), hsCRP assay was used. The lower detection limit for
hsCRP is approximately 0.04mg/L, with a study of healthy donors indicating a median of
hsCRP levels being 0.8mg/L, ranging between 0.07 – 29mg/L (Nanri et al., 2007).
Therefore, use of hsCRP enabled the detection of CRP levels an order of magnitude
lower than traditional assays allowing the ability to better detect a local, low grade
inflammatory component (Pearle et al., 2007).
Pre analysis screening using the Kolmogorov-Smirnov test was performed to investigate
the distribution of all variables. Accordingly, Ln transformation was used to overcome
skewness and kurtosis in the dependent variable of interest (hsCRP). A RMANOVA
evaluated the changes in hsCRP over time and time x % mROM, with the LSD post hoc
test evaluating any differences based on the observed main effect. Effect sizes (Cohen’s
“d”) were also calculated for pre-, post-, and 24h post intervention hsCRP for the three
stretching interventions comparing 30 vs. 60% mROM, 30 vs. 90% mROM, and 60 vs.
90% mROM for our main outcomes of interest (i.e. hsCRP). In addition, a secondary
analysis (linear regression) was conducted in order to investigate the association
between % mROM (independent variable) and hsCRP. With level of significance set at p
< 0.05 all statistical analyses were performed via SPSS (version 20.0, SPSS Inc., USA).
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4.3 – RESULTS
All participants were involved in all three interventions. There were no withdrawals from
the study. Figure 4.3 highlights the involvement of the participants in the study and
analysis.
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4.3.1 - RMANOVA
The RMANOVA revealed that Mauchly’s test for sphericity was violated, for both time (p
= 0.001), X2(5) = 13.140, p < 0.05, and for time x % mROM (p = 0.001), X2(5) = 15.923,
p < 0.05. With the degrees of freedom corrected using the Greenhouse-Geisser
estimates of sphericity for time (ε = 0.566) and time x % mROM (ε = 0.547), significance
was revealed for time (p = 0.011) but not for intensity (p = 0.506). Tests betweensubjects effects was significant (p = 0.001). With time as the observed main effect, an
LSD post hoc analysis reported significant differences between 30% and 90% (p =
0.004) and 60% and 90% (p = 0.034), but not between 30% and 60% (p = 0.089). A
comparison of the means of the Ln transformed values for hsCRP for 30% mROM post
(0.475 ± 0.242mg/L) and for 60% mROM post (0.567 ± 0.237mg/L) as well as for 30%
mROM 24h post (0.461 ± 0.232mg/L) and for 60% mROM 24h post (0.578 ±
0.198mg/L) were quite similar. However, the hsCRP concentration for 90% mROM post
(0.957 ± 0.595mg/L), and 90% mROM 24h post (1.036 ± 0.669mg/L) increased (Table
4.1,).
4.3.2 - Effect Sizes
4.3.2.1 - Pre-intervention hsCRP
Comparison of effect sizes for pre-intervention hsCRP suggests a low effect (“d” = -0.39;
95% CI, -0.56 to -0.22) when comparing 30 vs. 60% mROM. However, when comparing
30 vs. 90% as well as 60 vs. 90%, both comparisons revealed a high effect size (“d” = 1.09; 95% CI, -1.63 to -0.56 and “d” = -0.97; 95% CI, -1.5 to -0.43, respectively),
suggesting that 90% mROM is associated with a higher inflammatory response (Table
4.1). It is important to note that the interpretation of these results should be interpreted
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with caution, as some discrepancies in the raw data i.e. baseline hsCRP values in the
30% and 60% conditions were lower compared to the 90% condition, a fact that may
have influenced the RMANOVA. Please see Table 4.1, which presents these data.
4.3.2.2 – Post intervention hsCRP
Effect sizes post condition for hsCRP suggest a low effect (“d” = -0.39; 95% CI, -0.56 to
-0.21) when comparing 30 vs. 60% mROM implying that no major differences exist
between these two conditions in terms of the inflammatory response. However, the
comparison between 30 vs. 90% indicates a high effect size (“d” = -1.11; 95% CI, -1.67
to -0.54), suggesting that as the intensity of the stretch increases, so does the
inflammatory response. Interestingly, a high effect size (“d” = -0.99; 95% CI, -1.6 to 0.43) was also observed when comparing 60 vs. 90% (Table 4.1).
4.3.2.3 – 24h post intervention hsCRP
Effect sizes 24h post intervention hsCRP suggest a low effect size (“d” = -0.46; 95% CI,
-0.63 to -0.30) when comparing 30 vs 60% mROM. However, when comparing 30 vs
90% as well as 60 vs. 90% mROM, high effect sizes were observed (“d” = -0.95; 95%
CI, -1.75 to – 0.15 and “d” = -0.86; 95% CI, -1.7 to -0.06, respectively), suggesting a
pronounced inflammatory response in response to stretching intensity (Table 4.1).
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TABLE 4.1 – Mean (SD) measurement and Effect Sizes for hsCRP
Mean (SD) for hsCRP (mg/L)
Effect sizes (95% CI) between condition comparisons
30 %
60 %
90%
30 vs. 60%
30 vs. 90%
60 vs. 90%
Pre
0.48 (0.25)
0.57 (0.24)
0.96 (0.59)
-0.39 (-0.56 to -0.22)
-1.09 (-1.63 to -0.56)
-0.97 (-1.5 to -0.43)
Post
0.48 (0.24)
0.57 (0.24)
0.96 (0.59)
-0.39 (-0.56 to -0.21)
-1.11 (-1.67 to -0.54)
-0.99 (-1.6 to -0.43)
24h post
0.46 (0.23)
0.58 (0.19)
1.04 (0.67)
-0.46 (-0.63 to -0.30)
-0.95 (-1.75 to -0.15)
-0.86 (-1.7 to -0.06)
Key: Cohen’s “d” cut off points for effect size interpretation: small effect ≥ 0.20, medium effect ≥ 0.50, and large effect ≥ 0.80
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4.3.3 – Secondary Analysis - Linear Regression Report 30 – 90 % mROM and 60 – 90%
mROM
4.3.3.1 – hsCRP post
Linear regression analysis was used to test if the % mROM significantly predicted
participants’ hsCRP concentrations. In the analysis of 30% versus 90% mROM, the
results of the regression analysis indicated that % mROM explained 23.68% of the
variance in hsCRP [R2 = 0.24, F(1, 20) = 6.204, p = 0.022). Percentage mROM
significantly predicted hsCRP [Beta = 0.241, t(21) = 2.491, p < 0.022]. Therefore, when
comparing 30% to 90% mROM, we would expect an average increase in hsCRP of
0.24mg/L (Figure 4.4).
For the 60% compared to 90% regression analysis, % mROM explained 16.95% of the
significantly predicted hsCRP [Beta = 0.390, t(21) = 2.021, p < 0.057]. Therefore, when
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2.5
hsCRP (mg/L) post
2
1.5
1
0.5
R² = 0.2368
0
20
30
40
50
60
%mROM
70
80
90
100
Unstandardized B = 0.251mg/L
R2 Linear = 0.24
FIGURE 4.4 – Linear regression between 30 and 90% mROM hsCRP post
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2.5
hsCRP (mg/L) post
2
1.5
1
0.5
R² = 0.1695
0
50
55
60
65
70
75
%mROM
80
85
90
95
R2 Linear = 0.17
FIGURE 4.5 – Linear regression between 60 and 90% mROM hsCRP post
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4.3.3.2 – hsCRP 24h post
For the 30% compared to 90% regression analysis, % mROM explained 26.62% of the
significantly predicted hsCRP [Beta=.288, t(21) = 2.693, p < 0.014]. Therefore, when
The results of the regression analysis comparing 60% to 90% mROM indicated that %
mROM explained 19.12% of the variance in hsCRP [R2 = 0.19, F(1, 20) = 4.729, p =
0.042). Percentage mROM significantly predicted hsCRP [Beta = 0.458, t(21) = 2.175, p
< 0.042]. Therefore, when comparing 60% to 90% mROM, we would expect an average
increase in hsCRP of 0.46mg/L (figure 4.7).
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3
hsCRP (mg/L) 24h post
2.5
2
1.5
1
0.5
R² = 0.2662
0
20
30
40
50
60
%mROM
70
80
90
100
R2 Linear = 0.27
FIGURE 4.6 – Linear regression between 30 and 90% mROM hsCRP 24h post
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3
hsCRP (mg/L) 24h post
2.5
2
1.5
1
0.5
R² = 0.1912
0
50
55
60
65
70
75
%mROM
80
85
90
95
R2 Linear = 0.19
FIGURE 4.7 – Linear regression between 60 and 90% mROM and hsCRP 24h post
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4.4 DISCUSSION
This study investigated the effects of different percentages of mROM on hsCRP, a
biomarker for inflammation. The results suggest that stretching between 30% and 60%
mROM did not elicit an increase in blood hsCRP concentrations, with the mean values
being quite similar for both post and at 24h post intervention. When comparing 30% to
90% mROM and 60% to 90% mROM there was a marked increase in the concentration
of hsCRP levels for both post and 24h post stretch interventions. Effect sizes indicated
that a pronounced inflammatory response was observed when comparing 30 to 90 and
60 to 90 %mROM, however, caution is needed since a discrepancy with values for 90%
was detected at baseline. The secondary analysis suggested a moderate positive
relationship for both hsCRP post and 24h post.
The extent of muscle tissue disruption is reliant on the conjunction between the intensity
and the severity of the exercise (Tiidus and Ianuzzo, 1983). With regard to stretch
injuries it has been proposed that the initial mechanical insult of the stretch is followed
by a secondary or collateral injury that is coincident with neutrophil invasion (Toumi et
al., 2006). Neutrophils play a critical role in acute inflammation through removal of
necrotic tissue or cellular debris and release of cytokines to modulate chemotaxis
(Tidball, 1995). Neutrophils cause the secondary injury with the damaged myofibers
responsible for the production of IL-6, a ubiquitous intercellular cytokine associated with
the control and co-ordination of immune responses (Toumi et al., 2006). Since IL-6
features prominently in the expression of CRP (Gabay, 2006, Kilicarslan et al., 2013),
the marked increase in concentration of hsCRP post and 24h post associated with 90%
mROM, vs. 30% mROM and 60% mROM (confirmed by both the RMANOVA and the
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effect sizes) may suggest a possible association of intense stretching and inflammation
in lieu of strain to the muscle tissue. It is important to note that baseline mean hsCRP
for 30% and 60% conditions revealed normal baseline values, however, this did not
seem to be the case for 90%, where the mean hsCRP was considerable higher (please
see Table 4.1). This anomaly may have been the result of various different factors that
may have influenced the expression of circulating hsCRP, such as stress prior to the
intense stretching intervention, infection, lack of sleep, alcohol consumption prior to
assessment or other factors. Unfortunately, we were not able to identify which one of
these reasons resulted in this discrepancy, as all participants reported that they indeed
followed the recommendations provided by the principal researcher the day prior to the
assessment while they were all apparently healthy (infection free). Therefore, our
results should be treated with caution.
Studies referring to stretching intensity (Marschall, 1999, Wyon et al., 2009, Wyon et al.,
2013) demonstrate a disagreement, with some reporting a benefit for increased ROM
occurring following low intense stretching (Wyon et al., 2009, Wyon et al., 2013),
whereas at least one study observed that maximum stretching leads to significant
increases in ROM (Marschall, 1999). Regardless of what is reported about stretching
intensity, what is consistent is that intensity remains difficult to measure quantitatively
explaining the plethora of stretching articles referring to duration and frequency, which
are easily quantifiable (Apostolopoulos et al., 2015a). Assessing the degree of the
intensity of a stimulus is highly variable (Melzack and Katz, 1999), with the
transformation of the intensity of a physical stimulus into a perception following the
power law proposed by Stevens (Baliki et al., 2009). Perception is regarded as an
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internal, outwardly directed, ‘adaptive’ or ‘matching’ response to stimulation, generated
within the organizing system (McKay, 1963). According to Stevens’s law, sensation
magnitudes grow as power functions of stimulus intensities that produce them,
describing the relationships between human sensations as well as other subjective
impressions and the physical stimuli evoking them (Zwislocki, 2009).
In this study, our secondary analysis revealed a moderate positive correlation between
the independent variables (30%, 60%, and 90% mROM) and the dependent variable
(hsCRP) . The increase in the concentration of hsCRP associated with the different
percentages of mROM may potentially suggest a method for quantifying stretch
intensity using a blood biomarker. More research is needed to verify such a hypothesis.
The responses observed herein are related to stretch intensity rather than stretch
volume. Stretch volume refers to the total time under stretch with stretch intensity
alluding to how “hard” the stretch is, suggesting the length that the muscle is elongated
(Young et al., 2006). The extent, to which the muscle is elongated, i.e., the tension
generated during the stretch, may potentially be responsible for the onset of
inflammation, since the stretch volume for all the different intensities (30, 60, & 90%
mROM) was similar (5 sets at 60s with 10s rest between sets).
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4.5 – SUMMARY
The current data revealed that increases in % mROM are associated with a progressive
increase in hsCRP. Since the production of hsCRP has been linked to inflammation,
intensities between 30% - 60% mROM are less likely to cause such side effects.
Although these results were confirmed by our collective analyses (RMANOVA and
Effect Sizes), these results should be treated with caution, due to some unexpected
discrepancies in the baseline hsCRP values between the three conditions.
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5
STUDY THREE
THE EFFECT OF
DIFFERENT STRETCH
INTENSITIES ON
RECOVERY FROM
MUSCLE DAMAGE: A
RANDOMIZED TRIAL
Chapter 5 – The Effect of Different Stretching Intensities on
Recovery from Muscle Damage: A Randomized Trial
In study one we observed that IS may associate with a pronounced inflammatory
response, with study two suggesting that stretching between 30 – 60% of an
individual’s mROM did not elicit inflammation. Given that the practical application of
stretching includes the amelioration of some post-training effects, and which may
include control of inflammation, the focus of the third study was to investigate the
effects of LiS vs. HiS with regard to recovery from muscle damage.
Delayed onset of muscle soreness refers to acute muscle damage in response to
eccentric muscle contractions, as well as exhaustive and unaccustomed exercise or
intensities (Kanda et al., 2013). The intensity of discomfort associated with DOMS
increases within the first 24h post exercise, peaking between 24 and 72h, eventually
disappearing five to seven days post exercise (Talag, 1973, Armstrong, 1984). This
perception of soreness is due to the activation of free nerve endings around muscle
fibers serving as receptors of noxious stimuli associated with muscle damage
(Byrnes and Clarkson, 1986). In addition to soreness, the eccentric activity can
cause structural damage to muscle altering its function and joint mechanics,
observed with the release of CK (Rowlands et al., 2001).
Delayed onset muscle soreness results in a functional limitation, a restriction in the
ability to perform an activity within the normal range for an individual (Vasudevan,
1993). This temporary impairment leads to an increased risk of injury (Vasudevan,
1993, Saxton et al., 1995) described in terms of: loss of maximum voluntary force
production (Clarkson et al., 1992, Newham et al., 1983c, Newham et al., 1987),
changes in the contractile properties of muscle (Newham et al., 1983c), as well as
increased levels of muscle proteins representing damage (i.e. CK) (Clarkson et al.,
1992, Nosaka and Clarkson, 1992, Newham et al., 1987, Peake et al., 2005a). This
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disruption is influenced by three factors; the magnitude of the response, the previous
history of muscle use, and the injury specific interactions between muscle and the
invading inflammatory cells (Tidball, 2005). Therefore, the ability to alleviate this
process may partly reside in the act of diminishing the magnitude of inflammation
and the inflammatory response.
The time course for inflammation is dependent upon the exercise mode, intensity
and/or duration (MacIntyre et al., 1995). Injuries causing inflammation are
characterized by both a mechanical and a biochemical response (Armstrong, 1984).
The mechanical is characterized by a ‘local reaction’ at the site of damage, resulting
in the accumulation of leukocytes (i.e., neutrophils and macrophages) with the
magnitude related to both the intensity and duration of the exercise (Peake, 2002,
Robson et al., 1999, Castell et al., 1989, Peake et al., 2005a, Tiidus and Ianuzzo,
1983). Neutrophils are present within hours post exercise remaining for up to 48h
(MacIntyre et al., 2000, Stupka et al., 2001, Malm et al., 2000, Beaton et al., 2002b,
Frenette et al., 2000), with macrophages appearing at 24h remaining for up to 14d
(Table 2.1) (Malm et al., 2000, Peake et al., 2005a, Beaton et al., 2002a, Beaton et
al., 2002b). The biochemical response is characterized by the release of proinflammatory cytokines leading to a ‘systemic reaction’ (Castell et al., 1989). The
neutrophils and macrophages are responsible for the production of these cytokines,
specifically IL-1β, TNF-α, and IL-6. These cytokines are part of a network in which
the expression of each is influenced by the other (Shizuo et al., 1990), and may be
responsible as to whether the inflammatory process is detrimental or beneficial to the
damaged muscle (Mahoney et al., 2008, Tidball, 1995). Of the three cytokines, IL-6
is a major mediator of APPs, characterized by the increased expression of CRP
(Kushner and Rzewnicki, 1994, Castell et al., 1989, Bauer et al., 1989, Castell et al.,
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1988). In response to muscle damage, the rate of increase in concentration of CRP
can be as high as 1000-fold suggesting that it plays a major functional role at sites of
injury serving as a marker for damaged membranes, (Kushner, 1982, Gotschlich,
1989, Volanakis and Narkates, 1981). In otherwords, CRP, which is not specific to
any disease, is more accurate and the best reflection of the APR compared to any
other biomarker/APP, in demonstrating inflammation and the degree of tissue
damage (Kilicarslan et al., 2013, Libby et al., 2002).
