MONITORING OF SKELETAL MUSCLE ISCHEMIA USING ...

MONITORING OF SKELETAL MUSCLE ISCHEMIA USING NEAR INFRARED SPECTROSCOPY

by

Babak Shadgan

M.D., Azad University of Tehran 1993 M.Sc., The University of London, 2001

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

(Experimental Medicine)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

March 2011

© Babak Shadgan, 2011

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ABSTRACT

Early diagnosis of acute limb muscle ischemia (LMI) is essential in order to avoid

serious, irreversible local and systemic complications resulting in loss of the limb or even

death. To date, techniques for monitoring LMI are limited by lack of a feasible and

reliable monitoring method.

Purpose:

The main purposes of this thesis were to examine the feasibility and convergent validity

of conventional and wireless near infrared spectroscopy (NIRS) for continuous

monitoring of skeletal muscle oxygenation and hemodynamics during transient and long-

term LMI and to investigate the predictive value of NIRS-derived data for evaluation of

limb muscle oxidative changes during tourniquet-induced LMI.

Methods:

Following a complete literature review (Chapter 2), forearm muscle oxygenation and

hemodynamics were studied in 10 healthy subjects using wireless NIRS instrumentation

during isometric muscle contraction and tourniquet-induced LMI (Chapter 3). In Chapter

4, changes in NIRS-derived leg muscle oxygenation and hemodynamics, in conjunction

with muscle oxidative changes, following tourniquet-induced LMI were investigated in

17 patients undergoing surgery for ankle fracture. In Chapter 5, the effect of

electromagnetic interference (EMI) from 3 commonly used surgical instruments on NIRS

signals were investigated using a mathematical method of signal analysis.

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Results:

Chapter 2: No validated monitoring method for early detection of acute LMI was

revealed. Chapters 3-4: Wireless and conventional NIRS data were consistent with

muscle ischemia and reperfusion. Chapter 4: An average of 43.2±14.6 minutes of

tourniquet-induced ischemia led to a 172.3±145.7% (range: 10.7-363.3%) increase in

muscle protein oxidation (P<0.0005). Changes in NIRS-derived muscle oxygenated and

total-hemoglobin were both negatively correlated, while reoxygenation-rate was

positively correlated (P<0.05) to muscle protein oxidation. Chapter 5: EMI from 3 OR

instruments was found to have no effect on NIRS signals (P<0.01).

Conclusions:

NIRS is a feasible method for continuous monitoring of limb muscle oxygenation and

hemodynamics during transient and long-term tourniquet-induced ischemia. Tourniquet-

induced LMI of 21-74 minutes leads to oxidative muscle damage. A significant negative

association between the extent of tourniquet-induced oxidative damage and changes in

NIRS-derived local muscle oxygenated blood volume was found. EMI of commonly used

orthopaedic surgical instruments does not affect NIRS signals.

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PREFACE

This thesis provides an introductory chapter, followed by a published literature

review and three studies. Chapter 1 provides an introduction to, and rationale for, the

studies included in this thesis. Chapter 2 is a published review of the literature that

describes acute compartment syndrome and diagnostic methods thereof. This chapter has

been published in a peer-reviewed journal:

Shadgan B, Menon M, O'Brien PJ and Reid WD. Diagnostic techniques in acute

compartment syndrome of the leg. Journal of Orthopaedic Trauma. 22(8):581-587, 2008.

Chapters 3, 4 and 5 are original studies that report on the feasibility and

convergent validity of NIRS for monitoring of limb muscle oxygenation and

hemodynamics, and diagnosis of LMI in clinical settings. Chapters 3 and 5 have been

published in peer-reviewed journals as:

Shadgan B, Reid WD, Gharakhanlou R, Stothers L and Macnab A. Wireless near-

infrared spectroscopy of skeletal muscle oxygenation and hemodynamics during exercise

and ischemia. Spectroscopy. 23(5):233-241, 2009.

Shadgan B, Molavi B, Reid WD, Dumont G, Macnab AJ. Do radio frequencies of medical

instruments common in the operating room interfere with near-infrared spectroscopy

signals? Proceeding. SPIE, Vol. 7555, 755512;doi:10.1117/12.842712, 2010.

Chapter 4 has been submitted for publication in a peer-reviwed journal as:

Shadgan B, Harris RL, Reid WD, Jafari S, Powers SK, O’Brien PJ. Monitoring of

tourniquet-induced skeletal muscle injury by near infrared spectroscopy during

orthopaedic trauma surgery.

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I was the primary author of all manuscripts and responsible for study design, data

collection, performance, data analysis and manuscript preparation of all chapters.

I would like to acknowledge that Dr. Luke Harris assisted me with muscle biopsy

preparation of the study in Chapter 4. Muscle biopsy analysis of the study in Chapter 4

was carried out in collaboration with Dr. Scott Powers at the University of Florida. I

would also like to acknowledge that Mr. Behnam Molavi assisted me in study design and

data analysis of the study in Chapter 5.

The studies in Chapters 4 and 5 of this thesis received institutional research ethics

approval from the University of British, Columbia Clinical Research Ethics Board (UBC

CREB number: H07-02934) and Vancouver Costal Health Authority, Clinical Trials

Administration Office (research study approval number: V08-0029). Study in Appendix

III also received institutional research ethics approval from the University of British

Columbia, Clinical Research Ethics Board (UBC CREB number: H07-011070).

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TABLE OF CONTENTS

ABSTRACT ........................................................................................................................ ii

PREFACE .......................................................................................................................... iv

TABLE OF CONTENTS ................................................................................................... vi

LIST OF TABLES ........................................................................................................... xiii

LIST OF FIGURES ......................................................................................................... xiv

LIST OF ABBREVIATIONS .......................................................................................... xvi

ACKNOWLEDGEMENTS ............................................................................................. xix

DEDICATION .................................................................................................................. xx

CHAPTER 1 – INTRODUCTION .................................................................................... 1

1.1 Introduction ........................................................................................................... 2

1.1.1 Pathophysiology of Skeletal Muscle Ischemia .............................................. 2

1.1.1.1 Definition ................................................................................................. 2

1.1.1.2 Limb Muscle Ischemia ............................................................................. 3

1.1.1.3 Ischemia-Reperfusion Injury ................................................................... 3

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1.1.2 Exercise-Induced Limb Muscle Ischemia ...................................................... 5

1.1.3 Clinical Consequences of Ischemia ............................................................... 6

1.1.3.1 Acute Compartment Syndrome ................................................................ 6

1.1.3.2 Tourniquet-Induced Muscle Ischemia ..................................................... 8

1.1.3.3 Diagnosis and Monitoring of Limb Muscle Ischemia ........................... 10

1.1.4 Near Infrared Spectroscopy ......................................................................... 12

1.1.4.1 The Science of NIRS ............................................................................. 13

1.1.4.2 Chromophores of Interest ...................................................................... 14

1.1.4.3 NIRS Instrumentation ............................................................................ 14

1.1.4.4 NIRS Variables ...................................................................................... 16

1.1.4.5 Validity of NIRS Measurements ............................................................ 18

1.1.4.6 NIRS in Clinic ........................................................................................ 20

1.1.4.7 The Advantages of NIRS in Clinical Studies ........................................ 21

1.1.4.8 The Limitations of NIRS ....................................................................... 22

1.1.4.9 Feasibility of NIRS Monitoring in the Operating Room ....................... 23

1.2 Rationale of the Thesis ........................................................................................ 25

1.3 Thesis Objectives ................................................................................................ 26

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1.4 Specific Aims ...................................................................................................... 27

1.5 Hypothesis ........................................................................................................... 29

CHAPTER 2 – DIAGNOSTIC TECHNIQUES IN ACUTE COMPARTMENT

SYNDROME OF THE LEG ............................................................................................ 31

2.1 Introduction ......................................................................................................... 32

2.2 Diagnosis ............................................................................................................. 35