Numerous investigators have attempted to identify treatments for DOMS or soft
tissue injury ranging from static stretching (Maxwell et al., 1988, Johansson et al.,
1999, High et al., 1989, Lund et al., 1998, Wessel and Wan, 1994), use of nonsteroidal anti-inflammatory drugs (NSAIDS) (Janssen et al., 1983, Hasson et al.,
1993, Hertel, 1997), massage (Lightfoot et al., 1997, Weber et al., 1994),
cryotherapy (Gullick and Kimura, 1996), as well as activity (Hasson et al., 1989). In
this study, we suggest the use of LiS as another modality for the recovery and
treatment from muscle damage, restoring muscle function without eliciting an
inflammatory response. Studies by Apostolopoulos et al., have observed that IS may
be potentially associated with inflammation (Apostolopoulos et al., 2015c,
Apostolopoulos et al., 2015b) with LiS (Apostolopoulos et al., 2015b) being less likely
correlated with inflammation as measured by the release of hsCRP. As such, the aim
of the present study was to investigate the effects of a LiS and HiS on the recovery
from muscle damage, by measuring for both primary (EPT and IPT (muscle
function)) and secondary (soreness level, hsCRP and CK) outcomes. It is assumed
that LiS would result in faster recovery from muscle damage with higher values
recorded for muscle function (EPT and IPT), as well as with a greater decrease in
160 | P a g e
soreness levels and in the concentrations of hsCRP and CK, compared to HiS and
Control.
5.2 – METHODS
Thirty recreationally active male participants (age: 25 ± 6yrs, mass: 83.1 ± 10.7kg,
height: 1.78 ± 0.68m) were recruited and assessed for four consecutive days: at
baseline, 24, 48 and 72h. A recreational athlete is referred to as an individual who
does not participate in an organized team or individual sport requiring systematic
training and regular competitions against others. Each participant read and signed
an informed consent form and answered a PARQ prior to the investigation (P. 282),
with the rights of each participant being protected. Ethical approval was granted by
the University of Wolverhampton Ethics Committee. Participants were advised to
refrain from any new forms of physical activity that may cause DOMS. Exclusion
criteria included any form of physical limitations with regard to their hips, knees and
ankles.
Prior to the test day, participants were randomized into a LiS (30 – 40% of the
individual’s maximum perceived stretch), HiS (70 – 80% of the individual’s maximum
perceived stretch), or a Control group, using an online randomization program
(www.graphpad.com). Unlike the previous two studies of this PhD project were the
participants and investigator had direct contact and monitoring was possible for
maintaining the correct intensity of the stretch, participants of this study performed
the stretches at home. Therefore, a range of stretching threshold was introduced to
provide the opportunity to our participants to interpret the stretch sensation; this
161 | P a g e
approach closely simulates clinical and sport performance approaches (the patient or
the athlete stretches to a volitional range of ROM). By presenting both a range and a
description of what the stretch should feel like (adjectives of sensation), it afforded
the best opportunity to convey the magnitude of the stretch needed to be performed.
This represents a form of “magnitude production” in which participants can perform a
given task (in our case stretches between 30-40% and 70-80% of ROM) based on
their interpretation (Stevens, 1971). However, this is indeed a different approach
than that adopted in our two previous studies and thus it may represent a
methodological limitation. This has been now recognized and discussed in chapter
seven (Section 7.12 – Methodology P. 218 – 220).
On arrival to the biomechanics laboratory of the University of Wolverhampton,
participants’ anthropometric measurements (mass (kg) and height (m)) were taken
prior to the collection of a 5ml blood sample, drawn from the basilic, cephalic or the
median veins, of the participant’s forearm. Since four blood samples were collected
within four consecutive days, it was decided that these samples would be drawn
from alternate forearms in order to allow for some recovery time for the veins (Fig.
5.1).
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FIGURE 5.1 – Methodology Schematic of Intervention(s)
163 | P a g e
5.2.2.1 – Pre Muscle Damage tests
Prior to the MDP, all participants were assessed at baseline for EPT and IPT of the
right quadriceps with use of a Kin Com isokinetic dynamometer (Kin Com,
www.kincom.com, East Ridge, Tennessee, USA). Familiarization consisted of five
submaximal repetitions for each strength condition followed by a minute rest prior to
the maximal effort for both EPT and IPT. During maximal effort, participants were
verbally encouraged to generate the greatest effort possible for three repetitions,
with the highest value recorded for baseline. These tests were repeated again at 24,
48, and 72h.
Eccentric peak torque was measured at 1.05rads/sec (60deg/sec), with the right
quadriceps being moved through a range starting at 20 progressing to 100 degrees
of knee flexion. Isometric peak torque was measured with the participant’s knee
being held at 60 degrees of knee flexion by the Kin Com.
5.2.2.2 – Muscle Damage Protocol
Following a 5min rest period after maximal efforts for both EPT and IPT, participants
were familiarized and exposed to the MDP. This was an adaptation of Paschalis et al.
(Paschalis et al., 2007), consisting of six sets of 10 eccentric repetitions at a speed of
1.05rads/sec with a 2min rest between sets, with the right quadriceps moved through
a range from 20 to 100 degrees of knee flexion. Preceding muscle damage,
alignment of the dynamometer rotational axis and the right knee joint rotation axis
(lateral femoral epicondyle) was set for each participant, with each participant’s upper
body and left quadriceps strapped in. The familiarization process consisted of a
submaximal repetition cycle of 1 set of 10 repetitions based on the parameters cited
for the damage protocol above. After a minute rest, participants started the MDP.
164 | P a g e
Unfortunately, we did not collect data from the isokinetic dynamometry for the
quantification of muscle damage (i.e. reduction in force output from the first to the
sixth set). This approach was not actually suggested by the authors who conducted
similar research (Paschalis et al., 2007). Nevertheless, this omission is now
acknowledged in the limitations section of the thesis (Section 7.12 – Methodology, P.
219). However, to ensure standardization of the procedures of the muscle damaging
protocol for each participant of each group (LiS, HiS, and Control), force output was
closely monitored on the Kin Com display. Verbal encouragement was also provided
to each participant by individuals not associated with the study in order to minimize
any bias during the six sets of 10 repetitions of the MDP.
Upon completion, participants were asked to rate their soreness on a numerical
rating scale, anchored at 'no soreness' (zero) to 'extreme sore' (10). Participants
returned to the laboratory at 24, 48, and 72h post muscle damage for both EPT and
IPT tests, a collection of a 5ml blood sample and the recording of their perceived
soreness levels prior to the muscle function tests.
5.2.3 – Post Muscle Damage Protocol Stretching Exercises
Both the LiS (30 – 40% of the individual’s maximum perceived stretch) and HiS (70 –
80% of the individual’s maximum perceived stretch) groups performed the exact
same stretching exercises and routine bilaterally, for the hamstring, hip flexor and
quadriceps muscle groups (in that order) before going to bed that night (Figures 5.2
– 5.4). Although only the quadriceps was exposed to the MDP, this muscle has an
antagonistic relationship to the hamstring muscle group, with both muscles in turn
being affected and related to the amount of hip flexion (Worrel et al., 1989). Each
participant was instructed both visually and verbally by the investigator about the
proper execution of each stretch. In order to confirm compliance, each participant for
165 | P a g e
both the LiS and HiS group were given a booklet with instructions as how to perform
each stretching exercise (Appendix LiS (P. 286 – 290) and HiS (P.291 – 295)). In
addition, for the three consecutive days following MDP, each participant met with the
principle investigator and was questioned as to whether they performed their
respective exercises at the appropriate intensity. They were asked to both describe
and demonstrate the stretching exercises to determine whether they had a good
comprehension about the proper execution of the stretching exercises.
Each stretch was supported with use of a bench or pillow, allowing for better isolation
and targeting of the muscle group of interest. Supporting the muscle may minimize
muscular contraction from occurring during the stretch. This was confirmed by a
study which compared a standing hamstring stretch to a supine lying stretch. It was
observed that the supine stretch isolated the hamstring muscle better, was more
comfortable, but more importantly facilitated a better relaxation response during the
stretch (Abdel-aziem et al., 2013). With the duration of each stretch being 60s, and
the frequency being, three times per muscle group per side (6min), the stretching
routine lasted approximately 18min. The intensity of the stretch for each group
differed. Using a 10 point numerical rating scale, anchored at ‘no pain’ (zero) to
‘extreme pain’ (10), the LiS group was instructed to hold the stretch at an intensity
level of a three or four out of 10, eliciting a warm gentle feeling. The HiS group was
instructed to hold each stretch at an intensity level of seven or eight out of 10,
creating a feeling of discomfort with slight pain. Each percentage was based on an
individual’s maximum perceived stretch.
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FIGURE 5.2 – Hamstring Stretch
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FIGURE 5.3 – Hip Flexor Stretch
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FIGURE 5.4 – Quadriceps Stretch
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5.2.4 – Biomarkers
Approximately 5ml of the participant’s blood was drawn at baseline and for each
consecutive day after the muscle damaging protocol from the basilic, cephalic or
median veins of the arm of choice by a certified phlebotomist. Blood was collected in
a vacutainer (BD Vacutainer, K2E EDTA). After sitting for 10min, allowing the blood
to clot at room temperature, it was spun at 3000rpms in a centrifuge (SciQuip Sigma
2-6E) for 10min, in order to separate the plasma from the rest of the blood
components.
In order to analyze serum CK a VITROS CK Slide method (Ortho-Clinical
Diagnostics Inc., Rochester, NY Vitros Chemistry Products Ref – 847 9396) was
employed. The VITROS CK Slides and VITROS Chemistry Products Calibrator Kit 3
were used in order to prepare the samples. These were then analyzed by the
VITROS Chemistry System (5, 1FS). A drop of a participant’s sample (~11μL) was
deposited on the VITROS CK Slide. The slide consists of five layers with certain
layers containing certain active ingredients (see Table 5.1).
TABLE 5.1 – Contents of VITROS CK Slide
Layer
1. Upper slide mount
2. Spreading layer
3. Reagent Layer
Contents
•
•
•
•
•
•
•
•
N-acetylcysteine
Buffer, pH 7.0
Adenosine diphosphate
Glycerol, magnesium acetate
Glycerol kinase, leuco dye
Peroxidase
Glycerophosphate oxidase
Creatine phosphate
4. Support layer
5. Lower slide mount
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A drop of a participant’s sample (~ 11μL) was deposited on the CK Slide. This was
evenly distributed by the spreading layer to the underlying layers with use of Nacetylcysteine used to activate the CK. The CK catalyzed the conversion of creatine
phosphate and Adenosine Di-Phosphate to creatine and ATP. In turn, in the
presence of glycerol kinase, glycerol was phosphorylated to L-α-glycerophosphate
by ATP. Oxidation of L-α-glycerophosphate to dihydroxyacetone phosphate and
hydrogen peroxide occurred in the presence of L-α-glycerophosphate oxidase.
Finally, leuco dye was oxidized by hydrogen peroxidase in the presence of
peroxidase to form a dye. With use of the VITROS 5,1FS Chemistry system, during
incubation, which lasted approximately 5min at a temperature of 37°C, reflection
densities were monitored at a wavelength of 670nm. The rate of change in the
reflection density was converted to the enzyme activity and recorded. The coefficient
of variation for CK was 3.7%.
To determine serum hsCRP a FlowCytomix Simplex Kit (eBioscience – BMS8288FF
San Diego, CA, USA), with a Beckman FC500 flow cytometer (FCM) was used. The
flow cytometer measures the light scatter and fluorescence emission properties of
individual cells in each sample (Aghaeepour et al., 2013). It is used routinely in both
research and clinical labs to study normal and abnormal cell function and structure,
as well as diagnose and monitor human disease and responses to therapy,
respectively (Aghaeepour et al., 2013). Flow cytometry is well adapted to measure
soluble analytes (a compound of interest in an analytical procedure) such as
variations in cytokines, and CRP in the sera and plasma samples of both health and
non-healthy individuals (O'Hara et al., 2011a, Prunet et al., 2006). The advantage of
using flow cytometry procedures is that it allows for the detection of several
171 | P a g e
functional characteristic of a cell simultaneously (O'Hara et al., 2011a, Green et al.,
2011).
The cytometer measures beads coated with antibodies specifically reacting with
each of the analytes to be detected. After determining the number of microwell strips
required to test the desired number of samples plus appropriate number of wells
needed for running blanks and standards, a 50µl assay buffer was added to the filter
plates to pre-wet the wells. Rows 1 and 2 of the microplate were designated for
standards with 25µl of standard mixture dilutions added from rows 1 to 7 of the plate,
with 25µl of assay buffer added to the blank wells below row 7. A 25µl standard
mixture dilution was added to the first well of column 3 for cytometer setup.
Afterwards 25µl of each analyte sample for that particular microplate was added to
the designated wells. Biotin-conjugate mixture was added to all the designated
samples including the remaining blank wells. The wells were covered with adhesive
film and an aluminium foil to protect from light and the microplate was incubated at
room temperature (18° to 25°) for 2h on a microplate shaker at 500rpm. After
incubation, the adhesive film was removed and the wells were emptied using the
vacuum filtration manifold whereupon 100µl assay buffer was added to the microwell
strips and emptied again. A 100µl assay buffer with 50µl of streptavidin-PE solution
was added to all the wells including the blank wells. The microplate was again
covered with adhesive film, and protected from light with an aluminum foil and
incubated for a further 1h on a microplate shaker at 500rpm. Using a vacuum
filtration manifold the wells were emptied after the adhesive film was removed with
100µl assay buffer added to each microwell strip and emptied with the vacuum
filtration manifold followed by the addition of 200µl assay buffer to each well. The
172 | P a g e
contents of each well were mixed with repeated aspiration and ejection and each
200µl sample was transferred to a separate acquisition tube and filled up to 500µl,
with an addition of 300µl of assay buffer. The samples were then measured using a
flow cytometer. The coefficient of variation for hsCRP was 6.4%.
Routine pre-analyses screening using the Kolmogorov-Smirnov test was performed
to investigate the distribution of all the variables. Accordingly, Ln transformation was
used to overcome skewness and kurtosis in biomarker variables (CK and hsCRP). A
primary analysis consisting of a two way ANOVA with repeated measures for time (0,
24, 48 and, 72h) and conditions (LiS, HiS, and Control) was performed on the
variables for muscle function (EPT and IPT), soreness level, and blood biomarkers
(CK and hsCRP). In addition, the data for all three conditions (LiS, HiS, and Control)
were analyzed - using a contemporary magnitude based inferences approach - to
detect small effects of practical performance (Hopkins, 2007). This approach
establishes the likelihood that the intervention will have a beneficial, trivial or harmful
effect. To determine the smallest meaningful change in performance a Cohen unit of
0.2 was employed. Where the chance of benefit and harm are both > 0.5% the effect
is deemed unclear. Qualitative descriptors were then assigned to the quantitative
percentile scores as follows: 25 – 75% possible, 75 – 95% likely, and >99% most
likely (Batterham and Hopkins, 2005, Hopkins, 2002). A secondary analysis was
conducted with use of a univariate analysis to explore the association between the
dependent variables for muscle function and the blood biomarkers, with the fixed
factors of time and conditions. The level of significance was set at p < 0.05. All
statistical analyses were performed via SPSS (version 20.0 SPSS Inc. Chicago,
Illinois, USA).
173 | P a g e
5.3 – RESULTS
All participants were randomized into three groups, LiS (n = 10), HiS (n = 10), and
Control (n = 10) with each receiving the intended intervention, the MDP based on
Paschalis et al. .(Paschalis et al., 2007) There were no withdrawals from the study.
Figure 5.5 highlights the involvement of participants and analysis in the study.
Please note with regard to Tables 5.3 – 5.5 (magnitude based inferences approach),
these show the mean percentage changes in muscle function (EPT and IPT), muscle
soreness and blood biomarkers (CK and hsCRP) across the three time periods
(baseline to 24h, baseline 48, and baseline to 72h) for the LiS, HiS, and Control
groups and statistics for the difference in the changes.