2.2.1 Pressure Measurement .................................................................................. 36

2.2.2 Biomarkers .................................................................................................... 39

2.2.3 Magnetic Resonance Imaging ....................................................................... 41

2.2.4 Ultrasound ..................................................................................................... 42

2.2.5 Scintigraphy .................................................................................................. 43

2.2.6 Laser Doppler Flowmetry ............................................................................. 44

2.2.7 Near Infrared Spectroscopy .......................................................................... 44

2.2.8 Pulse Oximetry .............................................................................................. 47

2.2.9 Hardness Measurement Techniques ............................................................. 47

2.2.10 Direct Nerve Stimulation ............................................................................ 48

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2.2.11 Vibratory Sensation .................................................................................... 48

2.2.12 Tissue Ultrafiltration ................................................................................... 49

2.3 Summary ............................................................................................................. 49

CHAPTER 3 – WIRELESS NEAR INFRARED SPECTROSCOPY OF SKELETAL

MUSCLE OXYGENATION AND HEMODYNAMICS DURING EXERCISE AND

ISCHEMIA ....................................................................................................................... 51

3.1 Introduction ......................................................................................................... 52

3.2 Materials and Methods ........................................................................................ 56

3.2.1 Instrumentation ............................................................................................. 56

3.2.2 Subjects ......................................................................................................... 57

3.2.3 Experimental Protocol .................................................................................. 57

3.2.4 Data and Statistical Analysis ........................................................................ 59

3.3 Results ................................................................................................................. 60

3.4 Discussion ........................................................................................................... 64

3.5 Conclusions ......................................................................................................... 66

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CHAPTER 4 – MONITORING OF TOURNIQUET-INDUCED SKELETAL MUSCLE

INJURY BY NEAR INFRARED SPECTROSCOPY DURING ORTHOPAEDIC

TRAUMA SURGERY ...................................................................................................... 67

4.1 Introduction ......................................................................................................... 68


4.2.1 Subjects ......................................................................................................... 71

4.2.2 Experimental Overview ................................................................................ 71


4.2.4 Biopsy Collection and OxyBlot Analysis ..................................................... 75

4.2.5 Statistical Method ......................................................................................... 75

4.3 Results ................................................................................................................. 76

4.3.1 Descriptive Characteristics ........................................................................... 76

4.3.2 Tourniquet ..................................................................................................... 76

4.3.3 Cardiovascular .............................................................................................. 76


4.3.5 Muscle Biopsy .............................................................................................. 78

4.3.6 OxyBlot vs. NIRS Regression ...................................................................... 80

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4.4 Discussion ........................................................................................................... 83

4.5 Conclusions ......................................................................................................... 91

CHAPTER 5 – DO RADIO FREQUENCIES OF MEDICAL INSTRUMENTS

COMMON IN THE OPERATIVE ROOM INTERFERE WITH NEAR INFRARED

SPECTROSCOPY SIGNALS? ........................................................................................ 92

5.1 Introduction ......................................................................................................... 93


5.3 Results ................................................................................................................. 97

5.4 Discussion ........................................................................................................... 98

5.5 Conclusions ....................................................................................................... 100

CHAPTER 6 – CONCLUSIONS .................................................................................. 101

6.1 Overview ........................................................................................................... 102

6.2 Hypotheses’ Conclusions .................................................................................. 103

6.3 Significance ....................................................................................................... 107

6.4 Study Strengths and Limitations ....................................................................... 109

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6.5 Future Directions .............................................................................................. 110

6.5.1 Effect of Ischemia on Skeletal Muscle Lipid Peroxidation ........................ 110

6.5.2 Advancing the Clinical Use of NIRS .......................................................... 111

6.5.3 Early Diagnosis of ACS in High-Risk Patients Using NIRS ...................... 112

6.5.4 Monitoring of Limb Muscle Oxygenation and Hemodynamics During

Tourniquet-Induced Ischemia ............................................................................. 114

6.5.5 Developing Safer Tourniquet Systems Using NIRS ................................... 115

6.5.6 Monitoring the Effects of Ischemic Preconditioning by NIRS ................... 116

REFERENCES .............................................................................................................. 118

APPENDIX I – INFORMED CONSENT FORMS ...................................................... 149

APPENDIX II – PAPER: MOTION ARTIFACT REMOVAL FROM MUSCLE NIR

SPECTROSCOPY MEASUREMENTS ........................................................................ 166

APPENDIX III – PAPER: STERNOCLEIDOMASTOID MUSCLE OXYGENATION

AND HEMODYNAMIC RESPONSE TO INCREMENTAL INSPIRATORY

THRESHOLD LOADING MEASURED BY NEAR INFRARED SPECTROSCOPY 171

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LIST OF TABLES

Table 1.1: Current diagnostic methods of limb muscle ischemia……………………….12

Table 1.2: Overview of overall objectives and hypotheses of the studies in this thesis...30

Table 2.1: Advantages of diagnostic methods of acute compartment ………………….50

Table 3.1: Mean (±SD) physical characteristics of subjects undergoing wireless NIRS

monitoring of forearm muscle during isometric muscle contractions and tourniquet-

induced muscle ischemia……………………………………………………………...…60

Table 3.2: Mean (±SD) changes of O2Hb, HHb and tHb along with TSI% during

10, 30 and 50% of MVC and ischemia…………………………………………………..62

Table 5.1: The frequency of use of each device evaluated in each of the operative cases

studied………………………………………………………………………….………..97

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LIST OF FIGURES

Figure 1.1: Lateral fasciotomy wound of an ACS of leg………………………………....8

Figure 1.2: A NIRS set up……………………………………………...………………..15

Figure 1.3: Pattern of changes in O2Hb, HHb, tHb and Hbdiff (A), calculation of the

half-recovery time (B), calculation of the reoxygenation rate (C)……………………….18

Figure 2.1: The algorithm shows the application of ACS diagnostic method…………..36

Figure 3.1: The wireless instrument positioned over the flexor digitorum superficialis

muscle for study of muscle oxygenation and hemodynamics…………...........................58

Figure 3.2: The pattern of change in chromophore concentration (O2Hb and HHb) and

tHb and TSI% over the experimental protocol in a representative subject………………61

Figure 3.3: An example of a typical pattern of tourniquet induced muscle ischemia…...63

Figure 4.1: A summarized overview of the experimental protocol………………...…...73

Figure 4.2: NIRS operation during surgery in operation room.…….…………...…........74

Figure 4.3: Chromophore concentration changes for O2Hb and HHb, and NIRS variables

of tHb and Hbdiff, shown in a representative tibialis anterior muscle before, during and

after thigh tourniquet inflation……………..………………………………………….....78

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Figure 4.4: Raw oxidized protein volume in peroneus tertius samples at the beginning

(Pre) and end (Post) of tourniquet inflation……………………………………………...79

Table 4.5: Percent increase in peroneus tertius protein oxidation in men and women.....80

Table 4.6: Scatter plot and regression line showing the correlation between ΔO2Hb and

Pre-Post changes in muscle protein oxidation…….……………………………………..81

Table 4.7: Scatter plot and regression line showing the correlation between ΔtHb and

Pre-Post changes in muscle protein oxidation………………..………………………….82

Figure 4.8: Scatter plot and regression line showing the correlation between

reoxygenation rate and Pre-Post changes in muscle protein oxidation…..…………...….83