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TABLE 5.2 – Mean (SD) Measurement for EPT, IPT, and Soreness
EPT
Conditions
0h
24h
IPT
Soreness
48h
72h
0h
24h
48h
72h
0h
24h
48h
72h
LiS
247.5
(62.0)
229.6
(62.8)
244.3
(53.1)
263.1
(61.9)
207.6
(40.2)
196.4
(46.2)
209.5
(47.0)
222.3
(47.9)
6.0
(1.4)
5.0
(1.3)
2.9
1.2
(1.2) (0.4)
HiS
218.2
(59.7)
173.4
(35.6)
208.0
(44.7)
195.9
(31.9)
181.3
(41.2)
163.5
(41.7)
172.7
(50.1)
186.6
(39.1)
5.9
(1.8)
5.7
(2.2)
3.7
2.4
(1.4) (1.3)
Control
214.8
(52.7)
196.2
(49.8)
179.4
(42.8)
200.6
(65.6)
185.1
(55.2)
165.1
(49.5)
169.6
(50.6)
172.8
(55.4)
5.9
(1.8)
5.7
(1.9)
4.0
2.2
(1.3) (1.3)
Key: LiS = Low Intensity Stretching; HiS = High Intensity Stretching; EPT = Eccentric Peak Torque; IPT = Isometric Peak Torque
175 | P a g e
TABLE 5.3 – Changes in Creatine Kinase (CK), high sensitivity C-reactive protein (hsCRP), perceived muscle Soreness, Eccentric
Peak Torque (EPT) and Isometric Peak Torque (IPT) in Low Intensity Stretching (LiS) compared to Control and qualitative
inferences about the intervention effects
Variable
Time
CK
hsCRP
†
Soreness
EPT
IPT
LiS % change
Baseline-24h
Control %
change
34.0(23.7)
LiS vs. Control ES
Qualitative inference
52.7 (171)
LiS vs. Control %
change
14.0 (-36.9, 105.8)
0.18 (-0.62, 0.98)
Possibly harmful (48%)
Baseline-48h
14.5 (35.6)
41.2 (68.7)
23.2 (-11.9, 72.8)
0.28 (-0.17, 0.74)
Possibly beneficial (63%)
Baseline-72h
10.4 (25.5)
8 (45.2)
-2.2 (-23.3, 24.8)
-0.03 (-0.36,0.30)
Possibly trivial (69%)
Baseline-24h
9.7 (65.6)
15.7 (42.1)
5.5 (-24.9, 48.1)
0.05 (-0.26, 0.36)
Baseline-48h
-13.9 (91.7)
-9.0 (52.1)
5.7 (-31.2, 62.3)
0.05 (-0.34, 0.44)
Baseline-72h
-26.2 (114.2)
-25.7 (51.9)
0.7 (-38.1, 63.9)
0.01 (-0.44, 0.45)
Post-MDP-24h
-4.3 (31.8)
-18.3 (28.2)
-14.6 (-30.4, 4.7)
-0.47 (-1.08, 0.14)
Likely beneficial (78%)
24h-48h
-28.8 (35.5)
-44.5 (67.8)
-22.0 (-44.2, 8.9)
-0.74 (-1.74, 0.25)
48h-72h
-51 (69.8)
-56.7 (67.7)
-11.6 (-41.2, 32.8)
-0.37 (-1.58, 0.84)
Baseline-24h
-9.1 (11.7)
-7.4 (11.8)
1.9 (-6.5, 11.1)
0.07 (-0.25, 0.39)
Baseline-48h
-16.9 (16.7)
-0.4 (14.3)
19.8 (7.1, 34.1)
0.67 (0.25, 1.09)
Very likely beneficial (97%)
Baseline-72h
-8.9 (16.6)
6.8 (10.2)
17.2 (5.9, 29.6)
0.59 (0.21, 0.96)
Very likely beneficial (96%)
Baseline-24h
-11.7 (19.4)
-6.2 (8.2)
6.3 (-4.7, 18.6)
0.22 (-0.18, 0.62)
Baseline-48h
-9.4 (22.6)
0.3 (12.8)
10.8 (-2.9, 26.4)
0.37 (-0.11, 0.85)
Baseline-72h
-8.2 (24.4)
6.8 (9.8)
16.4 (1.8, 33.0)
0.55 (0.06, 1.03)
Key: MDP = muscle damaging protocol
* For CK, hsCRP and soreness beneficial = greater decrease with LiS vs. Control, and not beneficial means a greater increase with LiS vs. Control. For EPT
and IPT beneficial means a greater increase with LiS vs. Control, and not beneficial means a greater change with LiS vs. Control.
†
Muscle soreness values were compared immediately post-MDP to 24h post-MDP, 24h post-MDP to 48h post-MDP, and 48h post-MDP to 72h post-MDP.
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TABLE 5.4 – Changes in Creatine Kinase (CK), high-sensitivity C-reactive protein (hsCRP), perceived muscle Soreness, Eccentric
Peak Torque (EPT) and Isometric Peak Torque (IPT) in High Intensity Stretching (HiS) compared to Control and qualitative
inferences about the intervention effects
Variable
Time
CK
hsCRP
Soreness†
EPT
IPT
HiS % change
Baseline-24h
Control %
change
34.0 (23.7)
HiS vs. Control ES
72.5 (92.0)
HiS vs. Control %
change
28.8 (-13.1, 90.8)
0.39 (-0.22, 1.00)
Baseline-48h
14.5 (35.6)
60.5 (85.6)
40.2 (-4.7, 106.2)
0.52 (-0.07, 1.12)
Likely harmful (82%)
Baseline-72h
10.4 (25.5)
83.4 (123.8)
66.2 (2.8, 168.5)
0.78 (0.04, 1.52)
Baseline-24h
9.7 (65.6)
-18.7 (35.8)
-25.9 (-46.7, 2.9)
-0.31 (-0.65, 0.03)
Baseline-48h
-13.9 (91.7)
-26.2 (116.2)
-14.2 (-50.8, 49.4)
-0.16 (-0.73, 0.41)
Baseline-72h
-26.2 (114.2)
-25.2 (86.9)
1.4 (-41.1, 74.3)
0.01 (-0.54, 0.57)
Post-MDP-24h
-4.3 (31.8)
-6.7 (35.9)
-2.4 (-22.3, 22.4)
-0.05 (-0.49, 0.40)
Possibly Trivial (55%)
24h-48h
-28.8 (35.5)
-32.7 (56.8)
-5.4 (-30.0, 27.7)
-0.11 (-0.70, 0.48)
48h-72h
-51.0 (69.8)
-37.0 (47.7)
28.7 (-10.5, 85)
-0.49 (-0.22, 1.20)
Baseline-24h
-9.1 (11.7)
-19.3 (32.2)
-11.1 (-25.1, 5.4)
-0.49 (-1.19, 0.22)
Baseline-48h
-16.9 (16.7)
-3.8 (20.2)
15.7 (1.4, 32.1)
0.60 (0.06, 1.15)
Baseline-72h
-8.9 (16.6)
-8.3 (27.7)
0.6 (-14.2, 18.1)
0.03 (-0.63, 0.69)
Baseline-24h
-11.7 (19.4)
-10.3 (12.6)
1.6 (-9.7, 14.4)
0.06 (-0.37, 0.48)
Baseline-48h
-9.4 (22.6)
-6.0 (14.5)
3.7 (-9.4, 18.8)
0.13 (-0.36, 0.62)
Baseline-72h
-8.2 (24.4)
-22.5 (138.1)
-15.6 (-49.4, 41.0)
-0.61 (-2.45, 1.23)
†
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TABLE 5.5 – Changes in Creatine Kinase (CK), high sensitivity C-reactive protein (hsCRP), perceived muscle Soreness, Eccentric
Peak Torque (EPT) and Isometric Peak Torque (IPT) in Low Intensity Stretching (LiS) compared to High Intensity Stretching (HiS)
and qualitative inferences about the intervention effects
Variable
Time
CK
Baseline-24h
hsCRP
†
Soreness
EPT
IPT
HiS % change
LiS vs. HiS %
change
-11.5 (-54.3, 71.4)
LiS vs. HiS ES
72.5 (92.0)
LiS %
change
52.7 (171)
-0.16 (-1.01, 0.70)
Baseline-48h
60.5 (85.6)
41.2 (68.7)
-23.0 (-54.8, 31.1)
-0.34 (-1.03, 0.35)
Baseline-72h
83.4 (123.8)
8 (45.2)
-41.1 (-64.3, -2.9)
-0.68 (-1.33, -0.04)
Likely Beneficial (90%)
Baseline-24h
-18.7 (35.8)
15.7 (42.1)
42.5 (10.3, 84.2)
0.31 (0.09, 0.54)
Baseline-48h
-26.2 (116.2)
-9.0 (52.1)
23.2 (-24.6, 101.5)
0.19 (-0.25, 0.62)
Baseline-72h
-25.2 (86.9)
-25.7 (51.9)
-0.6 (-34.5, 50.8)
-0.01 (-0.38, 0.37)
Post-MDP-24h
-6.7 (35.9)
-18.3 (28.2)
-12.5 (-29.6, 8.7)
-0.27 (-0.71, 0.17)
Possibly Beneficial (61%)
24h-48h
-32.7 (56.8)
-44.5 (67.8)
-17.5 (-43.5, 20.2)
-0.39 (-1.16, 0.38)
48h-72h
-37.0 (47.7)
-56.7 (67.7)
-31.3 (-51.9, -1.8)
-0.76 (-1.49, -0.04)
Baseline-24h
-19.3 (32.2)
-7.4 (11.8)
14.7 (-3.3, 36.1)
0.50 (-0.12, 1.12)
Baseline-48h
-3.8 (20.2)
-0.4 (14.3)
3.5 (-8.7, 17.4)
0.13 (-0.33, 0.58)
Baseline-72h
-8.3 (27.7)
6.8 (10.2)
16.4 (0.3, 35.2)
0.55 (0.01, 1.09)
Baseline-24h
-10.3 (12.6)
-6.2 (8.2)
4.6 (-3.3, 13.2)
0.18 (-0.13, 0.49)
Baseline-48h
-6.0 (14.5)
0.3 (12.8)
6.8 (-3.3, 17.9)
0.26 (-0.13, 0.65)
Baseline-72h
-22.5 (138.1)
6.8 (9.8)
37.8 (-16.9, 128.6)
1.27 (-0.73, 3.27)
†
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5.3.1 – Analysis - Strength and Soreness
5.3.1.1 – Eccentric Peak Torque
For EPT, results revealed that time x condition was significantly different (p = 0.009). An
LSD post hoc analysis revealed a significant difference between LiS vs. both HiS (p =
0.039), CI .95 = 2.4547 (lower) 91.9953 (upper), p < 0.05 and Control conditions (p =
0.035), CI ,95 = 3.5797 (lower) 93.1203 (upper), p < 0.05. The comparisons between HiS
and Control were not significant (all p > 0.05) (Table 5.2, 5.6a and 5.6b). However,
when using the magnitude based inferences approach comparing the effect of LiS to
both Control and HiS, for the time period(s), baseline-48h, and baseline-72h, LiS was
very likely beneficial (97% and 96% respectively) compared to Control (Table 5.3), with
the LiS effect being likely beneficial (87%) between baseline and 72h compared to HiS
(Table 5.5). When comparing the effects of HiS to Control, HiS effect was likely harmful
(76%) from baseline to 24h (Table 5.4).
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FIGURE 5.6a – Eccentric Peak Torque
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FIGURE 5.6b – Eccentric Peak Torque (normalized)
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5.3.1.2 – Isometric Peak Torque
The results indicated no significance for time x condition (p = 0.185) (Table 5.2, Figures
5.7a & 5.7b). However, when using the magnitude based inferences approach which
compared the effect of LiS to both Control and HiS, for the time period baseline to 72h,
LiS was likely beneficial (89%) compared to Control (Table 5.3), and possibly beneficial
(82%) compared to HiS (Table 5.5). When comparing the effects of HiS to Control, HiS
was possibly harmful (65%) comparing baseline-72h (Table 5.4).
FIGURE 5.7a – Isometric Peak Torque
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Figure 5.7b – Isometric Peak Torque (normalized)
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5.3.1.3 – Soreness
No significant differences were detected for soreness for time x condition (p = 0.553).
(Table 5.2, Figures 5.8a & 5.8b). However, using the magnitude based inferences
approach it was found that LiS was likely beneficial (78 and 76% respectively)
compared to Control (Table 5.3), as well as likely beneficial (90%) compared to HiS for
the 48-72h time period (Table 5.5). Comparing the effects of HiS to Control, HiS was
likely harmful for the time period 48-72h (90%) (Table 5.4). In addition, table 5.6
suggests that LiS appears to be associated with a faster decrease in soreness
compared to both the HiS and Control groups.
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TABLE 5.6 – Mean, SD, and Percent Change for Soreness post muscle damage
Conditions
LiS
HiS
Control
Time Period
Mean (SD)
Post MDP
6.00 (1.41)
24h post MDP
5.00 (1.33)
48h post MDP
2.90 (1.19)
72h post MDP
1.20 (0.42)
Post MDP
5.95 (1.80)
24h post MDP
5.70 (2.21)
48h post MDP
3.70 (1.42)
72h post MDP
2.40 (1.27)
Post MDP
5.90 (1.79)
24h post MDP
5.70 (1.88)
48h post MDP
4.00 (1.33)
72h post MDP
2.20 (1.32)
% difference
between 24h &
post DOMS
% difference
between 48h &
post DOMS
% difference
between 72h &
post DOMS
16.6%
52%
80%
4.2%
32.7%
60%
3.4%
32%
63%
Key: LiS = Low Intensity Stretching; HiS = High Intensity Stretching; MDP-Muscle Damage Protocol; DOMS = Delayed Onset Muscle Soreness
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FIGURE 5.8a – Soreness
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FIGURE 5.8b – Soreness (normalized)
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5.3.2 – Analysis - Blood Biomarkers
5.3.2.1 – Creatine Kinase
Repeated measures ANOVA demonstrated significant differences for time x condition (p
= 0.037) for CK, however, the LSD post hoc analysis revealed no significant differences
between conditions (Table 5.7, Figure 5.9). No consistent effect was found between LiS
and Control over the three time periods using the magnitude based inferences approach
(Table 5.3). Comparing LiS to HiS, LiS was likely beneficial for the time period baseline
– 72h (Table 5.5). However, the effects of HiS to Control, between baseline to 48h, and
baseline to 72h, HiS was likely harmful (82 and 91%) (Table 5.4).
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TABLE 5.7 – Mean and SD Measurements for CK and hsCRP (Ln Transformed & Raw Data)
Blood Biomarkers
Conditions
LiS
CK (U/L)
hsCRP (mg/L)
0h
24h
48h
72h
0h
24h
48h
72h
5.64 (0.66)
6.29 (0.73)
5.99 (0.67)
5.72 (0.57)
0.02 (1.28)
0.17 (1.14)
-0.72 (1.31)
-0.28 (1.22)
348.1 (263.8)]* [672.5 (418.9)]* [479.8 (298.7)]* [349.8 (190.1)]* [1.73 (1.66)]* [1.93 (2.08)]* [1.63 (1.74)]* [1.32 (1.57)]*
HiS
5.23 (0.73)
5.78 (0.58)
5.70 (0.77)
5.84 (0.87)
0.59 (0.97)
0.38 (0.93)
0.28 (0.99)
0.29 (0.76)
[239.4 (183.6)]* [400.1 (247.4)]* [433.2 (406.3)]* [553.9 (627.8)]* [2.88 (3.54)]* [2.49 (3.62)]* [2.01 (2.38)]* [1.83 (1.98)]*
Control
5.51 (0.65)
5.80 (0.56)
5.65 (0.71)
5.61 (0.58)
0.07 (1.00)
0.16 (0.84)
-0.08 (0.85)
-0.24 (0.76)
[297.9 (187.1)]* [380.9 (215.6)]* [362.5 (311.6)]* [332.6 (217.3)]* [1.58 (1.29)]* [1.60 (1.28)]* [1.23 (0.89)]* [1.07 (0.79)]*
Key: *Raw Data; LiS = Low Intensity Stretching; HiS = High Intensity Stretching; CK = Creatine Kinase; hsCRP = high sensitivity C-reactive protein
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FIGURE 5.9 – CK in relation to Time
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5.3.2.2 – hsCRP
With regard to hsCRP, no significance was revealed for time x condition (p = 0.585)
(Table 5.7, Figure 5.10). The magnitude based inferences approach revealed
inconsistent effects of LiS compared to Control and HiS for all the time periods (Table
5.3, and 5.5). It should be noted that HiS started from a higher baseline than both LiS
and Control, suggesting a possible flaw in the methodology of the study, particularly the
MDP as well as the compliance of the participants. When comparing HiS to Control, it
was observed that for the time-period baseline to 24, HiS was possibly beneficial (71%)
(Table 5.4).
FIGURE 5.10 – hsCRP in relation to Time
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5.3.3 – Secondary Analysis – Relationship Muscle Function and Biomarkers
A univariate analysis was performed to explore the association between the dependent
variables for muscle function (EPT and IPT), and the blood biomarkers (CK & hsCRP),
while adjusting for conditions (LiS, HiS, and Control) and time (0, 24, 48, and 72h). It
was observed that when comparing the means for all three conditions, for the same
amount of injury or inflammation, the LiS produced significantly higher strength values
(EPT and IPT) in relation to CK and hsCRP (CK: p<0.001 and hsCRP: p = 0.005) (Table
5.8, Figures. 5.11-5.14).