Figure 6.1: A wireless NIRS instrument monitors vastus lateralis muscle oxygenation

and hemodynamics during lower limb trauma surgery…………………………………112

Figure 6.2: Schematic presentation of a hypothetical NIRS set-up for monitoring the

anterior compartment of a fractured leg……………..………………………….………114

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LIST OF ABBREVIATIONS

ACS............... Acute compartment syndrome

BMI............... Body mass index

CCD............... Charge-coupled device

CCO............... Cytochrome–c-oxidase

CECS.............Chronic exertional compartment syndrome

CDF............... Cumulative distribution function

CI................... Confidence interval

CK................. Creatine phosphate

COPD.............Chronic obstructive pulmonary disease

CSA............... Cross sectional area

CT.................. Computerized tomography

CW.................Continuous wave

DPF................ Differential path length factor

EMI….……...Electromagnetic interference

ESR............... Erythrocyte sedimentation rate

FABP............. Fatty acid binding protein

FEV1.............. Forced expiratory volume during the first second

FIR................. Finite impulse response

fNIRS............. Functional near infrared spectroscopy

FVC............... Forced vital capacity

GHz................ Gigahertz

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Hb….………. Hemoglobin

Hbdiff….……. Hemoglobin difference

HHb.….……..Deoxygenated hemoglobin

IC……..……..Intercostal muscle

ICP……..…... Intracompartmental pressure

ICU….……... Intensive care unit

IEC…….……International electrotechnical commission

IMA…….….. Ischemic modified albumin

IOD…….….. Interoptode distance

IPC….………Ischemic preconditioning

IR…….…….. Ischemia reperfusion

KHz….……...Kilohertz

LDF….…….. Laser Doppler flowmetry

LED…….….. Light emitting diodes

LMI…….….. Limb muscle ischemia

Mb…….…….Myoglobin

mmHg…….... Millimeters of mercury

MRI…….….. Magnetic resonance imaging

MVC….……. Maximum voluntary contraction force

NIR….……... Near infrared

NIRI……..…. Near infrared spectroscopy imaging

NIRS.............. Near infrared spectroscopy

Nm................. Nanometers

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O2................... Oxygen

O2Hb.............. Oxygenated hemoglobin

O2Mb............. Oxygenated myoglobin

OR................. Operating room

PPLL............. Pulsed phase-locked loop

PT.................. Peroneus tertius

RFI................ Radio frequency interference

SCM............... Sternocleidomastoid muscle

SD.................. Standard deviation

SpO2.............. Pulse oximeter oxygen saturation

SPSS.............. Statistical package for the social sciences

SRS................ Spatially resolved spectroscopy

SSP.................Skin surface pressure

TA……..…… Tibialis anterior

tHb…….….... Total hemoglobin

TSI…............. Tissue saturation index

VL…………...Vastus lateralis muscle

WBC...………White blood cell

Λ.……………Wavelength

Δp…..………. Delta pressure

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ACKNOWLEDGEMENTS

I would like to extend my heartfelt gratitude to my supervisor, Dr. Darlene Reid and my supervisory committee, Dr. Bill Sheel and Dr. Peter O’Brien whom without their outstanding mentorship this endeavor would not have been possible. I am sincerely thankful to Dr. Reid, whose encouragement and guidance from the initial to the final level enabled me to develop my learning and guided me to be a confident and independent researcher. Dr. Sheel showed me the characters of a successful scientist and Dr. O’Brien reminded me that it is quite possible to be a busy clinician and an effective researcher at the same time. I could never launch the tourniquet study in the Vancouver General Hospital without his support and leadership. Besides my supervisory committee members, I was lucky to further benefit from two wonderful mentors who provided the specific research direction and inspired me greatly along the way. I could not have progressed in the field of optical medicine without directions and supports of Dr. Andrew Macnab and Dr. Lynn Stothers. I have learned a lot from Dr. Macnab, not only about near infrared spectroscopy in which he is definitely a legendary figure, but how to be an innovative clinical researcher. I should also express my sincere appreciation to Dr. Vincent Duronio, for his diligent direction and constant supports at the UBC Experimental Medicine Program. I would like to thank my colleagues at the Muscle Biophysics Laboratory of Vancouver Costal Health Research Institute; Marc Roig, Luke Harris, Bahareh H.Ghanbari, Jennifer Rurak, Jenny Ying, Cristiane Yamabayashi and Ada Woo for their helps, contributions to my projects as well as their willingness and friendship. All volunteers and patients who participated in our clinical experiments deserve a sincere appreciation for their generosity and courage towards developing research for the good of future generation. I wish to acknowledge the British Columbia Lung Association, Micheal Smith Foundation for Health Research, Canadian Orthopaedic Foundation and Trauma Division of UBC Department of Orthopaedic Surgery for their academic supports and financial assistance during my study. I would like to end the acknowledgement by confirming this famous quotation: “The more I learn, the more I realize how little I know and how much more there is to learn”.

xx

DEDICATIONS

This thesis is dedicated to my wife, Shaya for standing by me through my ups and downs from twenty-two years ago, to my daughters, Armita and Atrina who have always borne all my absence from many occasions with a smile, and to my mother, father and brother for their endless love and support throughout my life.

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CHAPTER 1

Introduction

2

1.1 Introduction

1.1.1 Pathophysiology of Skeletal Muscle Ischemia

1.1.1.1 Definition

The term “ischemia” is derived from the Greek words of isch (restriction) and

hema (blood), meaning “restriction of blood”. In medicine, ischemia describes a critical

condition of “local and temporary deficiency of blood supply to tissue, chiefly due to

constriction of blood vessels” (Kent, 2007). If left untreated, the resultant lack of oxygen

and nutrients leads to tissue damage and ultimately the death of all cells downstream of

the blockage (Eliason & Wakefield, 2009). Ischemia can affect any number of regions

within the human body from the brain to the lower extremities. This thesis will focus on

ischemia of the limb muscles.

Limb muscle ischemia (LMI) occurs frequently as a result of trauma, a host of

diseases, and surgical procedures such as tourniquet applications (Blaisdell, 2002). In all

of these conditions, risk of a partial or complete vascular obstruction exists due to: a) a

vascular rupture, b) an intravascular obstruction by thrombosis or emboli, or c) a vascular

obstruction due to interstitial or external pressure on the vasculature. High intensity of

voluntary isometric muscle contraction can also induce a transient LMI, which can impair

muscle function if sustained for some minutes but does not result in significant muscle

changes.

3

1.1.1.2 Limb Muscle Ischemia

Reduction of blood flow servicing a muscle leads to a number of detrimental

consequences within the affected cells. The usual aerobic metabolism within cells will

switch over to anaerobic metabolism and if prolonged, leads to production and

intracellular accumulation of lactate, hydrogen ions and free oxygen radicals, depletion of

cellular ATP, leakage of extracellular calcium into muscle cells and release of

intracellular potassium and phosphate into the extracellular space (Knochel, 1993;

Eliason & Wakefield, 2009; Walker, 2009). Muscle damage resulting from these changes

range from mild and reversible to severe, irreversible cellular injury and death. Ischemic

duration and the muscle region affected are the primary determinants of the severity of

muscle ischemic injury and the permanent sequelae.

Salvage of ischemic muscles from irreversible damage and necrosis depends

wholly on timely restoration of blood flow, however, reperfusion following prolonged

muscle ischemia, particularly during the first minutes thereof, risks further increasing

muscle damage through the inflammatory response associated with oxidative stress

(Griosotto et al., 2000; Blasidell, 2002). This secondary damage, as discussed in the next

section, is referred to as ischemia-reperfusion injury (IR-injury).

1.1.1.3 Ischemia-Reperfusion Injury

In 1944, Bollman and Folk reported that periods of limb ischemia greater than 3

hours resulted in systemic “fatal shock” upon reperfusion (Bollman & Flock, 1944). In

4

other words, it has been known for over half a century that the metabolic state of

ischemic muscle is linked not only to the duration of ischemia, but also to the recovery of

ischemic muscles upon reperfusion. Today, it is well documented that the hazardous

effects of IR-injury on muscle are due to a complex cascade of cellular events (Rubin et

al., 1996) including activation of leucocytes, up-regulation of inflammatory cytokines

(Boros & Bromberg, 2006), activation of the complement cascade (Pemberton et al.,

1993), activation of the antithrombin-III and protein-C pathways (Christodoulou et al.,

2004), activation of calpain proteases (Koh & Tidball, 2000) and formation of reactive

oxygen species (Li & Jackson, 2002). These metabolic consequences ultimately result in

tissue cell membrane damage and rupture of the capillary endothelia, leading to local and

systemic tissue damage (Appell et al. 1997; Eliason & Wakefield, 2009; Mathru et al.,

2007). Much like the initial ischemic insult, the consequences of reperfusion are

proportional to the duration of the ischemia and range from reversible mild cellular

changes to skeletal muscle dysfunction and force loss. Further, severe cases can lead to

irreversible local muscle damage and ultimately, systemic inflammation and multi-organ

dysfunction, which are often life-threatening conditions.

Of particular interest for the purpose of this thesis, we examined two conditions of

blood flow limitation: 1) transient LMI induced by isometric contractions and 2) longer

term LMI induced by tourniquet applied during ankle surgery. Using these two models,

the induction of ischemia can be more carefully controlled. The most serious ischemic

condition in orthopedic trauma is acute compartment syndrome (ACS) and hence, was the

underlying reason for the thesis work.

5

1.1.2 Exercise-Induced Limb Muscle Ischemia

Static muscle contraction of forearm muscles can induce skeletal muscle

ischemia. Many investigations have shown that isometric contraction of skeletal muscle

causes blood flow impairment and transient muscle ischemia due to an increase in

intramuscular pressure that compresses the capillaries within the contracting muscle

(Humphreys & Lind, 1963; Lind & McNicol, 1967; Sjogaard et al., 1986; Kagaya &

Homma, 1997; Kahn et al., 1998; van Beekvelt et al., 2002a).

Low oxygen supply due to local blood flow restriction causes dramatic changes in

skeletal muscle metabolism. In addition to restriction of blood supply, local oxygen

availability during muscle contraction will lag behind the local oxygen consumption,

intensifying muscle deoxygenation (van Beekvelt et al., 2002b). Hypoxia reduces the rate

of adenosine triphosphate (ATP) hydrolysis, the cornerstone of energy production, and

switches the metabolic balance from aerobic to anaerobic in order to maintain cellular

activity and muscle force capacity (Arthur et al., 1992; Clanton, 2007). Anaerobic

metabolism leads to metabolic acidosis, impairment of muscle excitation-contraction

coupling and energy depletion that ultimately causes early muscle fatigue (Allen et al.,

1992; Abe et al., 2006; Lanza et al., 2006).

Impaired muscle force production associated with limb muscle fatigue can be a

limiting factor for muscle performance in the workplace as well as during many sports.

This is particularly important in activities that require; sustained contractions of limb

muscles for stabilization of body posture; controlled body movements such as limb

rotations; grip tasks, or supporting tools and sports equipment in a static position (Tesch

6

& Karlsson, 1984; Magnusson, 1997; Murthy et al., 2001; Davey et al., 2002; Reilly et

al., 2008). Investigating the effect of limb muscle ischemia on muscle performance is

therefore of high importance in both exercise and occupational sciences.

Controversy exists in the literature concerning the intensity of isometric

contraction that impairs limb muscle blood flow. Using different methods, it has been

reported that one minute of isometric contraction across a range of intensities, from 10%

to 70%, of muscle voluntary contraction (MVC) can lead to complete muscle ischemia in

healthy individuals (Humphreys & Lind, 1963; Murthy et al., 1997; Kagaya & Homma,

1997; Chapter 3 of this thesis). Direct monitoring of local muscle oxygenation and

hemodynamics during exercise at different work intensity levels is needed to contribute

to a better understanding of the physiology of skeletal muscle in health and disease.

1.1.3 Clinical Consequences of Ischemia

1.1.3.1 Acute Compartment Syndrome

ACS is a clinical condition characterized by an increase of pressure within a

compartment (an enclosed anatomical space within a limb) resulting in a lack of local

perfusion to the tissues within this space due to increased interstitial pressure,

compression, and thus, blockage of the vasculature. If untreated, ACS results in

irreversible ischemic damage to the tissues within the affected compartment (McQueen,

2006; Kostler et al., 2004, Shadgan et al., 2010c). ACS is most common in the lower

limb, specifically the anterior and lateral compartments of the leg. Roughly 36% of all

7

ACS cases are associated with tibial fractures and another 23% are due to blunt soft tissue

trauma (McQueen et al., 2000). The epidemiology, pathophysiology, clinical features and

diagnosis of ACS are discussed in Chapter 2 of this thesis.

Optimum management of ACS is primarily contingent on early diagnosis and a

prompt surgical fasciotomy of all affected compartments in view of compartmental

decompression and restoration of the circulation (Figure 1.1). Worthy of note, as

discussed in section 1.1.1.1, restoration of blood flow to the limb after the ischemia can

release activated leucocytes and their toxic metabolites may increase the local swelling

and tissue pressure, which may even amplify muscle ischemia and worsen the condition.

Therefore, delay in diagnosis of ACS of even a couple of hours can result in serious,

irreversible local and systemic complications including paralysis, muscle necrosis with

possible rhabdomyolysis resulting in loss of the limb or even death (Weinman, 2003;

Malinoski et al., 2004). Therefore, the ability to early diagnose an ACS, prior to the onset

of irreversible ischemic changes, is crucial to prevent permanent disability (McQueen et

al., 2006; Kostler et al., 2004; Finkelstein et al., 1996).

Current practice of ACS diagnosis is based on clinical observation of symptoms

and measurement of intracompartmental pressure, both of which are important predictors

but not always sufficient for an early and accurate ACS diagnosis (see details in Chapter

2). As such, access to an accurate, reliable and noninvasive method for direct monitoring

of limb muscle hemodynamics in high-risk patients would be of great benefit to the early

diagnosis and treatment of ACS in orthopaedic and emergency medicine.

8

Figure 1.1. Lateral fasciotomy wound of an ACS of the leg.

1.1.3.2 Tourniquet-Induced Muscle Ischemia

While ACS is a pathologic condition with a high risk of ischemia, ischemic

conditions are sometimes deliberately induced for the purposes of clinical and surgical

interventions. During orthopaedic surgery of the extremities for instance, a pneumatic

tourniquet is routinely used to obstruct blood circulation in order to avoid intraoperative

bleeding. Based on the current established guidelines, increasing the tourniquet pressure

to 250 mmHg in the upper extremity and 300 mmHg in the lower extremity obstructs

arterial flow sufficiently to create a bloodless surgical field (Odinsson & Finsen, 2006)

but also renders the limb muscle distal to the tourniquet at risk of ischemic damage.

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The relationship between altered blood flow of the extremities and tissue injury

has been recognized since the early 20th century when Harvey Cushing introduced the

first pneumatic tourniquet in 1904 and the first applications of a pneumatic tourniquet

were employed during the First World War (Bayliss, 1919). Since these early uses of

tourniquets, investigations of tourniquet-induced skeletal muscle ischemic injury have

been carried out in animal models and human patients. These investigations revealed a

considerable drop in cellular ATP stores along with increases in skeletal muscle lactate

over 60 to 90 minutes of limb ischemia, which almost completely recovered to pre-

ischemic values over reperfusion periods of only 5 to 10 minutes (Haljamae & Enger,

1975). All the while, several investigations have reported that roughly 2 hours or more of

tourniquet-induced limb ischemia followed by 2 hours of reperfusion leads to increases in

both pro-inflammatory signals and adhesion molecules locally, in the affected muscle, as

well as systemically, in the plasma (Huda et al., 2004; Mathru et al., 2007). As such, 90

minutes is the currently accepted standard for the maximum tourniquet time that should

be sustained before the cuff must be released to allow reperfusion of the ischemic limb. It

is estimated that more than 15,000 pneumatic tourniquets are used during surgical

procedures every day in North America (McEwen & Casey, 2009).

Tourniquet-related complications are broadly divided into two main subgroups,

neurological and ischemic complications. Neurological complications associated with

pneumatic tourniquet, including sensory and motor disturbances, are well known and

thoroughly discussed in the literature (Odinsson & Finsen, 2006; Middleton & Varian

1974; Ochoa et al., 1971).