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TABLE 5.8– Relationship between Muscle Function (EPT and IPT) and Blood Biomarkers (CK & hsCRP). Results
reported as means (95%CI)
CK (U/L)†
hsCRP (mg/L)‡
Conditions
EPT
IPT
EPT
IPT
LiS
244.06 (277.44 to 260.68)
205.42 (191.34 to 219.49)
247.73 (231.41 to 264.04)
211.06 (197.16 to 224.96)
HiS
199.91 (183.43 to 216.39)
177.82 (163.86 to 191.78)
195.84 (179.37 to 212.30)
172.07 (158.04 to 186.10)
Control
198.75 (182.28 to 215.23)
174.89 (160.93 to 188.84)
199.16 (182.87 to 215.46)
174.99 (161.10 to 188.88)
Key: LiS = Low Intensity Stretching; HiS = High Intensity Stretching; EPT = Eccentric Peak Torque; IPT = Isometric Peak Torque; CK = Creatine
Kinase; hsCRP = high sensitivity C-reactive protein; CI = Confidence Interval
†
Covariates appearing in the model are evaluated at the following values for CK = 5.73
Covariates appearing in the model are evaluated at the following values for hsCRP = 0.109
‡
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400
350
300
EPT (Nm)
250
200
150
100
HiS
LiS
CONTROL
Linear (HiS)
R² = 0.22
R² = 0.1395
Linear (LiS)
Linear (CONTROL)
50
0
0.00
2.00
4.00
6.00
8.00
hsCRP (mg/L)
10.00
12.00
14.00
FIGURE 5.11 – Relationship between EPT and hsCRP
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350
300
IPT(Nm)
250
200
150
100
HiS
LiS
CONTROL
Linear (HiS)
Linear (LiS)
Linear (CONTROL)
50
0
0.00
10.00
20.00
30.00
hsCRP (mg/L)
R² = 0.001
R² = 0.2409
R² = 0.042
40.00
50.00
FIGURE 5.12 – Relationship between IPT and hsCRP
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400
350
300
EPT(Nm)
250
200
150
100
HiS
LiS
CONTROL
Linear (HiS)
Linear (LiS)
Linear (CONTROL)
50
0
0.00
500.00
1,000.00
1,500.00
R² = 0.002
R² = 0.0938
2,000.00
2,500.00
CK (U/L)
FIGURE 5.13 – Relationship between EPT and CK
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350
300
IPT(Nm)
250
200
150
100
HiS
LiS
CONTROL
Linear (HiS)
R² = 0.0006
Linear (LiS)
Linear (CONTROL) R² = 0.248
50
0
0
500
1,000
1,500
2,000
2,500
CK (U/L)
FIGURE 5.14 – Relationship between IPT and CK
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5.4 – DISCUSSION
This study investigated the effects of different stretching intensities; LiS, HiS, and no
stretching (Control) on muscle function, measured as either EPT and IPT, soreness, as
well as blood biomarkers for muscle damage (CK) and inflammation (hsCRP) post
MDP. A RMANOVA revealed significant effects for both EPT and CK comparing LiS,
HiS and Control, with none observed for IPT, soreness, and hsCRP. The magnitude
based inferences approach which looked at the beneficial, trivial, and harmful effects of
all the three conditions (LiS, HiS, and Control) on muscle function (EPT and IPT),
soreness, and blood biomarkers (CK and hsCRP), revealed that LiS was associated
with a beneficial effect on producing a higher EPT sooner compared to HiS and Control.
In addition, LiS had a beneficial effect on CK (a decrease in concentration) compared to
HiS. Unlike RMANOVA, the magnitude based inference approach indicated that LiS
was beneficial for producing higher IPT as well as being associated with a decrease in
soreness compared to both HiS and Control. However, with regard to the blood
biomarkers, the magnitude based inferences approach suggested that LiS was
inconsistent compared to both Control and HiS.
As expected all three groups exhibited a decrease in both EPT and IPT from 0 to 24h
suggesting that the eccentric MDP was effective, as previously described (Paschalis et
al., 2007). However, a significant difference was observed only for EPT for LiS as
opposed to both HiS and Control groups. In specific, at 48h LiS approached and
surpassed the pre muscle damage values continuing to increase to 72h, and this was
significantly different in comparison to both the HiS and Control groups. Results using
the magnitude based inferences approach comparing the effects of LiS to both HiS and
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Control, also suggested that LiS had a beneficial effect for increasing EPT compared to
both HiS and Control potentially suggesting a greater recovery post muscle damage.
During the same time period (48 to 72h) the HiS group decreased in EPT values. A
possible explanation may be that since this group continued to stretch to discomfort and
pain, this may continue to attenuate recovery. Previous studies have demonstrated that
high intensity stretching may have an adverse effect on the body (McClure et al., 1994,
Jacobs and Sciacia, 2011) and may be potentially associated with inflammation
(Apostolopoulos et al., 2015b, Apostolopoulos et al., 2015c). Interestingly, the Control
group in EPT surpassed HiS approaching 72h. Although no significant difference was
seen with IPT, LiS exceeded baseline values prior to 48h as compared to HiS, which
reached and surpassed these values closer to 72h. This difference in effect was also
suggested by the magnitude based inferences approach, which observed that from
baseline to 72h, LiS was beneficial for causing an increase in IPT, compared to HiS and
Control. In fact, when comparing HiS to Control it suggested that HiS was potentially
harmful, in otherwords, it may be responsible for causing a decrease in IPT, thereby
associated with a slower recovery from muscle damage. Similar to EPT, the LiS group
was potentially responsible for a faster recovery post muscle damage for IPT. This trend
warrants further investigation. A possible explanation may be that since isometric
contraction involves tension development with no change in muscle length (Ryschon et
al., 1997), the intensity level of the LiS group (30 – 40% of a maximum perceived
stretch) may have assisted the muscles recovery allowing it to generate greater IPT
earlier. Therefore, LiS appears to have a beneficial effect with regard to generating
greater EPT and IPT following muscle damage. This is an interesting result since
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strength loss is more pronounced and longer lasting following exercise involving
eccentric muscle actions as opposed to concentric and isometric actions (Byrne et al.,
2001). Based on previous reports (Jacobs and Sciacia, 2011) we hypothesized that
inflammation may be the drive behind this phenomenon, however, this is not supported
by the present data as no significant changes in hsCRP were observed. The magnitude
based inferences approach observed an inconsistent effect with regard to LiS vs.
Control and HiS. A possible explanation for this observation is that the extent of the
inflammatory response may be dependent upon the muscle groups used (Peake et al.,
2005c), the amount of muscle mass recruited during the eccentric exercise (Peake et
al., 2005c), and the exercise (Akimoto et al., 2002) itself. For example, a study by
Akimoto et al. (Akimoto et al., 2002), indicated that downhill, and endurance running
accounted for a greater increase in CRP compared to a bicycling ergometer exercise at
an intensity of 80% VO 2max. Therefore, since participants were exposed to a non-weight
bearing eccentric exercise (i.e., seated on the Kin Com), with only the knee extensors
being stressed, this may account for the observed discrepancy.
A significant change in the concentration of CK, an intramuscular enzyme increasing in
the blood post muscle damage (Nosaka et al., 1991), was observed for all three
independent variables which was statistically significant. As expected concentration for
CK increased for all the conditions from 0 to 24h, confirming the effectiveness of the
MDP. From 24 to 72h, both the LiS and the Control group registered a decrease in CK
concentration, with both approaching baseline values. Interestingly, instead of observing
a decrease in the HiS group after 24h this group continued to register an increase in CK
concentration compared to the LiS and Control groups (Table 5.4, Figure 5.9). No
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consistent effect was observed when comparing LiS to Control using the magnitude
based inferences approach for all three time periods (Baseline to 24, 48, and 72h), with
LiS being more likely beneficial compared to HiS from baseline to 72h for causing a
decrease in the concentration of CK. In addition, HiS compared to Control was likely to
have a more harmful effect from baseline to 48h and baseline to 72h suggesting that
HiS may be responsible for not observing a decrease in the concentration of CK. This
continued expression in CK concentration affiliated with the HiS group, may potentially
be due to the use of the intense stretching protocol. This protocol required participants
to stretch to discomfort and slight pain at an intensity level of 70 – 80% of a maximum
perceived stretch (Appendix P. 291 – 295), as opposed to the LiS group which stretched
at an intensity level of 30 – 40% of a maximum perceived stretch (Appendix P. 286 –
290). This observation is supported by the statistically significant decrease in EPT, with
the HiS group observed to having a harmful effect (a decrease in peak torque values)
for both EPT and IPT compared to Control according to magnitude based inference
approach. Data from other studies reveal that prolonged periods of stretching to pain
and discomfort has been associated with an adverse response (Brand, 1984, McClure
et al., 1994). According to Nosaka et al., muscle has the ability to recover contractile
function with the adaptation effect varying among the indicators of damage and duration
of the adaptation effect (Nosaka et al., 1991).
Both groups performed the same stretching exercises (hamstring, hip flexor, and
quadriceps), with each being held for 60s and repeated for three times per muscle
group per side. The only difference between LiS and HiS was the stretching intensity.
Although soreness levels were not statistically significant between the conditions, use of
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the magnitude based inferences approach indicated that LiS had a beneficial effect on
deceasing soreness post MDP compared to both HiS and Control. In contrast, the HiS
group revealed an increase in soreness for the time period between baseline to 72h
compared to Control. This is also confirmed in Table 5.6, with LiS associated with a
substantial decrease in soreness (80%) vs. Control (63%), and HiS (60%), respectively.
Since participants of the HiS group were instructed to stretch to discomfort with slight
pain, this potentially may account for the observed effect on the slightly higher soreness
level associated with HiS. Previous data suggest that prolonged strength loss is
indirectly the most valid way to assess for changes in the magnitude of the injury with
time. (Faulkner et al., 1993, Warren et al., 1999) . Since HiS was associated with having
an adverse effect on muscle function (EPT and IPT), stretching at a higher intensity (70
– 80% of a maximum perceived stretch) may be treated with caution.
In this study, both the LiS and HiS groups performed supported stretches. With the body
supported during the stretch, using a bench or other implements, this minimized the
activation of potential stabilization reflexes, allowing the stretched muscle to relax. In a
study by Abdel-aziem et al. comparing a standing hamstring stretch to a supine lying
stretch, the supine stretch isolated the hamstring muscle better, was more comfortable,
but more importantly facilitated a better relaxation response during stretching.(Abdelaziem et al., 2013). Since both groups performed supported stretches, at the same
duration and frequency, the difference was the intensity of the stretch. Therefore, use of
a supported LiS may prove to be more favourable for recovery from muscle damage,
since the muscle tissue is relaxed physically as well as neurologically. However, more
research is needed to further investigate the relationship supported LiS and recovery.
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The secondary analysis which compared strength variables (eccentric and isometric
peak force) to blood biomarkers (CK and hsCRP), observed that LiS produced higher
scores for both EPT and IPT for the same amount of muscle damage (CK) and
inflammation (hsCRP) (Table 5.8). Interestingly, the Control group was associated with
a greater torque output vs. the HiS group for hsCRP. These results suggest that LiS
may be potentially responsible for mitigating an adverse effect on the response of the
muscle over time in relation to an injury and/or physical damage. By minimizing the
impact of the damage to the muscle, this allows for greater repair. Since intensity of
stretching is important, previous studies have demonstrated that too much force applied
during the stretch may result in an inflammatory response (Brand, 1984, McClure et al.,
1994, Jacobs and Sciacia, 2011, Apostolopoulos et al., 2015c, Apostolopoulos et al.,
2015b), possibly explaining the difference in the force output observed between LiS and
HiS groups.
Injuries are characterized by an initial mechanical and a secondary biochemical
response, with the latter peaking one to three days post initial injury (Tidball, 1995,
Tidball, 2005, Armstrong, 1984). The mechanical injury is related to the disruption of the
individual sarcomeres after stretches of activated muscle fibers (Brown and Hill, 1991,
Faulkner et al., 1993, Friden et al., 1983). This results in an immediate loss of force
production due to uncoupling of the excitation/contraction force producing and/or
transmitting structures of the muscle (Toumi and Best, 2003), explaining the observed
initial loss of force relative to EPT and IPT.
The biochemical injury is facilitated and enhanced by the increased concentration of
both the neutrophils and macrophages in response to muscle damage (Closa and
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Folch-puy, 2004, Peake et al., 2005c). According to Tidball, the extent to which
neutrophils promote muscle injury is determined by the acute use of the muscle at the
time of injury in conjunction with the intensity and severity of the exercise (Tidball,
2005). The neutrophils and macrophages are responsible for promoting inflammation by
releasing pro-inflammatory cytokines (IL-1β, TNFα and IL-6) (Gresnigt et al., 2012,
Tidball, 1995). IL-6, features prominently in the release of CRP, a principal downstream
mediator of the APR (Pradhan et al., 2001, Gabay, 2006, Kilicarslan et al., 2013, Akira
et al., 1990), with both IL-6 and CRP being markers of systemic inflammation (Pradhan
et al., 2001). The release of CRP is indirectly related to muscle damage, since IL-6 has
been positively correlated to the release of CK (Bruunsgaard et al., 1997).
The continued increase in the concentration of CK (Table 5.7, Figure 5.9) may be
suggestive of a continued release of IL-6, although a significant difference in hsCRP
(inflammation) was not observed in this study. According to the magnitude inferences
based approach, intense stretching associated with HiS might continue the expression
of muscle damage, since HiS was associated with having a harmful effect (i.e., no
decrease in CK concentration) from baseline to 48 and to 72h compared to Control, with
LiS having a beneficial effect (i.e. a decrease in CK) from baseline to 72h. Both LiS and
Control expressed a decrease in CK after an initial rise from 0 to 24h post muscle
damage (Table 5.4, Figure 5.9). With LiS, the low stress/strain on the muscle tissue
may be responsible for mitigating muscle damage suggesting a faster return to baseline
values (Table 5.7, Figure 5.9). However, no consistent effect was observed comparing
LiS to Control based on the magnitude based inferences approach, suggesting that
doing no stretching (Control) might be better than LiS with regard to mitigating CK. More
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investigation is required. The rise in CK in relation to HiS may be suggestive of a
mechanical strain, which may be greater than the ability of the muscle to resist,
specifically after damage was already imposed on the muscle post DOMS protocol.
According to the magnitude based inferences approach, HiS had a harmful effect (i.e.
an increase in CK concentration) compared to Control between baseline to 48 and
baseline to 72h, with LiS having a beneficial effect (i.e., a decrease in CK concentration)
from baseline to 72h compared to HiS. Alternatively, since the effect of LiS compared to
Control was inconsistent, with HiS compared to Control associated with a harmful effect,
this may suggest that use of LiS compared to HiS, would be a better choice for recovery
from muscle damage. This recommendation is further supported since the LiS group
was observed to have a beneficial effect with both muscle function (EPT and IPT), and
soreness.
The continued rise in CK in HiS group suggests that the stretch intensity (70 to 80% of
maximum perceived stretch) may be responsible for a continued muscle strain, which
may be greater than the ability of the muscle to resist, specifically after damage was
caused post DOMS protocol. This strain may be associated with the disruption of the
contractile apparatus of the muscle (myofibrils) (Baker et al., 2004, Mair et al., 1995). In
response to this disruption an increase in neutrophil concentration occurs further
promoting muscle injury (Tidball, 2005). As suggested in the literature review above, the
disruption of the myofibrils is associated with the disruption of the homeostasis of Ca2+
(Belcastro et al., 1998). Although Ca2+ is important for the primary process of
contraction and relaxation (Berchtold 2000, Tate 1991), a variety of Ca2+ binding
proteins such as calpains exist, which are not directly involved with this process, but are
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important for muscle plasticity and performance (Berchtold et al., 2000, Suzuki et al.,
2004). Calpains have been associated with the proteolysis of skeletal muscle proteins
(i.e. α-actinin, tropomyosin, and desmin) following exercise (Belcastro et al., 1998,
Takahasi, 1990), with exercise induced protease linked to an increase in leukocyte
populations following strenuous bouts of exercise (Camus et al., 1992, Field et al.,
1991, Gabriel et al., 1992). This Ca2+ binding protein is associated with both the I- and
Z- band regions of the sarcomere, with higher concentration observed at the Z-disk than
anywhere else in the myofibril (Kumamoto et al., 1992). Kunimatsu et al. revealed that
the peptides released in response to the activity of calpain were chemoattractive for
neutrophils without producing any cytotoxic effects (Kunimatsu et al., 1993, Kunimatsu
et al., 1995, Kunimatsu et al., 1989). This release of neutrophils at the site of injury
promote and regulate the synthesis of IL-6 (Griendling et al., 2000), with IL-6 as
suggested in section 2.7.2.3 of the thesis, being the primary mediator of the APP, CRP
(Akira et al., 1990, Akira et al., 1993, Streetz et al., 2001). Although an effect was not
observed with regard to LiS in relation to hsCRP, according to Tilg et al., the role of
CRP depends on the experimental system, for this APP exerts specific functions that
are part of a complex mechanism to control the homeostasis of the organism (Tilg et al.,
1997). Further research is needed.
Both IL-6 and CRP exhibit an immunosuppressive and anti-inflammatory effect, with
CRP exhibiting a role in the induction of anti-inflammatory cytokines in circulating
monocytes and suppression of pro-inflammatory cytokines in macrophages (Tilg et al.,
1997). Neutrophil expression is determined by the acute use of the muscle at the time of
injury, believed to exacerbate an injury delaying the regeneration of the muscle tissue
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(Pizza et al., 2001). Since, the magnitude of the damage is related to the intensity of the
activity causing the injury (Tidball, 1995, Tidball, 2005), it is plausible that the low
intensity level (30 – 40% of a maximum perceived stretch) associated with LiS may
influence the recovery and healing of the muscle tissue. This may account for the
observed increase in the strength performances. Further, the elevated levels of
neutrophil concentration during passive stretching, have been interpreted as a response
to a minor injury (Pizza et al., 2001). However, further more research is needed.