10

Unlike ischemic conditions arising from physiologic origins, experimental data

demonstrate that the extent of tourniquet-induced muscle damage and related

complications, be they neurological or ischemic in origin, are dependent on a number of

different factors beyond tourniquet time. These include tourniquet cuff design (Younger

et al., 2004), the shape and circumference of the limb (Tredwell et al., 2001), muscle

fiber type composition and aerobic capacity (Yamada et al., 2006), patient gender

(Tiidus, 2000) and the anesthetic agents utilized (Mathru et al., 2007). A method for

continuous monitoring of muscle ischemia in muscles distal to the tourniquet during

tourniquet inflation will therefore facilitate surgeons’ decision making about appropriate

tourniquet time on a per patient basis rather than relying on a universal and fixed

tourniquet time guidelines for all cases.

1.1.3.3 Diagnosis and Monitoring of Limb Muscle Ischemia

Although ischemia is widely known to induce muscle damage, the extent of

damage suffered by different patients in response to similar ischemic windows is highly

variable. While it is well documented that limb muscle ischemia for more than 90

minutes is associated with significant damage of IR-injury secondary to reperfusion

(Bollman & Flock, 1944; Sapega et al., 1985; Artacho-Perula et al., 1991; Odinsson &

Finsen, 2006) Appell et al. (1993) demonstrated that even as little as 15 minutes of

ischemia can predispose limb muscles to IR-injury too. Limiting the duration and extent

of the initial ischemic insult can therefore serve not only to protect limb muscles from

ischemic damage, but can also mitigate IR-injury upon reperfusion. It is for this reason

11

that prompt diagnosis of limb muscle ischemia is paramount for the viability of the limb

and the well being of the patient.

Diagnosis of acute LMI is largely based on clinical evaluation of the signs and

symptoms, which are then to be confirmed by a reliable diagnostic method. Classic

clinical features of acute limb ischemia important for initial diagnosis include pain, cold

and pallor of the limb, pulse deficit, numbness, and sensory deficit that may progress to

motor loss. However, these initial symptoms require further evaluation to determine the

etiology, severity, and reversibility of the condition in order to assess its urgency and

decide on a subsequent course of management (McPherson & Wolfe, 1992). To

complicate matters further, children, unconscious or critically ill patients or patients who

are under anesthesia are typically unable to give a clear history or participate in a clinical

examination making clinical diagnosis often difficult. Lastly, the etiology, severity and

the level of the limb muscle ischemia, as well as the individual tolerance and perception

of the patient, can mask or blunt symptoms of ischemia causing diagnosis to be delayed

thus greatly increasing the risk of significant morbidity. The methods available for

diagnosis of LMI can be divided into three main groups: a) those that detect the source(s)

of the limb ischemia; b) those that evaluate limb circulation; and c) those that measure the

biomarkers of skeletal muscle ischemia (Table 1.1).

Each of these diagnostic techniques has both advantages and disadvantages. Of

greatest importance however, is that there exists no ideal monitoring method for patients

who are at high risk of acute limb muscle ischemia. This, as yet unidentified diagnostic

technique should ideally be noninvasive, sensitive, specific, quantifiable, stable, user

friendly, portable, inexpensive, and capable of real time, continuous monitoring at patient

12

bedside (Dunn, 1990). One possible technique that complies with most of these criteria is

near infrared spectroscopy (NIRS).

Table 1.1. Current diagnostic methods of limb muscle ischemia.

Evaluation & Measurement of:

Source of the Limb Ischemia Limb Circulation Ischemic Biomarkers

Intracompartmental pressure Duplex Ultrasonography Creatine kinase

Doppler Sonography Myoglobin

Laser Doppler Flowmetry Fatty acid binding protein

Angiography Ischemic modified albumin

Computed tomographic angiography Lactate (pH)

Magnetic resonance angiography

Angioscopy

1.1.4 Near Infrared Spectroscopy

NIRS is a non-invasive optical technology that uses energy from light in the near-

infrared spectrum to monitor changes in local tissue oxygenation (oxygen delivery,

consumption, utilization) and hemodynamics (local blood volume) in real time (Delpy et

13

al., 1997; Ferrari et al., 2004). This technology is widely applied as a research and

clinical monitoring tool (Gagnon et al., 2005; Wolf et al., 2007), and there exist

comprehensive reviews of its application, instrumentation, measurement methods, and

limitations in the literature (Delpy et al., 1988; van der Sluij et al., 1997; Boushel et al.,

2001; van Beekvelt et al., 2001a; Ferrari et al., 2004; Gagnon et al., 2005a; Wolf et al.,

2007).

1.1.4.1 Science of NIRS

The science of NIRS is hinged on some of the fundamental principles of optics

and photonics as they relate to the transmission of light through living tissues and the

absorption of light by tissue chromophores. NIRS units use lasers or diodes that transmit

pulses of multiple wavelengths of light into tissues, and optical sensors that detect

returning photons. When NIR light is transmitted through tissue, some is irretrievably lost

due to scattering and some is absorbed by compounds other than the chromophores of

interest. Only a small proportion of the original photons transmitted can be detected

returning from the tissue. The changes in absorption at discrete wavelengths generate raw

optical data that can be converted by mathematical software algorithms into real-time

concentration changes for each chromophore using a modification of the Beer-Lambert

law (Delpy et al., 1988; van Beekvelt et al., 2001b).

14

1.1.4.2 Chromophores of Interest

The principal chromophores of interest in physiological and clinical studies using

NIRS are oxygenated (O2Hb) and deoxygenated (HHb) species of hemoglobin (Hb),

which each have a distinct extinction coefficient (absorption characteristic) across the

NIR spectrum. Other tissue chromophores such as water, myoglobin or cytochrome c

oxidase (CCO) also absorb light differently across the NIR spectrum depending on their

redox status (Cooper & Springett, 1997). Hb and its muscular counterpart myoglobin

(Mb) exhibit similar absorption spectra thus limiting distinction between the two signals.

However, the contribution of Mb to the NIRS signal is minimal and therefore does not

affect the Hb measurements (Mancini et al., 1994; Boushel et al., 2001; Ferrari et al.,

2004; Neary, 2004).

1.1.4.3 NIRS Instrumentation

The basic continuous wave (CW) NIRS equipment is composed of the following

components: 1) signal generator including at least one pulsed laser diode capable of

generating multiple wavelengths (720-920 nm) for the chromophores being sampled, 2)

fibreoptic bundles that transmit light from the light source to a tissue interface and back

to the hardware, 3) tissue interface in the form of a patch or probe including at least two

mounted optodes, 4) photon counting hardware, 5) computer connected to the photon

counting hardware with software containing algorithms for converting raw optical data

into chromophore concentrations, and for storing and displaying data and 6) visual

15

display on which NIRS data are typically displayed graphically against time. Figure 1.2

shows the main components of a NIRS system.

Figure 1.2. A NIRS instrumentation system configured for transcutaneous monitoring.

Depth of penetration of NIRS is limited to half the interoptode distance i.e. a 30 mm

interoptode distance would provide a depth of penetration of ~ 15 mm.

Monitoring a significant amount of the tissue of interest within the field of view

depends on how NIR light penetrates the tissues. Although light is scattered widely once

below the skin surface, the field interrogated effectively via NIRS approximates a banana

shaped area between the emitter and the sensor (Cui et al., 1991) (Figure 1.2). Machines

can provide several options including a choice of: multiple wavelengths; more than one

16

data channel for comparison of multiple sites (van der Sluijs et al., 1997), displaying a

tissue oxygen saturation index (TSI%) from the ratio of O2Hb to total tissue Hb; spatial

data by using a regional map using arrays of emitters and receivers (Obrig & Villringer,

2003). Currently most NIRS instruments use lasers as and require fibreoptic cables to

transmit light to and from the patient. Light emitting diodes (LED’s) are an alternative

light source which can be combined with Bluetooth® to provide wireless capacity. More

detail on wireless NIRS prototypes can be found in Chapter 3.

1.1.4.4 NIRS Variables

The nature of changes in NIRS-derived chromophore concentrations in response

to physiologic alterations such as vascular occlusion, muscle contraction or movement

provides important information about the physiological condition of the muscle of

interest. As discussed above, based on differences in their absorption characteristics

across the NIR spectrum, NIRS can monitor changes in the concentrations of O2Hb and

HHb simultaneously. Further, changes in total hemoglobin (tHb), the sum of O2Hb and

HHb concentrations, can be calculated and offer insight into changes in local blood

volume in the tissue of interest (Boushel et al., 2000b; 2002; van Beekvelt et al., 2002a).

NIRS can also inform about local blood flow within the muscle of interest through

measurement of the rate of change in local blood volume. For instance, within the first

seconds of a tourniquet-induced venous occlusion, the rate of increase in tHb within the

muscles distal to the tourniquet indicates the local blood flow (van Beekvelt et al.,

1999a). More specific indexes of muscle blood flow can be obtained by measuring the

17

rate of change in the concentration of an intravenous tracer, such as indocyanine green,

(Boushel et al., 2000b).

The difference between changes of O2Hb and HHb concentrations, a value

abbreviated as (Hbdiff) is interpreted as an index of tissue oxygenation, especially when

the tHb concentration remains unchanged (Grassi et al., 1999; Kirkpatrick et al., 1997;

Tachtsidis et al., 2007). Rate of muscle deoxygenation, as measured by the rate of

decrease in Hbdiff, following a complete arterial occlusion provides information about the

rate of muscle oxygen consumption (mVO2) of the muscles distal to the occlusion level, a

value expressed in units of µM.s-1 (De Blasi et al, 1994). This measure provides

important information for the evaluation of muscle metabolism status. Likewise, these

same measurements can be used to provide information about the quality of muscle

recovery upon reperfusion.

The time required for a half recovery of O2Hb from maximum deoxygenation at

the end of the ischemic period to maximum reoxygenation level during hyperemia is

referred to as the “half-recovery time” and is viewed as an index of tissue O2Hb influx

and oxygen consumption after reperfusion (Chance et al., 1992). Further, the rate of

increase in O2Hb concentration during the first 3 seconds of reperfusion is referred to as

the “reoxygenation rate” and is interpreted to be an index of the speed at which recovery

starts upon reperfusion (Figure 1.3). Finally, reactive hyperemia is a term used to

describe a transient increase in tissue blood volume following a period of ischemia

secondary to vasodilatation in response to the tissue hypoxia. This index is used to

evaluate the effect of ischemia on the muscle vasculature function by measuring the

amount by which tHb increases upon tourniquet release and the time required for

18

increased tHb to return to the baseline (Chance et al., 1992; McCully et al., 1994; van

Beekvelt et al., 1999a; 2001b; 2002; Harel et al., 2008).

Figure 1.3. A) Patterns of change in O2Hb, HHb, tHb and Hbdiff during tourniquet-

induced leg muscle ischemia during trauma surgery (Chapter 4), B) calculation of the

half-recovery time, the time needed for half recovery of O2Hb from maximum

deoxygenation to maximum reoxygenation and C) calculation of the reoxygenation rate,

the rate of increase in O2Hb during the first 3 seconds immediately after reperfusion.

1.1.4.5 Validity of NIRS Measurements

Recent literature summarizes the validity of many NIRS-derived measures

through comparison with other standard measures such as magnetic resonance

plethysmography (Wickramasinghe et al., 1992), pulse oximetry (Watkin et al., 1999),

19

Doppler ultrasonography (Grubhofer et al., 2000), positron emission tomography

(Niwayama et al., 2000), phosphorus magnetic resonance spectroscopy (Sako et al.,

2001), venous oxygen saturation (Mancini et al., 1994; Grubhofer et al., 1997; Buuank el

al., 1998) and electromyography (Yamada et al., 2008). Confidence in the physiologic

information generated using NIRS in muscle studies is rooted in the consistency and

reproducibility of the patterns of change in chromophore concentration observed using

NIRS in different skeletal muscles, in studies conducted by different investigators, and

when using NIRS equipment from different manufacturers. It should however be noted

that while relative changes in chromophore concentration are consistent between studies

and equipment, the magnitude of change varies between data sets using different NIRS

hardware and software (Boushel et al., 2001; van Beekvelt et al., 2002a; Ferrari et al.,

2004; Wolf et al., 2007; Hamaoka et al., 2007).

Reports of poor correlation between some standard measures and NIRS-derived

entities also exist in the literature and warrant mention. Costes et al. (1996), Hicks et al.

(1999) and MacDonald et al. (1999) have reported that NIRS was insensitive compared

to venous oxygen saturation (via analysis of deep vein blood samples) of the same limb

during muscle exercise under normoxic conditions. In those studies however, good

correlations were reported between tissue oxygen saturation measured via NIRS versus

venous oxygen saturation under hypoxic conditions. However, the most important flaw of

the previous three studies is that they did not use a NIRS device that could accurately

measure the tissue oxygen saturation. Costes et al., Hicks et al. and MacDonald et al. all

used an identical model of CW NIRS device (Runman CWS-2000, NIM Inc.) to compare

changes in the “tissue oxygen saturation” with deep venous oxygen saturation.

20

Measuring the absolute amount of local muscle oxygen saturation requires a more

complex NIRS instrumentation with a spatially–resolved configuration (Rolfe, 2000;

Boushel et al., 2001; Ferrari et al., 2004) and a greater number of sensors and

wavelengths. A second limitation of most validation reports is that NIRS-derived

parameters are often compared to a surrogate measure that only reflects one component

of the NIRS parameter as discussed at length by Hamaoka et al. (2007). For example,

O2Hb reflect a combination of tissue, venous and arterial concentrations of oxygenated

hemoglobin whereas the previous three studies validated O2Hb against venous oxygen

saturation. Hamaoka et al. (2007) also explains that the oxygenation gradient between

arterioles and venules is larger under normoxic than under hypoxic conditions. Thus, the

larger arteriolar contribution to O2Hb during normoxia may mask or hinder detection of

contributions by the venous component. The methodology used in these studies might

therefore have negatively confounded their observations. Further studies are warranted to

investigate the validity and accuracy of NIRS measures in muscle studies in varying

physiological conditions and in different skeletal muscle groups.

1.1.4.6 Clinical Applications of NIRS

During the past three decades, many studies applied NIRS for the assessment of

brain (Brazy et al., 1985; Wyatt et al., 1986; Al-Rawi, 2005; Petrova & Mehta, 2006) and

muscle (De Blasi et al., 1993; 1994; Homma et al., 1996; Ferrari et al., 1997)

oxygenation and hemodynamics in health (Wolf et al., 1997; Ferrari et al., 2004; Gagnon

et al., 2005a), disease (Allen et al., 1992; Boushel et al., 2001; Nakayama et al., 2001;

21

Asgari et al., 2003; van den Brand et al., 2004; Ward et al., 2006; Hamaoka et al., 2007),

and exercise science (Bhambhani et al., 1999; Neary et al., 2004; Kell & Bhambhani,

2008). These reports have generated considerable promising evidence that support the

continued use of NIRS towards an increasing number of applications. Recent research

has explored a broad range of potential clinical applications of NIRS for diagnosis of

pathologies and for continuous monitoring of tissue hemodynamics during surgical

operations, in the intensive care unit and at the bedside (Boldt, 2002). Studies include

evaluation of neurological conditions such as subdural hematomas and acute cerebral

infarction (Gagnon et al., 2005b; Kahraman et al., 2006) and evaluation and monitoring

of tissue status in conditions such as skin flaps (Scheufler et al., 2004; Gravvanis et al.,

2006), burns (Sowa et al., 2001, 2006; Cross et al., 2007), trauma (Gentilello et al., 2001;

van den Brand et al., 2004), bladder dysfunction (Stothers et al., 2008; Shadgan et al.,

2010b) and cancer (Asgari et al., 2003). At the cellular level, NIRS has yielded some

important findings in metabolic and mitochondrial myopathies (van Beekvelt et al.,

1999b).