5.5 – SUMMARY
In study three, it was observed that use of LiS was associated with a beneficial effect for
muscle function (EPT and IPT) as well as soreness compared to both HiS and Control,
as revealed by the magnitude based inferences approach. However, though there was
a significant difference with regard to CK for all three conditions, it was revealed that
comparing LiS to Control for both the blood biomarkers (CK and hsCRP), no consistent
effect was found between LiS and Control across the three time periods, as well as for
hsCRP relative to HiS. Further research is required to elucidate these mechanisms. IL-6
is the primary mediator for the release of CRP; measurement of this cytokine may
provide further insight with regard to the effect of LiS and HiS in relation to inflammation
and the inflammatory response, since this study observed that LiS was associated with
a beneficial effect for muscle function and soreness. Such an investigation may provide
insights into the potential role of LiS for the recovery and healing of traumatized muscle
tissue.
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6
SUMMARY DISCUSSION
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
Chapter 6 – Summary Discussion
The magnitude of the force applied to the joint during a stretching exercise is defined
as the intensity of the stretch (Jacobs and Sciacia, 2011). The three studies of this
PhD specifically observed how stretching intensity, in the form of either a low or high
intensity stretch, influenced the inflammatory response. To investigate these effect,
CRP in the form of hsCRP and the pro-inflammatory cytokines IL-1β, IL-6, and TNFα involved in the regulation of the immune response, haematopoiesis and
inflammation in the blood (Akira et al., 1990) were measured. All three studies were
the first to measure the effects of stretch intensity through blood and enzyme
biomarkers for inflammation in humans. A brief summary of our findings can be
found in Table 6.1 below.
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TABLE 6.1 – Summary of Studies
STUDY
n
DESIGN
L
One
Two
Three
12
11
30
M
Randomized
within Subject
Crossover
Trial
[Control & IS]
Randomized
within Subject
Trial
[Three stretch
interventions
30, 60 & 90%
mROM]
BLOOD BIOMARKERS
Inflammation
STRETCH
INTENSITY
H
X
X
X
X
hsCRP
X
IL-1β
X
IL-6
X
TNF-α
MD
CK
A significant increase in the concentration of hsCRP was
observed when comparing IS to Control conditions. This was
confirmed with effect sizes looking at between conditions. Within
comparisons also revealed a small increase in hsCRP for IS vs.
Control conditions.
X
X
Randomized
Clinical Trial
[10 x LiS, HiS,
& Control]
X
X
RESULT
X
Both low (30 %mROM), and medium (60 %mROM) intensity
stretching did not elicit an inflammatory response, with
concentration for hsCRP being quite similar and not significant. A
pronounced inflammatory response was observed when
comparing 30 to 90 and 60 to 90 %mROM, however, caution is
needed since a discrepancy with values for 90% was detected at
baseline. In addition, our secondary analysis revealed that for
every increase in % mROM there was a moderate increase in
hsCRP.
LiS showed a significant increase in EPT compared to both HiS
and Control. As well, magnitude based inferences approach
revealed that LiS was associated with a positive effect for both
IPT and soreness levels compared to Control and HiS, with HiS
associated with having a harmful effect compared to Control.
With regard to blood biomarkers, LiS was associated with
inconsistent effects compared to Control and HiS for all three
time periods. Our secondary analysis revealed that, for the same
amount of muscle damage the LiS group produced higher
strength scores for both EPT and IPT.
Abbreviations: n number of participants; L (Low); M (Medium); H (High); hsCRP (high sensitivity C-reactive protein); IL-1β (interleukin-1β); IL-6 (interleukin-6);
TNFα (Tumor necrosis factor alpha); MD (Muscle Damage); CK (Creatine Kinase)
210 | P a g e
6.2 – ACUTE INFLAMMATORY RESPONSE TO STRETCHING
The first study investigated whether IS causes acute inflammation. Twelve
participants were involved in a randomized crossover trial participating in both
conditions: IS and Control. The muscles stretched were the hamstrings, gluteals, and
quadriceps. The duration of each stretch was 60s with each muscle group being
stretched for three sets. The stretching intensity was equivalent to an eight out of 10
on a numerical rating scale (NRS), with zero representing no pain and 10 extreme
pain. At this level, participants experienced some pain and discomfort. Blood was
collected pre-, immediately post and at 24h post interventions and analyzed for
hsCRP (primary outcome), IL-1β, IL-6, and TNFα. A significant 14-fold increase in
the concentration levels of hsCRP post treatment (24h) was observed similar to
those observed in aggressive forms of physical activities such as rugby (Cunniffe et
al., 2011, Lund et al., 2011), plyometric exercise (Chatzinikolaou et al., 2010), or a
long duration run (Kim et al., 2007). This increase was interesting since a study by
Kim et al., observed a 23-fold increase in hsCRP measured during the latter half of a
200km ultra-marathon race (Kim et al., 2007). Though we are not claiming that IS is
similar to intense physical activities of high duration or eccentric exercise, this rise in
the concentration of hsCRP was an interesting observation. This APP has been
observed to interact with both humoral and cellular effector systems of inflammation,
inducing a pro-inflammatory effect, (Ballou and Lozanski, 1992, Cermak et al., 1993,
Volanakis, 2005). In addition, effect sizes between and within groups also
suggested, that IS was associated with an increase in hsCRP concentration
compared to the Control condition.
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6.3 – STRETCH INTENSITY VS. INFLAMMATION: A DOSE DEPENDENT
ASSOCIATION?
Study two, was a continuation from study one, with the intention of observing three
stretching intensity interventions based on the participants mROM. This study was a
randomized within subject design with all 11 participants being exposed to three
stretch intensity conditions calculated at 30%, 60%, and 90% of their mROM for the
right hamstring muscle. The percentages were chosen to represent a low (30%
mROM), medium (60% mROM), and high (90% mROM) stretching intensity. Blood
was collected prior to, immediately after, and at 24h post interventions, and analyzed
for hsCRP. Based on the results for hsCRP in relation to the % mROM, it was
demonstrated that a stretching intensity between 30% and 60% mROM did not
induce significant increases in the concentration of hsCRP at 24h. An increase in the
concentration of hsCRP was observed when comparing 30 to 90 and 60 to 90%
mROM. However, caution should be exercised with the interpretation of these results
since an anomaly was observed at baseline with the high stretching intensity
condition (90% mROM). In addition, our secondary analysis revealed that for every
increase in % mROM there was a moderate increase in hsCRP. However, although
these results provide for the first time some evidence for the associations of
inflammation and stretching intensity, anomalies in the baseline hsCRP suggest
further research is required before forming any firm conclusions.
212
6.4 – THE EFFECT OF DIFFERENT STRETCH INTENSITIES ON RECOVERY
FROM MUSCLE DAMAGE: A RANDOMIZED TRIAL
With study three, the intention was to investigate whether the body recovers faster
with the use of either a LiS or HiS following a bout of muscle damage as measured
by muscle function tests (EPT and IPT), blood biomarkers, and soreness levels. The
protocol used to induce muscle damage resulted in, as expected, DOMS (Paschalis
et al., 2007).
In this study 30 participants were randomized into a LiS, HiS, and Control groups,
with the LiS group stretching at an intensity level of 30 to 40% of a maximum
perceived exertion vs. 70 to 80% for the high intensity stretching group. A
measurement for both EPT and IPT was performed prior to the DOMS protocol in
order to establish baseline values. These measurements were repeated again at 24,
48, and 72h. Soreness levels were taken immediately post MDP and repeated daily
prior to both EPT and IPT retests. Statistical significance was observed for EPT
comparing LiS to both HiS and C, with no significance observed for either IPT or
soreness levels.
However, an effect was observed using the magnitude based inference approach for
LiS compared to both Control and HiS for muscle function (EPT, IPT), and soreness.
With regard to EPT, LiS was very likely beneficial to have an effect from baseline to
48 and baseline to 72h compared to Control, Similarly, a very likely beneficial effect
was also observed from baseline to 72h for LiS compared to HiS. When comparing
HiS to Control, HiS was observed to have a harmful effect from baseline to 24h
compared to Control.
Comparing LiS to both HiS and Control for IPT, a likely beneficial effect was seen for
LiS compared to Control from baseline to 72h with a possibly beneficial effect for LiS
213
compared to HiS for the same time period. Similar to EPT, comparing the effects of
HiS to Control, HiS was possibly harmful from baseline to 72h.
The magnitude based inferences approach revealed that LiS had an effect on
soreness compared to both HiS and Control, with LiS being likely beneficial
compared to Control post muscle damage to 24h, and between 24 – 48h, as well as
likely beneficial compared to HiS for 48 – 72h period, for decreasing soreness.
Comparing the effects of HiS to Control, HiS was likely harmful for the time period 48
– 72h, suggesting that HiS was more likely to cause further damage compared to
Control.
Besides evaluating for force performance (muscle function) and soreness, blood was
collected prior to and subsequently post DOMS at 24, 48, and 72h, and analyzed for
both hsCRP and CK. Significance was observed only for CK and not for hsCRP
when comparing LiS, HiS, and Control. With regard to the blood biomarkers, the
magnitude based inferences approach revealed no consistent effect between LiS
compared to Control over the designated time-periods for CK. However, LiS was
likely to have a beneficial effect compared to HiS from baseline to 72h. Alternatively,
comparing HiS to Control, HiS was likely harmful from baseline to 48h and baseline
to 72h for CK. Similarly, inconsistent effects were revealed for LiS compared to
Control and HiS for all time periods for hsCRP, however comparing HiS to Control, it
was observed that from baseline to 24h that HiS was possibly beneficial. In turn, our
secondary analysis observed that for the same amount of inflammation and muscle
damage caused by the DOMS protocol, participants using LiS developed higher
force values compared to the HiS and Control.
214
6.5 – SUMMARY
With study one an increase in the concentration levels of hsCRP was associated
with IS compared to the Control condition, suggesting that IS may be potentially
linked to inflammation. Normally present in plasma in only trace amounts, a dramatic
increase in rate of synthesis of CRP has been observed with an inflammatory
stimulus in lieu of tissue damage and exercise (Kushner et al., 1989, Michigan et al.,
2011). Since stretching has been defined as an external force (Weerapong et al.,
2004), this mechanical stimulus is potentially responsible for causing damage to the
sarcolemma, since stretching has been observed to cause the release of neutrophils
in adult male mice (Pizza et al., 2002). Results from study two, suggest that a low
(30% mROM) or moderate (60% mROM) stretching intensity level does not alter the
circulating levels of hsCRP. Unfortunately, because of the anomaly observed at
baseline for 90% mROM, it is difficult to determine if the increase in hsCRP observed
between 30% to 90% mROM, and 60% and 90% mROM immediately post and at
24h post is due to HiS. In addition, in study three it was observed that the LiS group
was associated with a positive effect with regard to muscle function (EPT and IPT),
as well as soreness, with HiS having a potentially harmful effect compared to Control
(i.e., associated with a decrease in EPT and IPT, as well as an increase in
soreness). With regard to the blood biomarkers (CK and hsCRP), using the
magnitude inferences based approach LiS was associated with inconsistent effects
for all three-time-periods compared to both HiS and Control. More research is
therefore, required to highlight the mechanisms investigated within this thesis.
215
7
LIMITATIONS
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
Chapter 7 – Limitations of Studies
7.1 – LIMITATIONS OF STUDIES (Table 7.1)
7.1.1 – Blood Collection
A major limitation with regard to the studies of the thesis was in the design for the
collection of blood samples. Although hsCRP was measured in all the studies, and
study one did measure IL-1β, IL-6, and TNFα, the design for collecting the blood
samples missed the half-life of these cytokines; IL-1β, IL-6, and TNF-α. In the
literature the half-life for IL-1β, IL-6, and TNFα, are approximately 2.5h (Hazuda et
al., 1988), 2-4h (Pedersen, 2000, Rohde et al., 1997, Marino and Giotta, 2008), and
18.2min (Oliver et al., 1993), respectively. Since the immune response is regulated
by cytokines released by the innate immune cells (neutrophils, monocytes, and
macrophages) in response to stimulus (Lacy and Stow, 2011), these proinflammatory proteins are part of a cytokine network in which the expression of one
is influenced by another. In specific, IL-1 and TNFα are potent inducers of IL-6, with
IL-6 inversely regulating TNFα expression (Akira et al., 1990). The main
physiological function of IL-6 is the induction of the hepatic APR characterized by the
increased expression of APPs, specifically CRP (Gabay and Kushner, 1999, Bauer
et al., 1989). Therefore, although the blood collections failed to measure the half-life
for IL-6, the literature suggests that increased expression of CRP is due to a relevant
increase in IL-6. The absence of IL-6 prevents proper recovery (McFarland-Mancini
et al., 2010) since it is necessary for the resolution of inflammation as observed in
mice deficient in IL-6 (IL-6-/-) (Kopf et al., 1994). Although the IL-6-/- mice did not
express any developmental abnormalities they were unable to generate an APR
(Kopf et al., 1994). The induction of IL-6 due to injury or infection is believed to be a
very important in-vivo distress signal coordinating the activities of the hepatocytes,
macrophages and lymphocytes (Kopf et al., 1994), and accordingly, the relationship
217 | P a g e
between IL-6 and CRP represents the body’s mechanism for resolving the acute
inflammatory response. Therefore, we believe that the observations in the
expression of CRP observed herein, were due to relevant changes in IL-6 (Samols et
al., 2002, Akira et al., 1990, Fuller and Grenett, 1989, Streetz et al., 2001, Gruys et
al., 2005).
7.1.2 – Methodology
A methodological progression of the thesis with regard to stretching intensity, the
number of sets used with all three studies may be considered as another potential
limitation. Participants in study one were stretched at a high intensity level of 80% of
their maximum perceived exertion (slight pain and discomfort) for three sets. In study
two, although the investigation was focused on identifying a difference with regard to
various stretching intensities (low (30 %), medium (60%), and high (90%)) of mROM,
instead of using 80% for the high intensity stretching, which would have made it
consistent with study one, 90% was used. In addition, the number of sets were
increased from three to five sets. However, a study by Taylor et al. observed that the
first four stretches for either repeated stretching of the MTU to 10% beyond resting
length and to a set tension resulted in the greatest changes in the MTU (Taylor et al.,
1990). This study concluded that with repetitive stretching there was little alteration
of the MTU after four stretches implying that this is the minimum amount needed for
the greatest elongation of the MTU during stretching (Tidball, 2005). With study three
although 3 sets were performed for all the stretches (similar to study 1), a range was
introduced with regard to intensity for both LiS (30 – 40%) and HiS (70 – 80 %).
Whereas, this zone represents a methodological limitation, it should be emphasized
that it also provided a leeway with regard to the interpretation of the stretch
sensation. Unlike the other two studies were the participants and investigator had
218 | P a g e
direct contact and monitoring was possible with regard to attaining and maintaining
the correct intensity of the stretch, participants in study three performed the stretches
at home. By presenting both a range and a description of what the stretch should
feel like (refer to Appendix Stretching exercises for study three P. 286 – 295), it
afforded the best opportunity to convey the magnitude of the stretch needed to be
performed before going to sleep. According to psychophysical laws, this represents a
form of “magnitude production” in which the range provided is conveyed to a
participant who best adjusts their response to produce a match (Stevens, 1971).
Since no data from the isokinetic dynamometer was collected to specifically quantify
whether muscle damage occurred in the participants (i.e., comparing set 1 to set 6),
this may also represent a limitation. We were unable to account for the magnitude of
muscle damage between the experimental groups ensuring that both groups (LiS
and HiS) started from the same level of muscle damage. However, the choice to not
take into account these data, was influenced by the fact that the description of the
muscle damaging protocol was based on a study by Paschalis et al. (Paschalis et al.,
2007), which also did not quantify the extent of muscle damage. The observed
difference in baseline values for muscle function (EPT, IPT), and blood biomarkers
(CK, hsCRP) represents another potential methodological limitation. With regard to
muscle function, the LiS group started at a higher baseline value compared to both
HiS and Control (Figures 5.6 & 5.7). Similarly, the values for the blood biomarkers
also varied with HiS beginning at a lower baseline than both LiS and Control for CK,
and at a higher baseline for hsCRP. These discrepancies in baseline values suggest
a potential flaw with the MDP as well as the potential compliancy of the participants
to the instructions given prior to testing. It appears that the participants of the LiS
group were more compliant with regard to the muscle function tests. In turn, the
219 | P a g e
inability to control for the activities of the participants outside the laboratory setting
may account for the observed discrepancies in baseline. Participants were informed
not to partake in any activity or activities prior to muscle testing, except for the
stretching protocol, since these activities may influence the outcome of the study.
7.1.3 – Compliance
Limiting non-compliance amongst participants is essential for the proper execution
and completion of randomized clinical trials (Oba et al., 2006). Although measures
were taken to promote and maximize participant compliance with use of verbal and
written instruction(s), we realize that non-compliance represents a potential
limitation. Non-compliance is an inherent issue with research studies potentially
influencing the outcome of the intervention(s) compromising the research study
(Whitney and Dworkin, 1997). Low levels of compliance may lead to a lack of or a
reduction in power to detect the effects of the intervention potentially reporting on
false negatives that may cause the rejection of effective interventions (Campbell et
al., 2000, Moher et al., 2001a, Moher et al., 2001b). False negatives refer to a result
that fails to detect a true difference in the intervention or the effect (Brookes et al.,
2001). In addition, with less compliancy from the participants this can reduce the
external validity of a study, in otherwords, how generalizable can the results be from
the study (Kehoe et al., 2009).