1.1.4.7 Advantages of NIRS in Clinical Studies

As a clinical monitoring and diagnostic device, NIRS provides a number of

distinct advantages over other strategies for monitoring tissue hemodynamics. Of

particular note are: its non-invasive nature, non-toxic energy source, and its ability to

monitor a range of physiologic changes continuously, in real time for prolonged periods

at the bedside. NIRS can also be used in conjunction with other diagnostic or therapeutic

22

technologies without risk of electromagnetic interaction (See Chapter 5 for a discussion

of this topic). It is also user-friendly, portable and inexpensive.

1.1.4.8 Limitations of NIRS

Limitations relevant to clinical applications of NIRS include restrictions imposed

by the basic science principles underlying this technology. First of all, it is important to

recognize that, since the full extent of the field through which light scatters is always

unknown in vivo, measurement of the absolute concentration of each chromophore is not

possible using conventional NIRS. In fact NIRS can only estimate changes of

chromophore concentration by measuring changes in chromophore concentration from

baseline. As such, NIRS is most informative in situations when a temporary change in the

physiologic state or dynamic properties of the tissue can be induced or is anticipated.

This is exemplified by situations such as pressure-induced ischemia, muscle contraction

or exercise. (See appendix III, as an example of using NIRS for monitoring inspiratory

muscle function during incremental inspiratory threshold loading).

NIRS is also limited by the depth of light penetration into living tissue that is

restricted to approximately half of the distance between the emitter and receiver optodes.

This effectively caps the depth of penetration at roughly 40 millimetres (Homma et al.,

1996) and constrains non-invasive transcutaneous application of NIRS to the monitoring

of superficial tissues in patients with only minimal subcutaneous adipose tissue (Boushel

et al., 2001; van Beekvelt et al., 2001a). It has been shown that adipose tissue absorbes

and attenuates NIRS signals, however the precise in vivo influence of adipose tissue on

23

NIRS measurement remains uncertain (Homma et al., 1996; Niwayama et al. 2000).

Factors such as movement, ambient light and strong electromagnetic field can generate

artefact within the NIRS signal, which may disturb accurate reading of NIRS signals. A

new method of motion artifact removal from muscle NIRS measurements are examined

and described in Appendix II of this thesis. Attenuation of NIRS signals in subjects with

dark skin pigmentation (Wassenaar & van den Brand, 2005) or in the presence of blood

accumulation (hematoma) within the field of in vivo NIRS study or any change in the

tissue optical pathlength, e.g. stretching or compressing the tissue of interest or acute

hemodilution, can also adversely affect NIRS data collection (Duncan et al., 1995;

Yoshitani et al., 2007). Lastly, like other new technologies in biomedical research, NIRS

requires further specifically designed studies to examine the consistency and

reproducibility of NIRS-derived measurements for each specific clinical application.

1.1.4.9 Feasibility of NIRS Monitoring in the Operating Room

New therapeutic or diagnostic methods and instrumentations require initial

feasibility studies in view of proofing the concept and assessing the practicality of their

use in clinical settings as well as to determine any possible confounding factors or

procedural restrictions. While the feasibility of using conventional NIRS for monitoring

limb muscle oxygenation and hemodynamics has been extensively studied during

exercise (Chance et al., 1992; Bhambhani et al., 1999; Boushel et al., 2000a; van

Beekvelt et al., 2002a, Shadgan et al., 2008b), and clinically (Macnab, 2009), studies

investigating intraoperative and wireless NIRS monitoring of limb muscles are lacking.

24

Electromagnetic radiation from instruments currently used in the OR, especially

devices that emit electromagnetic frequencies between 10 kHz and 1 GHz, may interfere

with signals used for data recording by new instrumentation, such as NIRS (Silberberg,

1993; Segal et al., 1995). Furthermore, several other essential activities during surgical

procedures would disrupt or severely limit NIRS monitoring intraoperatively including:

1) compromising the sterile field during the surgical procedures due to the necessity of

placement of NIRS sensors on target muscles inside the sterile boundaries; 2) interference

of surgical procedure due to NIRS monitoring close to the incision site; 3) movement of

the limb by the surgical team during surgery; 4) difficulties posed by NIRS operational

parameters (such as preparation and probe fixation). Because NIRS monitoring is limited

to those muscles with only a thin layer of subcutaneous tissue, compromising the sterile

field and interference with surgical procedure were key feasibility issues that required

assessment. Regarding wireless NIRS, operational parameters and device placement

could limit patients’ tolerance of the device in non-operative studies. The device,

although small, may interfere with with the limb movement of interest. As well, the

strapping attachment to secure its position may cause discomfort or might not adequately

hold the NIRS device in position for the duration of the monitoring period.

Therefore, the feasibility study of NIRS application using a wireless device during

exercise, and using conventional NIRS during limb surgery is essential to evaluate

potential variables that could interfere with or confound NIRS signal detection or

recording in these applications. Feasibility of NIRS to monitor and detect LMI is a

primary focus of my thesis work and is discussed in detail in Chapters 3, 4 and 5,

respectively.

25

1.2 Rationale of the Thesis

Acute limb muscle ischemia is a limb-threatening condition requiring immediate

medical care. Successful remediation of this condition is dependent on early diagnosis in

order to prevent reversible ischemic damage of limb muscles from escalating to severe

ischemia-reperfusion injury and its associated permanent damage and necrosis of the

affected limb muscles. Currently, there exists no reliable monitoring method for the early

diagnosis of acute limb muscle ischemia.

NIRS demonstrated potential as a responsive and valid technique for monitoring

tissue oxygenation and the hemodynamic response to physiological and pathological

conditions that may alter regional blood flow, such as local changes in tissue interstitial

pressure, arterial obstruction and muscle contraction. As such, NIRS may be a valuable

tool to provide rapid, non-invasive and real-time, continuous monitoring of limb muscle

oxygenation and hemodynamics in patients at high-risk for acute limb muscle ischemic

conditions and therefore also provide a sound method for early diagnosis of these

conditions. However, despite all of the advantages of this optical technique, further

investigation of this technology is required before NIRS can be confidently introduced

into clinical diagnostic practice.

26

1.3 Thesis Purposes

The main purposes of this thesis are:

1. to examine the feasibility and convergent validity of CW NIRS for continuous

monitoring of skeletal muscle oxygenation and hemodynamics during transient

and long-term tourniquet-induced LMI.

2. to investigate the predictive value of NIRS-derived data for evaluation of limb

muscle oxidative changes during tourniquet-induced LMI.

3. to investigate the feasibility of NIRS for continuous monitoring of tourniquet-

induced limb muscle ischemia during ankle surgery without being affected by

EMI of medical devices commonly used in orthopaedic operation room.

27

1.4 Specific Aims

The specific aims of this thesis are to:

1. examine the feasibility of a CW wireless NIRS instrument to monitor forearm muscle

oxygenation and hemodynamics during muscle contraction and tourniquet-induced

ischemia (Chapter 3).

2. determine the internal consistency of the data obtained by the CW wireless NIRS

instrument during isometric contraction at different work intensities and transient

tourniquet-induced ischemia in healthy subjects (Chapter 3).

3. determine the intensity of sustained isometric muscle contractions that induces a

complete local muscle ischemia, using the NIRS instrument with spatially resolved

configuration (Chapter 3).

4. determine the internal consistency of NIRS measures by examining the relationship

between duration of tourniquet-induced ischemia (tourniquet time) and changes in NIRS-

derived skeletal muscle O2Hb, HHb, tHb, Hbdiff, half- recovery time, hyperemia interval

and reoxygenation rate before, during and after limb muscle ischemia (Chapters 3 & 4).