7.1.4 – Nutrition
Lack of controlling the nutritional intake prior to testing may also be regarded as a
potential limitation for all three studies. In otherwords, participants were not given a
list of foods to avoid post intervention and for the duration of the studies. According
to Esposito et al., an intake of macronutrients may produce oxidative stress and
220 | P a g e
inflammatory responses (Esposito and Giugliano, 2006). Macronutrient refers to
nutrients required in large amounts providing the energy necessary to carry out daily
activities as well as maintaining body functions (NZ, 2011). For instance, ingestion of
glucose has been associated with an increase in leukocytes and mononuclear cells
and in the amount of nuclear factor (NF)-κΒ (Dandona et al., 2005). Interestingly NFκΒ has been associated with regulating the activity of at least 125 genes most of
which are pro-inflammatory (Dandona et al., 2005).
7.1.5 – Mental Stress
In addition, the collection of blood for all three studies may have caused some
participants to be stressed. Although all participants willingly allowed the collection,
some did exhibit anxiety with the insertion of the vacutainer into their veins.
Depending on the nature of the stressor, evidence exists that psychological stress
may suppress or enhance immune functions (Maes et al., 1998, Kunz-Ebrecht et al.,
2003). In response to acute psychological stress, studies have suggested that proinflammatory cytokine production occurs (Maes et al., 1998, Steptoe et al., 2001,
Kunz-Ebrecht et al., 2003). According to Shephard, the acute stress response is
marked by an increase in IL-1ra and IL-6 which may take several minutes to evoke
(Shephard, 2002). The mental stress during the intense stretching of study one as
well as the high intense stretch intervention of study two may have also contributed
to the increased levels of the blood biomarkers. However, with the nature and the
design of these studies it would have been very difficult to control or eliminate this
mental stress.
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7.1.6 - Age
Since all three studies made use of recreationally active young males (average age
26 yrs), age may be considered as another limitation. Although the studies referred
to the intensity of the stretch and were not concerned with the increase or decrease
in ROM, it would have been interesting to observe if a difference existed in
considering age. According to Feland et al., age associated changes are observed in
the elderly, which include a decreased muscle mass associated with fiber atrophy
and hyperplasia, as well as motor unit remodelling (Feland et al., 2001a). The
decline of skeletal muscle mass occurs at an average rate of 4% per decade until 50
years of age and after, the loss is 10% per decade (Fielding, 1995). Therefore, it
would have been very interesting to observe how the magnitude and rate of
stretching intensity may affect the response of older muscle with regard to the
measurement of inflammatory biomarkers.
7.2 – SUMMARY
With regard to all three studies; a larger sample size, the use of methods or tools to
minimize non-compliance and mental stress, and observing the effects of the
interventions relative to a greater age range (i.e., elderly patients), would have
ensured a better representative distribution of the population in general. This would
have allowed the opportunity to make greater generalizations with the observed
results. In addition, the timing for the collection of blood samples coinciding with the
half-lives of the specific pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), would
have allowed for a better blood panel analysis. This analysis would have provided a
more thorough indication of the effect of the magnitude and the rate of stretching
intensity relative to inflammation and the inflammatory response(s).
222 | P a g e
TABLE 7.1 – Summary of Studies and Scheduled Time-Periods for Blood Collection
STRETCH
INTENSITY
BLOOD BIOMARKERS
Inflammation
STUDY
L
M
H
C hsCRP IL-1
MD
IL-6 TNFα CK
TIME PERIOD FOR BLOOD
COLLECTIONS
LIMITATION
Pre
Imm. 24h 48h 72h
post post post post
•
One
X
X
X
X
X
X
X
X
•
•
•
•
X
•
•
Two
X
X
X
X
X
X
•
•
•
•
X
•
•
Three
X
X
X
X
X
X
X
X
X
X
•
•
•
•
Blood was collected at time periods not
allowing for the proper measurement for IL-1β,
IL-6 & TNFα
Compliance
Nutrition
Mental Stress
Age (no elderly)
IL-6 was not measured
Methodological (Intensity level 90% vs. 80%
(study 1) & 5 vs. 3 sets (study 1 and 3))
Compliance
Nutrition
Mental Stress
Age (no elderly)
IL-6 was not measured
Methodological (Range of intensity 30-40% vs.
70-80% vs. 30%(study 2) and 80% (study1))
Compliance
Nutrition
Mental Stress
Age (no elderly)
Key: L = Low; M = Medium; H = High; C = Control; hsCRP = high sensitivity C-reactive protein; IL-1β = interleukin-1 beta; IL-6 = interleukin-6;
TNFα = Tumor necrosis factor alpha; MD = Muscle Damage; CK = Creatine Kinase
223 | P a g e
8
FUTURE RESEARCH
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
Chapter 8 – Future Research
Study one suggested that IS was more likely associated with inflammation (i.e.,
increase in hsCRP levels), with study two observing that no inflammation was
associated when stretching at either 30 and 60% of mROM. These results were also
confirmed by effect sizes. With study three, results from both RMANOVA and
magnitude based inferences approach suggested that LiS was beneficial for the
increase in EPT and IPT values as well as associated with a faster decrease in
soreness compared to both HiS and Control. These results with regard to muscle
function and soreness suggest that LiS is potentially linked to faster recovery times. In
the literature review, the concept of mechanotransduction was introduced, conceivably
suggesting that it may be the potential mechanism of how stretching (mechanical force),
may result in a biochemical response (inflammation). Calpain was introduced as the
interface between the mechanical disruption and the biochemical response providing a
plausible yet reasonable theory of stretching intensity and the onset or the mitigation of
the
inflammatory
response.
Further
research
specifically
focused
on
mechanotransduction, calpain, inflammation, and stretching intensity, may provide an
understanding of how this mechanical force, either high or low, may influence the
hierarchy of structures from the macro (muscle, tendons, MTU) to the micro (cells,
ECM), with regard to recovery from muscle damage. Such an understanding may
provide further insights to coaches and medical practitioners (physicians, therapists etc)
with regard to the potential effects and outcomes associated with LiS and HiS. Such
knowledge will allow for the better design of rehabilitation and recovery programs
minimizing further tissue damage. For instance, if inflammation is already present in the
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connective and soft tissue of the musculoskeletal system (i.e., rheumatoid (RA) and
osteo (OA) arthritis), it would be interesting to investigate whether LiS, both passively
and/or actively, may disrupt the inflammatory response. Mitigation of the inflammatory
response with use of LiS may provide an adjunct intervention to medication. In addition,
it is plausible that since the practice of LiS is not associated with pain and discomfort,
and since pain has been associated with mental/emotional stress (Gatchel, 2004,
Merskey and Bogduk, 1994, Melzack and Katz, 1999), that LiS may provide some relief
from this stress. Similar to physical stress, mental/emotional stress releases the proinflammatory cytokines IL-1, IL-6 and TNF-α (Steptoe et al., 2001). These proteins are
very responsive to mental/emotional stress suggesting their involvement in pathways
influencing the health of the individual(s) psychosocially (Steptoe et al., 2001).
8.2 – Further Research
8.2.1 – Further Research Based on Studies of the Thesis
Subject to the results of the three studies, further research is needed to continue to
investigate the effects of stretching intensity on acute inflammation, and recovery from
muscle damage. These investigations would benefit from the recruitment of a greater
population of participants. Although power calculations for both study one and two
indicated that 11 participants equates to 80% power based on the primary outcome
(hsCRP), unfortunately with study one missing data for hsCRP for three participants
resulted in the study being underpowered (n = 9).
An improved design with regard to blood collection (i.e. half-lives) for the proinflammatory cytokines (IL-1β, IL-6, and TNF-α) will provide a better understanding of
the biochemical response of stretching intensity and its influence on the inflammatory
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response. As indicated in section 2.7.2.2 of the thesis, these pro-inflammatory cytokines
form a collaborative network in which the expression of each influences and is
influenced by the other. For instance, IL-1β and TNF-α, are both potent induces of IL-6,
with IL-6 regulating the expression of TNF-α (Akira et al., 1990, Akira et al., 1993,
McGee et al., 1995). This synergistic relationship is an attempt to regulate the immune
response and the inflammatory reaction (Akira et al., 1990, Akira et al., 1993, Streetz et
al., 2001). Since financial constraints were an issue with this thesis regarding blood
analyses, future studies would focus mainly on blood collection coinciding with the halflives for only IL-6 (i.e. 2 – 4h) (Pedersen and Hoffman-Goetz, 2000) , and hsCRP (19 –
20h) (Marino and Giotta, 2008, Pepys and Hirschfield, 2003). According to the literature
in section 2.7.2.3, IL-6 is the primary mediator of the APR (Streetz et al., 2001, Gruys et
al., 2005, Ramadori et al., 1988), with both IL-1β and TNF-α unable to induce a full APR
(Akira et al., 1990). Since IL-6 acts as a messenger between the hepatocytes
synthesizing APPs, such as CRP, and the local site of damage, the measurement of
this cytokine would be the primary choice for future studies, both for the response of
acute inflammation as well as muscle damage. With regard to muscle damage, a
positive relationship between IL-6 and CK was demonstrated in a study by Bruunsgaard
et al. (Bruunsgaard et al., 1997).
The design of the three studies were intended to provide a logical progression
investigating the effects of IS (Study 1) and then comparing low, medium, and intense
stretching to probe how these different magnitudes of stretching may influence the
acute inflammatory response (Study 2). Based on the results of these studies, study
three was designed to investigate whether stretching intensity (i.e. low or high) may
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ameliorate muscle damage, soreness, and influence the inflammatory response, post
muscle damage. Although results from the studies revealed that intense stretching is
associated with an increase in hsCRP levels but not with low and medium intense
stretching, and that LiS was beneficial for aiding the recovery of muscle from muscle
damage, flaws within the methodology of the three studies prevent definitive
conclusions to be drawn. Intense stretching levels for all three studies were different
(80% study 1; 90% study 2; and a range for study 3, 70 – 80%). In addition, low
intensity levels for studies two and three were different (30% for study 2 and a range for
study 3, 30 – 40%). In turn, inconsistencies were apparent with regard to frequency
amongst the three studies. Participants in studies, one and three, were stretched and
performed three sets, respectively, while in study two they were stretched for five sets.
Although evidence in the literature suggests that 5 sets elicits a similar response to 3
sets, with no statistical difference existing between the two (Taylor et al., 1990), this
inconsistency represents a methodological flaw with the project. The only consistency
amongst the stretching protocol for all three studies was the duration of the stretching
(60s). Future studies would look at making certain that a consistency exists which may
allow for a better interpretation of the results since confounding factors with regard to
methods will be minimized. However, besides conducting future research to address the
issues of blood collection, participant size, and the inconsistencies regarding stretching
intensity and frequency for the three studies of the thesis, based on the results that
were obtained from this project, the sections below provide some suggestions for future
research projects.
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8.2.2 – Ultrasonography
Ultrasonography, a non-invasive technique, has been used to determine the stiffness
and hysteresis, elastic characteristics, and the viscoelastic properties of the human
tendon structures in-vivo in real time (Kubo et al., 2002, Fukashiro et al., 1995, Kubo et
al., 2001). In the past, research investigating the effect of stretching on the properties of
both connective and soft tissue were limited to animal studies (Stromberg and
Wiederhielm, 1969, Viidik, 1972, Taylor et al., 1990). The difficulty with these studies
were their applicability to humans because of the differences existing amongst species
(Ker et al., 1988). With ultrasonography, one may observe the response of the tissues
to the mechanical stress/strain within their natural environment. This provides real time
data of the stress/strain distribution along the tendon and aponeurosis allowing for a
better understanding of the structure and function of the tendinous tissues (Muramatsu
et al., 2001). A future study would be to investigate the response of the connective and
soft tissue to either LiS or HiS during real time. In study three it was observed that LiS
was associated with a faster recovery with regard to muscle function (EPT and IPT) and
a decrease in soreness (RMANOVA and magnitude inference based approach)
compared to both HiS and Control post muscle damage. According to Morse et al.
(Morse et al., 2008), the MTU is a complex structure consisting of tendon, muscle fibers
and connective tissue. Observing the response of the connective tissue and muscle
fibers in real time may provide answers as to why LiS was associated with a better
muscle function and recovery from soreness compared to HiS and Control.
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8.2.3 – Oxygen Free Radicals
Tissue damage is associated with an acute inflammatory response, with the influx of the
inflammatory cells (i.e. neutrophils and macrophages (pro-inflammatory)) promoting the
release of and activation of oxygen free radicals (Farber, 1994, Closa and Folch-puy,
2004). This damage results in either an increase in the rate of partially reduced oxygen
species or the weakening of the antioxidant defences of the cells (Farber, 1994). The
extent to which the inflammatory cells promote damage is determined by the acute use
of the muscle during the time of damage, in partnership with both the intensity and
severity of the exercise (Tidball, 2005).
Oxygen free radicals are toxic mediators promoting the generation of the proinflammatory cytokines (IL-1, IL-6, and TNF-α), regulating the synthesis of these
cytokines by modulating their nuclear factors (Griendling et al., 2000). The increased
expression of these cytokines amplify the initial cell response to tissue damage,
determining how quickly the body may recover from tissue damage. Results in study
one revealed that IS was associated with an increase in hsCRP, with low and moderate
stretching intensities of study two (30% and 60% mROM, respectively) suggesting that
at these intensities there was no increase in the concentration of hsCRP. Although an
anomaly was found at baseline for 90% mROM, values for hsCRP for 30 to 90 and 60
to 90% mROM post and at 24h post suggested an increase in the concentration of
hsCRP. This was confirmed by the primary statistical analyses RMANOVA and effect
sizes, as well as the secondary analyses. In the literature, the rise in the concentration
of hsCRP has been related to the local release of IL-6 at the site of injury, with its main
physiological function being the induction of the APR (Cunniffe et al., 2011, Moshage,
230 | P a g e
1997). This orchestrated response to tissue damage, is characterized by the increased
expression of CRP (Akira et al., 1990, Bauer et al., 1989). It would be interesting to
observe whether stretching intensity (high or low) may influence the release and
concentration of the neutrophils and macrophages responsible for oxidative stress in
humans, since with regard to passive stretching the release of neutrophils were
observed in adult male mice (Pizza et al., 2002).
8.2.4 – Musculoskeletal Disorders and Pain
Within the clinical setting, future research could investigate the influence of both LiS and
HiS with regard to musculoskeletal disorders such as, osteo- and rheumatoid arthritis,
and low back pain (Felson, 2000). These disorders consist of conditions where part of
the musculoskeletal system is injured or affected over time, with symptoms including
pain, dysfunction and/or discomfort in the bones, joints, muscles or surrounding
structures, with this being either acute or chronic (Verhagen et al., 2012). Aetiology is
related to a variety of activities or causes such as: sport (professional, elite, or
recreational), work, fractures, contusions, degenerative (osteo-arthritis), or systemic
(rheumatoid arthritis) diseases, as well as with increased ageing (i.e. osteo- and
rheumatoid arthritis) (Verhagen et al., 2012, Felson, 2000). On average it has been
reported that musculoskeletal pain accounts for between 13.5% and 47% of the general
population (Cimmino et al., 2011). If this trend continues, it will put a huge burden on the
public health care system.
Pain and loss of function is common to musculoskeletal disorders (Cimmino et al., 2011,
Verhagen et al., 2012, Atzeni et al., 2011), with pain being an index of the severity and
activity of the underlying condition, as well as a prognostic/therapeutic indicator and
231 | P a g e
determinant of health-resources use (Cimmino et al., 2011). According to IASP pain is,
an unpleasant sensory and emotional experience associated with actual or potential
tissue damage or described in terms of such damage (Crombie et al., 1999). Presently,
exercises aimed at reducing the risk of injury and pain associated with musculoskeletal
disorders include both land (walking, resistance, stretching), and aquatic (water-based
exercises, aquatic therapy or hydrotherapy) (Wang et al., 2001, Silva et al., 2008). The
design and prescription of these exercises adhere to the parameters of training as well
as the strain factors of stretching specifically: intensity (magnitude), as well as duration
and frequency (rate) (Mujika et al., 1995, Marschall, 1999). The complaint of pain and
swelling in the joints associated with several of the musculoskeletal disorders (i.e.
osteo- and rheumatoid arthritis) may determine the intensity, duration, and frequency of
the exercise (and/or stretching) performed.
In study one, IS was associated with a potential increase in hsCRP, with study two
observing no increase of hsCRP at both low and medium stretching intensities (30 and
60% mROM). Study three, suggested that LiS compared to HiS and Control was
affiliated with faster recovery and a decrease in muscle soreness, despite the reported
inconsistencies regarding LiS compared to Control with regard to the blood biomarkers
(CK, and hsCRP). However with regard to the measure of muscle damage (CK), LiS
was likely beneficial, compared to HiS, with HiS being likely harmful compared to
Control (magnitude based inferences approach). Since musculoskeletal disorder is
associated with pain, it would be interesting to investigate whether LiS would provide
firstly a relief from pain and in turn aide in the recovery of the tissue from the muscle
damage associated with musculoskeletal disorder.
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8.2.5 – Recovery and Regeneration
Recovery and regeneration refers to the procedure(s) followed by the body to restore
homeostasis and adapt its functions and system at a higher level (Koutedakis et al.,
2006). Training and muscle damage due to training disrupts this homeostasis, a major
response in professional and recreational sports (Li et al., 2001). Damage due to
training may affect the musculoskeletal system either directly (muscle lacerations and
contusions), or indirectly (ischemia, excessive stress or strain and neurological
dysfunction) (Kasemkijwattana et al., 1998, Garrett et al., 1987). Muscle damage due to
training causes a destruction in the plasmalemma of the tissue (Jarvinen and Lehto,
1993, Kalimo et al., 1997), activating an extracellular-regulated protein kinase cascade
initiating local inflammation (Aronson et al., 1998, Li et al., 2001). Maximizing athletic
performance is the balancing of the optimal amount of physical training with proper
recovery periods, allowing for the greatest adaptation from competition and training
(Gamble, 2006, Coutts and Aoki, 2009). To achieve this, coaches need to develop
physical and sport specific conditioning programs aimed at improving the performance
of the athlete, while minimizing damage and injury by avoiding the consequences of
negative overtraining (maladaptation) (Kentta and Hassmen, 1998, Berdej-del-Fresno
and Laupheimer, 2014, Apostolopoulos, 2004, Apostolopoulos, 2010). According to
Kentta et al., adaptive processes involve physiological, psychological, biochemical and
immunological symptoms which must be considered, independently and together, in
order to fully understand the processes of adaptation to load (Kentta and Hassmen,
1998). If the training load has an adverse effect on the muscle and connective tissue,
the athlete is continually predisposed to damage, disrupting the integrity of the tissue.
233 | P a g e
The body’s natural response is associated with a balance between an anabolic (i.e.
growth hormone and insulin-like growth factor) and catabolic (i.e. cortisol) hormonal
response (Meckel et al., 2009), with the release of pro-inflammatory cytokines (i.e. IL-6)
(Meckel et al., 2009, Nemet et al., 2002, Nieman et al., 2001).
In sports, for an athlete to improve their performance, the timing for recovery and
regeneration is very important to facilitate the anabolic process. With study three, we
observed that LiS was affiliated with a faster recovery since the LiS group generated
greater values for muscle function (EPT and IPT), and was associated with a faster
decrease in soreness compared to both HiS and Control (confirmed by magnitude
based inferences approach). Comparing HiS to Control the magnitude based inferences
approach suggested that HiS was likely to have a harmful effect on performance for
both EPT and IPT as well as being associated with an increase or continued soreness.
Both Faulkner et al. (Faulkner et al., 1993), and Warren et al. (Warren et al., 1999),
suggest that prolonged strength loss is indirectly the most valid way to quantitatively
assess for changes in the magnitude of the injury with time. HiS compared to LiS and
Control exhibited a prolonged strength loss with mean values for HiS not reaching
baseline values for EPT as compared to LiS, with the Control group performing better
than the HiS group. With respect to IPT, LiS was observed to exceed baseline values
sooner compared to both HiS and Control (Table 5.2).
Training and exercise have been associated with muscle pain, and soreness, with pain
experienced during or immediately post exercise, and soreness related to DOMS
occurring 24 – 48h post strenuous exercise (Miles and Clarkson, 1994). Therefore,
based on the results of study three a future study comparing LiS to HiS would
234 | P a g e
investigate whether LiS is better than HiS with regard to the recovery and regeneration
of the musculoskeletal system from various bouts of training load and intensity (low,
medium, high). In otherwords, whether the magnitude and the rate of stretching
expedites the healing process of the damaged tissue, allowing for better adaptation of
the body to the training load. Parallel to this study, it would investigate whether
stretching intensity may be considered as either an anabolic or catabolic catalyst, for
according to Li et al., currently no therapy exists producing complete regeneration and
100% full functional recovery (Li et al., 2001).
8.2.6 – Relaxation Response
The stretching exercises for both LiS and HiS groups were performed prior to going to
sleep. An interesting response voiced by the LiS participants was the better night’s
sleep they received performing the exercises. Unlike the HiS group, which stretched to
the point of discomfort with slight pain (refer to Appendix P. 291 – 295), the LiS group
performed a stretch similar to a warm gentle feeling (refer to Appendix P. 286 – 290).
Although the information conveyed was anecdotal, it prompted the need to investigate
whether LiS may promote a relaxation response.
The relaxation response refers to a hypothesized integrated feedback resulting in the
generalized decrease in activity of the sympathetic nervous system (SNS) (Beary and
Benson, 1974). This response is opposite to the fight-or-flight response, both
physiologically as well as psychologically (Bhasin et al., 2013). It is a physical state of
deep rest changing physical and emotional responses to stress as well as being
associated with a decrease in muscle tone (Benson, 2000). It has been observed that
the activation of the SNS facilitates inflammation through the induction of CRP and pro235 | P a g e
inflammatory cytokine (IL-1, IL-6 and TNF-α) production (Grebe et al., 2010, Elenkov et
al., 2005). Recent evidence indicated that the involvement of CRP and pro-inflammatory
cytokines factor in the pathogenesis of major depression, metabolic syndromes and
sleep disturbances (Elenkov et al., 2005). Sleep has a restorative role (Vyazovskiy and
Harris, 2013) providing the opportunity for processes such as the synthesis of
macromolecules (Mackiewicz et al., 2007), recuperation from oxidative stress or toxins
accumulated during wakefulness (Reimund, 1994, Inoue et al., 1995), or replenishment
of energy stores such as glycogen (Scharf et al., 2008, Benington and Heller, 1995).
Therefore, it would be very interesting to investigate if a relaxation response occurs with
use of LiS, and if so, how this may influence the quality of sleep and subsequently the
recovery of the athlete or the individual from training, muscle damage, and injury.
236 | P a g e
9
CONCLUSION
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
Chapter 9 – Conclusions
In this thesis, the magnitude of stretching intensity (low, medium, and high) was
investigated to determine if it might be a stimulus for acute inflammation as well as
recovery from muscle damage. The overall results from our primary and secondary
statistical analyses may suggest that intense stretching may cause inflammation, as
assessed by the acute phase reactant CRP; it may also prolong recovery from
muscle damage. In contrast, LiS was not observed to be associated with
inflammation while at the same time, it promoted quicker recovery from muscle
damage. However, specific limitations pertaining to the methodological design of the
three individual studies of the project, do not allow definitive conclusions to be
drawn. Given that LiS may have significant beneficial practical applications in both
sport and rehabilitation, further research is required in this area.
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10
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STRETCH INTENSITY
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INFLAMMATORY
RESPONSE
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field athletes. J Strength Cond Res, 22, 13-19.
WITROUW, E., MAHIEU, N., ROOSEN, P. & MCNAIR, P. 2007. The role of stretching
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WREN, T. A. L., BEAUPRE, G. S. & CARTER, D. R. 2000. Mechanobiology of tendon
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278 | P a g e
APPENDIX
STRETCH INTENSITY
and THE
INFLAMMATORY
RESPONSE
SECTION ONE – FORMS
Appendix - Forms
QUESTIONNAIRE FOR POTENTIAL PARTICIPANTS IN PROJECTS INVOLVING BLOOD
ANALYSIS
All information given will be treated with strictest confidentiality.
Answer all questions by ‘ticking’ the appropriate box.
Yes
No Not Known
1.
Are you receiving any medicines, dental treatment, have had
recent illness or attending hospital outpatients?
2.
Have you had any body piercing, acupuncture or have been
tattooed in the last 6 months?
3.
Have you ever been advised by a doctor not to give
blood?
4.
Are you or have you ever suffered from any of the following?
Allergies (Hay fever, Asthma etc)
Anaemia or other blood disorders
Brucellosis
Cancer
Diabetes
Epilepsy (fits)
Glandular fever (in last 2 years)
Heart disease
Hepatitis (jaundice) or been in contact with a case in the last 6 months
High blood pressure (except during pregnancy)
Kidney disease
Peptic ulcers
Stroke
Thyroid disease (goitre etc)
Tropical disease especially malaria
Venereal disease
5.
Is your lifestyle likely to place you at risk of HIV infection (AIDS)?
Please advise the test supervisor if you have travelled outside Europe
within the last 6 months and/or received travel vaccinations.
DECLARATION
I have had explained to me, and fully understand, the reasons for blood analysis during exercise testing.
I have not answered ‘Yes’ to any of the questions listed and to the best of my knowledge am fully eligible to
undertake blood testing and do so of my own free will.
If you have answered 'Yes' to any of the above questions please see test supervisor.
Signature of Participant:______________________________________________Dated:_______________
I hereby declare that I have read this form in its entirety and I understand that the questions asked have been
answered to my satisfaction.
Signature of Tester:______________________________________
281 | P a g e
Appendix - Forms
PRE-TEST QUESTIONNAIRE
Name:_______________________________________________________________________________
Date of Birth:_______________________________________ Age:________________
As you are to be a participant in this laboratory, would you please answer the following questions truthfully and
completely. The purpose of this questionnaire is to ensure that you are in a fit and healthy state to complete the
exercise test(s). Your co-operation in this is greatly appreciated. *Please delete where appropriate
ANY INFORMATION CONTAINED HEREIN WILL BE TREATED AS CONFIDENTIAL.
1.
How would you describe your present level of activity?
Sedentary moderately active active highly active*
2.
How would you describe your present level of fitness?
Very unfit moderately fit trained highly trained*
3.
How would you consider your present body weight?
Underweight ideal weightslightly overweight very overweight*
4.
Smoking habits:
Currently a smoker yes/no*
A previous smoker
smoker, how long since stopping?.............years
yes/no* of...…per day
If
previous
An occasional smoker yes/no* of ...........per day
A regular smoker
yes/no* of ...........per day
5.
Consumption of alcohol:
Do you drink alcoholic drinks? yes/no* If yes then do you: have the occasional drink? yes/no*
Have a drink every day?
yes/no*
Have more than one drink a day?yes/no*
6. Have you had to consult your doctor within the last 6 months? yes/no*
If yes, please give details to the test supervisor
7 Are you presently taking any form of medication?yes/no*
If yes, please give details to the test supervisor
8.Do you suffer, or have you ever suffered, from:
Asthma? yes/no*
Diabetes?yes/no*
High blood pressure? yes/no*
Bronchitis? yes/no*
Epilepsy?yes/no*
9.Do you suffer, or have you ever suffered from, any form of heart complaint?yes/no*
10.Is there history of heart disease in your family?yes/no*
11.Do you currently have any form of muscle or joint injury?yes/no*
12.Have you had any cause to suspend your normal training in the last two weeks? yes/no*
13.
Is there anything to your knowledge that may prevent you from successfully completing the tests that have
been outlined to you? yes/no*
I have read, understood and completed this questionnaire. Any questions I had were answered to my full
satisfaction.
Signature of Participant:______________________________________________ Date:_______________________
I hereby declare that I have read this form in its entirety and I understand that the questions asked have been
answered to my satisfaction.
Signature of Tester:________________________________________________________
282 | P a g e
Appendix - Forms
School of Sport, Performing Arts and Leisure
Primary researcher: Nikos Apostolopoulos BPHE Sports Medicine
Study: Acute Inflammatory Response to Stretching
Background: The critical issue with all activities is how strong (intensity), how long (duration) and how
often (frequency) should they be administered in order to notice a response. Static stretching is a form of
activity of great interest in both clinical and performance environments. Numerous studies have referred
to duration and frequency with few focused on intensity. In some studies static stretching has been shown
to induce significant increases in delayed onset muscle soreness (DOMS) and creatine kinase (CK)
amongst subjects unaccustomed to such exercise. Research has failed to substantiate that the soreness
associated with DOMS may be an inflammatory response even though the results and events associated
with DOMS suggest otherwise.
Purpose: The aim of this pilot study is to indicate whether intense static stretching can cause an acute
inflammatory response.
Methods: You will be randomly assigned to a Control or stretch session. During the Control session you
will be asked to relax for approximately 18min, however, during the stretch session your lower body
(hamstrings, glutes and quadriceps) will be subjected to an intense stretch for approximately 18min (6min
per side). Three static stretches will be performed in sequence, held for approximately 60s, and repeated
3 times per side. Blood will be collected from one of your forearms prior to, immediately post as well as
24h post Control and stretch session.
Confidentiality: All data will be strictly confidential and in line with the code of conduct of the British
Association of Sport and Exercise Sciences. Your data will be stored in a locked cabinet at the University
of Wolverhampton and eventually destroyed. All data will be recorded without names; a code will be
created to record the scores. The only people with access to the data will be the researchers.
You are free to withdraw from participating in this research and withdraw use of your data at any time
without any negative pressure or consequences.
Please place a cross in the box to confirm that:
1.
You have read and understand the information sheet for the above study and have had
opportunity to ask questions
2.
I understand that my participation is voluntary and that I am free to withdraw at any time,
without giving any reason.
3.
You have been free from any major injury during the past 3 months
Name of Participant:
Signature of Participant:
Date:
2013
Signature of Tester:
If you require further information, please contact: Nikos Apostolopoulos (University of Wolverhampton)
Telephone: 01902 51 5168 (9am-5pm), or email [email protected]
283 | P a g e
Appendix - Forms
Primary researcher: Nikos Apostolopoulos BPHE-Sports Medicine
Study:Stretch Intensity vs. Inflammation – A Dependent Association?
Background: A critical issue with all activities is at what physical force are adaptive or maladaptive benefits
manifested. The development of force in this case is in relation to the parameters of training that in and of themselves
may define it, specifically; intensity, duration, and frequency. Various combinations of all three may determine an
increase or decrease in performance and or quality of life. Of these parameters intensity potentially plays the most
important role however continues to remain the hardest to quantify, since it is perceptually determined. Belonging to
the causative factors of training, intensity possibly determines the quality of physical movement. For instance an
intense activity may place a great amount of stress on the body affecting proper movement and quality of life.
Overtraining, a result of improper stresses, predisposes the athlete to greater fatigue thereby increasing risk of injury.
Though improper prescription of intensity load may be maladaptive, the proper recommendation may aide in the
recovery of the body. Understanding this load is imperative for the recovery and regeneration of the musculoskeletal
tissue. Knowing what level of intensity activates the inflammatory system may provide the coach and medical
practitioner with an important and fundamental tool for the appropriate development of the athlete or increase an
individual's quality of life.
Purpose: The aim of this study is to determine at what percentage of a maximum perceived end range of motion
(max-ROM) determined by a straight leg raise test is the inflammatory system stimulated.
Methods: You will randomly be assigned to one of three stretch intensities (30%, 60%, or 90%) based on a
percentage of your max-ROM. For instance, if the max-ROM for your hamstring is 100 degrees (measured during a
straight knee leg raise test) you will be assigned to either a 30, 60 or 90 degree angle, respectively. You will be
subjected to five straight knee leg raises, each being held for 60s followed by a 10s rest in between. In turn, blood will
be collected from one of your forearms prior to, immediately post, as well as 24h post stretch session. The test will be
conducted once per week over three consecutive weeks ensuring that you have completed all three stretch
intensities.
Confidentiality: All data will be strictly confidential and in line with the code of conduct of the British Association of
Sport and Exercise Sciences. Your data will be stored in a locked cabinet at the University of Wolverhampton and
eventually destroyed. All data will be recorded without names; a code will be created to record the scores. The only
people with access to the data will be the researchers.
You are free to withdraw from participating in this research and withdraw use of your data at any time without any
negative pressure or consequences.
1.
You have read and understand the information sheet for the above study and have had opportunity to
ask questions
2.
I understand that my participation is voluntary and that I am free to withdraw at any time, without
giving any reason.
3.
You have been free from any major injury during the past 3 months.
Date:
2014
If you require further information, please contact: Nikos Apostolopoulos (University of Wolverhampton) Telephone:
01902 51 5168 (9am-5pm), or email [email protected]
284 | P a g e
Appendix - Forms
Primary researcher: Nikos Apostolopoulos BPHE-Sports Medicine
Study: The Effect of Different Stretch Intensities on Recovery from Muscle Damage
Background: Eccentric exercise can cause delayed onset muscle soreness (DOMS), a familiar experience for both
elite and novice athletes with symptoms ranging from muscle tenderness to severe pain. DOMS can effect muscle
force generation, as well as cause soreness and stiffness of the muscle tendon unit. A critical issue with DOMS has
been the ability to recover quickly for treatment strategies continue to remain uncertain. What is certain is that DOMS
is influenced by the intensity and duration of the exercise determining the severity of its onset. In turn, intensity,
duration, and frequency of the treatment and the recovery protocol may prove to be just as important. However,
intensity may be the most influential regards proper prescription for treatment. Improper intensity load may create
pain and possibly injury due to the magnitude of force imparted on the muscle-tendon unit. Understanding the
prescription of intensity load during treatment may provide clues and recommendations aiding in the recovery of the
soft and connective tissues. Proper intensity load may influence the inflammatory system decreasing its adverse
effects thereby increasing the performance of the athlete as well as increasing an individual's quality of life.
Purpose: The aim of this study is to determine whether the intensity of low intensity stretching vs. high intensity
stretching is more beneficial for the recovery of the soft and connective tissue following an eccentric exercise.
Methods: You will randomly be assigned to one of three groups; low intense stretching, high intense stretching,
and Control. Your right quadriceps will be subjected to an eccentric load with use of an isokinetic dynamometer. You
will perform 6 sets of 10 repetitions with a rest period of 2min in between each set. It is IMPORTANT that you
provide a maximum effort and resistance to the eccentric load. Prior to the test your height, age, and weight will be
recorded, and you will perform a peak eccentric and isometric effort test. In turn, blood will be extracted from one of
your forearms and the circumference of your right quadriceps will be measured pre and post exercise. After the test
those in the microstretching or active stretching groups will be shown the stretching exercises to be performed once
a day for four consecutive days. Total time for the stretch routine is approximately 18min. A booklet with the
stretching exercises and a daily diary will be provided to record your daily activity. Peak eccentric and isometric
measurements, blood collection, and quadriceps circumference measurements will be conducted 24, 48, and 72h
post eccentric load. In addition, a soreness assessment will be carried out in the form of point tenderness to touch
and with use of a numerical rating scale immediately post as well as 24, 48 and 72h after eccentric load.
Confidentiality: All data will be strictly confidential and in line with the code of conduct of the British Association of
Sport and Exercise Sciences. Your data will be stored in a locked cabinet at the University of Wolverhampton and
eventually destroyed. All data will be recorded without names; a code will be created to record the scores. The only
people with access to the data will be the researchers.
You are free to withdraw from participating in this research and withdraw use of your data at any time without any
negative pressure or consequences.
1.
You have read and understand the information sheet for the above study and have had opportunity to
ask questions
2.
I understand that my participation is voluntary and that I am free to withdraw at any time, without
giving any reason.
3.
You have been free from any major injury during the past 3 months.
Date:
2014
If you require further information, please contact: Nikos Apostolopoulos (University of Wolverhampton) Telephone:
01902 51 5168 (9 am - 5 pm), mobile: 07568 377 331 or email [email protected]
285 | P a g e
Appendix - Forms
STRETCHING EXERCISES FOR STUDY 3
LOW INTENSITY STRETCHING (LiS)
Appendix - Forms
EXERCISE #1 - HAMSTRING
Muscle(s) Being
Stretched
HAMSTRING MUSCLES
Description of how to perform LOW INTENSE
STRETCHING EXERCISE:
Place the left leg on the bench as shown in the
diagram with a pillow underneath the left knee. With
the right leg placed perpendicular to the bench, move
the upper body forward from the hip towards the left
knee with hands on either side. Hold the GENTLE
stretch for a FULL 60s and then switch sides.
REPEAT each stretch 3 times per side, for a total of
6min.
[Note: To lessen the intensity of the stretch you could
move the upper body backward, place a higher pillow
underneath the knee, or move the leg nearest the floor
slightly forward].
Variables of Intense Stretching Exercise:
INTENSITY:
30 – 40 % of a maximum perceived stretch [WARM GENTLE
FEELING]
DURATION:
60s
FREQUENCY: 3 times per muscle group once per day
Diagram:
287 | P a g e
Appendix - Forms
EXERCISE #2 - HIP FLEXOR
Muscle(s) Being
Stretched
HIP FLEXOR MUSCLES
Start with both knees on the ground making sure that
your hips and shoulders are square and that your
knees are shoulder width apart. Place a chair on the
side of the hip flexor that is going to be stretched. In
the diagram, this will be the right side. Place your right
hand on the chair. While supporting yourself with your
right hand, extend your left leg away from your body.
Make sure your lower back and upper body are
straight and you are not leaning forward. From this
position, lower your body to the ground making sure
that your left leg forms a 90-degree angle at your
knee. You should begin to feel a GENTLE stretch in
the right upper thigh hip region. Hold the stretch for a
FULL 60s and then switch legs. REPEAT each stretch
3 times per side, for a total of 6min.
INTENSITY:
DURATION:
FREQUENCY:
FEELING]
60s
3 times per muscle group once per day
Diagram:
288 | P a g e
Appendix - Forms
EXERCISE #3 - QUADRICEPS
Muscle(s) Being
Stretched
QUADRICEPS MUSCLE
Start on your left side with your body in the position as
shown in the diagram below. Make sure that your right
upper leg is resting on a pillow. Grab your right foot
with your right hand and begin to pull the lower leg
towards your right hamstring until you begin to feel a
GENTLE stretch in the right quadriceps. Make sure
that your right hip is positioned over the left hip during
the stretch insuring a proper quadriceps stretch. Hold
the stretch for a FULL 60s. Switch sides and repeat
the stretch for the left quadriceps. REPEAT each
stretch 3 times per side, for a total of 6min.
INTENSITY:
DURATION:
FREQUENCY:
FEELING]
60s
Diagram:
289 | P a g e
Appendix - Forms
SORENESS QUESTIONNAIRE:
Could you please CIRCLE THE NUMBER BELOW that best describes your
SORENESS LEVEL each day after the eccentric exercise? To determine this you need
to apply pressure on middle of the muscle belly of your quadriceps muscle with your
fingers.
POST ECCENTRIC EXERCISE
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY ONE (24h post eccentric exercise)
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY TWO (48h post eccentric exercise)
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY THREE (72h post eccentric exercise)
1
NO
SORENESS
2
3
4
5
SORE
6
7
8
9
10
VERYVERY
SORE
290 | P a g e
Appendix - Forms
STRETCHING EXERCISES FOR STUDY 3
HIGH INTENSITY STRETCHING (HiS)
Appendix - Forms
EXERCISE #1 - HAMSTRING
Muscle(s) Being
Stretched
HAMSTRING MUSCLES
Description of how to perform HIGH INTENSE
Place the left leg on the bench as shown in the
diagram with a pillow underneath the left knee. With
the right leg placed perpendicular to the bench, move
the upper body forward from the hip towards the left
knee with hands on either side. Hold the INTENSE
stretch for a FULL 60s and then switch sides.
6min.
[Note: To lessen the intensity of the stretch you could
move the upper body backward, place a higher pillow
underneath the knee, or move the leg nearest the floor
slightly forward].
INTENSITY:
70 – 80 % of a maximum perceived stretch [DISCOMFORT WITH
SLIGHT PAIN]
DURATION:
60s
FREQUENCY: 3 times per muscle group once per day
Diagram:
292 | P a g e
Appendix - Forms
EXERCISE #2 - HIP FLEXOR
Muscle(s) Being
Stretched
Description of how to perform HIGH INTENSE
HIP FLEXOR MUSCLES
Start with both knees on the ground making sure that
your hips and shoulders are square and that your knees
are shoulder width apart. Place a chair on the side of the
hip flexor that is going to be stretched. In the diagram,
this will be the right side. Place your right hand on the
chair. While supporting yourself with your right hand,
extend your left leg away from your body. Make sure
your lower back and upper body are straight and you are
not leaning forward. From this position, lower your body
to the ground making sure that your left leg forms a 90degree angle at your knee. You should begin to feel an
INTENSE stretch in the right upper thigh hip region.
Hold the stretch for a FULL 60s and then switch legs.
6min.
INTENSITY:
DURATION:
FREQUENCY:
SLIGHT PAIN]
60s
Diagram:
293 | P a g e
Appendix - Forms
EXERCISE #3 - QUADRICEPS
Muscle(s) Being
Stretched
QUADRICEPS MUSCLE
Start on your left side with your body in the position as
shown in the diagram below. Make sure that your right
upper leg is resting on a pillow. Grab your right foot
with your right hand and begin to pull the lower leg
towards your right hamstring until you begin to feel an
INTENSE stretch in the right quadriceps. Make sure
that your right hip is positioned over the left hip during
the stretch insuring a proper quadriceps stretch. Hold
the stretch for a FULL 60s. Switch sides and repeat the
stretch for the left quadriceps. REPEAT each stretch 3
times per side, for a total of 6min.
INTENSITY:
DURATION:
FREQUENCY:
SLIGHT PAIN]
60s
Diagram:
294 | P a g e
Appendix - Forms
SORENESS QUESTIONNAIRE:
Could you please CIRCLE THE NUMBER BELOW that best describes your
SORENESS LEVEL each day after the eccentric exercise? To determine this you need
to apply pressure on middle of the muscle belly of your quadriceps muscle with your
fingers.
POST ECCENTRIC EXERCISE
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY ONE (24h post eccentric exercise)
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY TWO (48h post eccentric exercise)
1
2
3
4
NO
SORENESS
5
6
7
8
9
SORE
10
VERYVERY
SORE
DAY THREE (72h post eccentric exercise)
1
NO
SORENESS
2
3
4
5
SORE
6
7
8
9
10
VERYVERY
SORE
295 | P a g e
Appendix - Forms
RELEASE OF IMAGE
I the undersigned _Alexander Dallaway_ hereby authorize Nikos Apostolopoulos to publish a
photograph taken of me on November 11, 2013, in the Biomechanics Laboratory of
Wolverhampton University for use in the thesis, Stretch Intensity and the Inflammatory
Response.
I hereby release and hold harmless Nikos Apostolopoulos from any reasonable expectation of
privacy or confidentiality associated with the image specified above.
I further acknowledge that my participation was voluntary.
I hereby release Nikos Apostolopoulos, and any third parties involved in the creation or
publication of this image, from liability for any claims by me or any third party in connection with
my participation.
Authorization
Printed Name:___Alexander Dallaway___
Signature:__
_
Date:___14/12/15____
296 | P a g e
Appendix - Forms
297 | P a g e
Appendix - Forms
SECTION TWO – SAMPLES OF RAW DATA FOR
STUDIES
Appendix – Raw Data For Studies
STUDY ONE
TABLE A: Age, Mass, Height, and hsCRP
PARTICIPANTS
1
2
3
4
5
6
7
8
9
10
11
12
AGE
(yrs)
MASS
(kg)
HGT
(m)
33
24
35
29
25
23
31
33
34
25
28
25
92
83
76
86
95
79
68
71
83
78
69
72
1.78
1.83
1.71
1.86
1.85
1.70
1.70
1.72
1.73
1.77
1.73
1.73
hsCRP – Control (mg/L)
hsCRP – Intense Stretching (mg/L)
Pre
Immediately
post
24h post
Pre
Immediately
Post
24h post
0.92
0.71
1.13
0.78
̶
0.74
1.80
0.86
0.63
1.22
1.03
0.33
0.95
̶
1.19
0.83
4.64
0.81
̶
0.69
1.30
0.70
0.64
0.76
0.92
0.71
1.13
0.78
̶
0.74
1.80
0.86
0.63
1.22
1.03
0.33
1.53
2.17
0.48
0.78
1.03
0.57
4.51
1.38
0.99
1.17
0.82
0.36
2.67
2.20
0.69
1.25
1.14
0.74
7.22
1.41
0.99
1.42
0.94
1.40
4.52
̶
7.08
1.83
1.19
0.81
10.00
1.57
1.34
1.79
5.73
3.20
299 | P a g e
STUDY TWO:
TABLE B: Age, Mass, Height, and hsCRP values for 30, 60 and 90% maximum ROM
Participant
Age
(yrs)
Mass
(kg)
Hgt
(m)
1
22
99
2
23
3
30% mROM
60% mROM
90% mROM
Pre
Post
24h post
Pre
Post
24h post
Pre
Post
24h post
1.91
0.97
0.91
0.53
1.01
0.98
0.95
1.55
1.67
3.83
77
1.70
0.68
0.71
0.58
0.47
0.44
0.64
1.41
1.29
0.71
27
91
1.79
0.26
0.27
0.42
1.23
1.24
0.83
1.51
1.44
1.20
4
20
89
1.83
1.09
0.97
1.08
1.07
1.04
0.80
2.99
3.34
3.89
5
32
94
1.80
0.80
0.75
0.45
1.10
1.14
1.20
0.49
0.56
0.96
6
25
88
1.83
0.28
0.27
0.57
0.20
0.22
0.29
4.54
4.90
2.43
7
41
72
1.75
0.31
0.28
0.39
0.69
0.68
0.61
0.31
0.33
0.38
8
21
87
1.78
0.59
0.64
0.45
0.67
0.71
1.36
3.14
3.57
2.62
9
25
87
1.76
1.61
1.70
1.84
1.62
1.61
1.32
5.81
5.87
9.90
10
20
82
1.76
0.25
0.28
0.24
0.52
0.47
0.46
0.28
0.28
0.42
11
25
73
1.73
0.36
0.41
0.38
0.37
0.37
0.50
0.51
0.47
0.54
300 | P a g e
STUDY THREE:
TABLE C: Eccentric Peak Torque (EPT) and Isometric Peak Torque (IPT) and Soreness
Values
Conditions
n
EPT(Nm)
IPT(Nm)
Soreness Level
0h
24h
48h
72h
0h
24h
48h
72h
0h
24h
48h
72h
HiS
1
2
3
4
5
6
7
8
9
10
208
194
303
321
186
173
270
189
136
202
168
147
188
143
160
173
257
204
150
144
223
168
256
250
178
216
238
256
130
165
183
186
188
200
178
204
269
226
163
162
178
144
222
230
191
151
229
208
113
147
147
114
194
202
161
154
204
228
117
114
146
130
212
216
164
161
221
254
112
111
172
155
221
231
166
180
243
220
131
147
1
7
6
6
6
6.5
6
7
7
7
1
8
6
6
6
7
7
5
8
3
1
4
3
4
3
3
3
5
6
5
1
2
3
2
2
2
1
2
4
5
LiS
1
2
3
4
5
6
7
8
9
10
316
279
182
149
192
314
211
320
237
275
310
217
176
169
170
323
178
307
197
249
326
222
179
176
221
316
210
282
274
236
332
273
176
176
243
360
221
317
270
263
272
154
187
159
187
264
198
238
201
216
268
162
177
128
162
260
180
242
182
203
275
177
207
133
194
296
203
212
218
180
275
186
191
163
207
320
211
255
225
190
7
8
7
5
5
5
8
4
6
5
6
6
6
4
4
5
5
2
6
6
2
3
3
3
4
1
2
2
4
5
1
1
2
2
1
1
1
1
1
1
Control
1
2
3
4
5
6
7
8
9
10
336
207
255
193
216
172
173
195
244
157
288
196
232
175
234
140
140
216
207
134
258
197
176
167
182
174
143
183
218
96
328
214
260
138
216
160
126
212
231
121
314
153
219
172
228
160
128
149
180
148
241
167
214
158
222
144
105
149
165
86
242
198
223
158
209
137
100
155
183
91
252
201
247
138
202
143
94
169
185
97
3
8
6
4
7
7
4
8
7
5
3
8
3
4
8
7
6
7
6
5
3
7
3
3
3
5
3
5
4
4
3
2
1
1
2
3
1
5
1
3
301 | P a g e
GRAPHS FOR PARALLEL GROUPS TRIAL (WILL HOPKINS MATERIAL)
1.0 – Creatine Kinase (CK) (U/L)
Control
HiS
1200
1000
CK (U/L)
800
600
400
200
0
-200
0
24
Time (h)
48
72
Figure 1 – High Intensity Stretching (HiS) vs. Control
302 | P a g e
Control
LiS
1200
1000
CK (U/L)
800
600
400
200
0
0
24
48
72
Time (h)
Figure2: Low intensity stretching (LiS) vs. Control
303 | P a g e
HiS
LiS
1200
1000
CK (U/L)
800
600
400
200
0
-200
0
48
24
72
Time (h)
Figure 3 – LiS vs. HiS
304 | P a g e
2.0 – high sensitivity C – reactive protein (hsCRP) (mg/L)
Control
HiS
7
6
hsCRP (mg/L)
5
4
3
2
1
0
-1
-2
0
24
48
72
Time (h)
Figure 1 – HiS vs. Control
305 | P a g e
Control
LiS
5
hsCRP (mg/L)
4
3
2
1
0
-1
0
48
24
72
Time (h)
Figure 2 – LiS vs. Control
306 | P a g e
HiS
LiS
7
6
hsCRP (mg/L)
5
4
3
2
1
0
-1
-2
0
48
24
72
Time (h)
307 | P a g e
3.0 – Eccentric Peak Torque (EPT) (Nm)
Control
HiS
300
250
EPT (Nm)
200
150
100
50
0
0
24
48
72
Time (h)
308 | P a g e
Control
LiS
350
300
EPT (Nm)
250
200
150
100
50
0
0
24
48
72
Time (h)
309 | P a g e
HiS
LiS
350
300
EPT (Nm)
250
200
150
100
50
0
0
24
48
72
Time (h)
310 | P a g e
4.0 – Isomteric Peak Torque (IPT) (Nm)
CONTROL
HiS
300
250
IPT (Nm)
200
150
100
50
0
0
24
48
72
Time (h)
311 | P a g e
CONTROL
LiS
300
250
IPT (Nm)
200
150
100
50
0
0
24
48
72
Time (h)
312 | P a g e
HiS
LiS
300
250
IPT (Nm)
200
150
100
50
0
0
24
48
72
Time (h)
313 | P a g e
5.0 – SORENESS LEVEL
Control
HiS
9
8
Soreness Level
7
6
5
4
3
2
1
0
Post Muscle
Damage
Protocol (MDP)
48
24
72
Time (h)
314 | P a g e
Control
LiS
9
8
Soreness Levels
7
6
5
4
3
2
1
0
Post MDP
48
24
72
Time (h)
315 | P a g e
HIS
LiS
9
8
Soreness Levels
7
6
5
4
3
2
1
0
Post MDP
24
48
72
Time (h)
316 | P a g e
SECTION THREE – SYSTEMATIC REVIEW
Appendix – Systematic Review
318 | P a g e
319 | P a g e
320 | P a g e
321 | P a g e
322 | P a g e
323 | P a g e
324 | P a g e
325 | P a g e
326 | P a g e
327 | P a g e
328 | P a g e
329 | P a g e
330 | P a g e
331 | P a g e
332 | P a g e
333 | P a g e
334 | P a g e
335 | P a g e
336 | P a g e
337 | P a g e
338 | P a g e
339 | P a g e
340 | P a g e
341 | P a g e
342 | P a g e

Apostolopoulos_PhD t... - Wolverhampton Intellectual Repository

Transcription

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