5. determine the relationship between duration of tourniquet time and skeletal muscle

protein oxidation (Chapter 4).

6. determine if NIRS-derived variables during tourniquet induced ischemia are predictive

of changes in skeletal muscle protein oxidation (Chapter 4).

28

7. determine if electromagnetic interference from the operation of three medical devices

commonly used in the orthopaedic operation room (surgical drill, surgical cutter and

portable X-ray) affect NIRS signals (Chapter 5).

29

1.5 Hypotheses

The main hypotheses of this thesis are:

1. Conventional and wireless NIRS will prove to be feasible methods for the continuous

monitoring of transient and long-term tourniquet-induced limb muscle ischemia

(Chapters 3 & 4).

2. Changes in muscle protein oxidation state in muscles distal to the tourniquet during

tourniquet-induced ischemia will correlate positively to tourniquet time and changes in

HHb and reoxygenation rate, and inversely to changes of O2Hb and tHb as monitored

using NIRS (Chapter 4).

3. NIRS signals will not be affected by EMI of medical devices that are commonly used

in the orthopedic operating room (Chapter 5).

An overview of the studies, objectives and hypotheses of this thesis are outlined in Table

1.2.

30

Table 1.2.

Overview of overall objectives and hypotheses of the studies in this thesis.

31

CHAPTER 2

Diagnostic Techniques in Acute Compartment

Syndrome of the Lower Leg, A Review Article *

* This chapter has been published in a peer-reviewed journal as:

Shadgan B, Menon M, O'Brien PJ and Reid WD. Diagnostic techniques in acute compartment syndrome of the leg. Journal of Orthopaedic Trauma. 22(8):581-587, 2008.

32

2.1 Introduction

Acute compartment syndrome (ACS) is a critical limb muscle ischemic condition

characterized by the increase of pressure within a closed anatomical space resulting in a

lack of local perfusion to the tissues within this space (Amendola & Twadel, 2003;

Kostler et al., 2004). If untreated, the lack of perfusion results in irreversible damage to

the tissues in the affected compartment. The results of an unrecognized or untreated

compartment syndrome of the lower leg include: pain, paralysis, paresthesia and muscle

necrosis with possible rhabdomyolysis. The potential disability associated with a

neglected compartment syndrome is usually irreversible.

Compartment syndrome has been reported in a wide variety of traumatic and non

– traumatic clinical scenarios. The most common injury resulting in a compartment

syndrome is a fracture of the tibial diaphysis due to the relatively high incidence of this

fracture as well as to the anatomical environment of closed fascial spaces found in this

area (McQueen et al., 1996a; McQueen et al., 2000; Chang et al., 2000; Amendola &

Twadel, 2003; Kostler et al., 2004; Court-Brown et al., 2006). This differentially affects

young, active individuals (Court-Brown & McBirnie, 1995; Court-Brown & Koval,

2006). Permanent disability in this particular group of patients can place a large burden

on the individual, society and, often, our medico-legal system (Giannoudis et al., 2002;

Bhattacharyya & Vrahas, 2004).

The reported incidence of compartment syndrome varies due to differing

diagnostic criteria, sampling methods, and patient populations (McQueen et al., 1996b;

Amendola & Twadel, 2003; Kostler et al., 2004; Hope & McQueen, 2004). Reported

33

incidence of compartment syndrome following tibial fractures ranges from 1.2% to

30.4% (McQueen et al., 1996a; Chang et al., 2000). The incidence is greater in males,

those under age 35, and can vary dependent on the method of fixation. Thirty-six percent

of all compartment syndromes occur after tibial diaphyseal fractures (Court-Brown &

Koval, 2006). Fractures of the tibial plateau only develop a compartment syndrome in

3.0% of cases (McQueen et al., 2000).

Treatment of a compartment syndrome consists of immediate and complete

fasciotomy of all fascial compartments involved. In the lower leg, this involves all four

anatomical compartments. Wounds are left open for a minimum of 48 hours, or until the

compartment syndrome is resolved. Direct closure of the fasciotomy wounds is

attempted, however, plastic surgical techniques are often required. Delay in the diagnosis

or treatment of the syndrome results in permanent disability. Therefore, the ability to

diagnose a compartment syndrome in a timely manner, prior to the onset of irreversible

ischemic changes, is crucial to prevent permanent disability (McQueen et al., 1996a;

Finkelstein et al., 1996).

The first sign of a compartment syndrome is excessive pain disproportionate to

the severity of injury in an at risk patient. If untreated, paresthesia and paralysis occur.

The timing between these symptoms is variable (Olson & Glasgow, 2005). Clinical

examination of the patient reveals palpable tightness, an increase in pain upon passive

stretch of the compartment involved, progressive paresthesia and eventually paralysis.

The clinical picture is variable and often only a few signs are present. Observation of a

patient with a developing compartment syndrome can lead to a delay in diagnosis and

treatment. This delay possibly contributes to permanent disability. Ischemic contracture

34

complicating tibial fractures has been estimated to occur in up to 2% of cases (Ellias,

1958). Only 13% of patients with paralysis at the time of their diagnosis recover from this

impairment (McQueen, 2006).

Giannoudis and colleagues (2002) reported a significant detriment in health

related quality of life, as measured by the EQ-5D (EuroQol) tool (The Euroqol Group,

1990), in patients who had undergone fasciotomy and required a skin graft or those who

had longer closure times after compartment syndrome of the leg. Vandervelpen et al.

(1992) showed that one in four patients undergoing leg fasciotomy reported late

functional disabilities. Fitzgerald et al. (2000), who reported on the sequelae of

fasciotomy wounds, found symptoms related to the skin wounds in up to 77% of patients

who had undergone fasciotomy of the upper or lower limb.

35

2.2 Diagnosis

Little debate exists as to the necessity of a thorough and immediate fasciotomy

once a compartment syndrome has been diagnosed. Nor is there disagreement when a

clear presentation of clinical signs of compartment syndrome is present in a high-risk

patient. However, in patients who cannot give a clear history or participate in a rigorous

clinical examination, diagnosis is often difficult (Matsen et al., 1980). This includes

children, those with concomitant neurological injury, the critically ill and patients under

prolonged general anesthesia. In these patients, intra-compartmental pressure

measurements have been used to screen for the development of compartment syndrome

when clinical examination is either unreliable or equivocal. Several modalities have been

investigated as possible diagnostic adjuncts in the early identification of an acute

compartment syndrome. A reliable screening tool to diagnose a developing compartment

syndrome would provide the opportunity to intervene early and avoid the sequelae of a

delayed diagnosis. Although further investigation is needed, several of these techniques

show promise (Figure 2.1).

36

Figure 2.1. This algorithm shows the application of ACS diagnostic methods based on

ACS pathophysiological stages.

2.2.1 Pressure Measurements

Whitesides and colleagues (1975) were the first to apply compartment pressure

measurement to the diagnosis of acute compartment syndrome. Since then, several

techniques have been described, each with limitations that restrict their reliability or

37

practical use. These include the needle manometer, the wick catheter, and the slit catheter

(Whitesides et al, 1975; Mubarak et al., 1976; Rorabeck et al., 1981). The STIC catheter

(Stryker) has become popular as a hand-held portable device that is easily used in a

variety of settings without the need for complex equipment. Continuous pressure

monitoring is available by attaching a reliable catheter to an arterial transducer system

(McQueen, 1996). This allows a continuous readout of the pressure in the compartment

and allows us to observe changes over time. Although there is some technical learning

required for its accurate use, compartment pressure measurements have been successfully

used clinically as an adjunct to clinical examination (McQueen et al., 1996b).

Traditionally, the diagnosis of a compartment syndrome has been on clinical

criteria, with objective pressure measurements used as an adjunct for equivocal cases

because the clinical picture is rarely complete. The pressure threshold that is diagnostic of

compartment syndrome has been debated at length. Experts have advocated fasciotomy

for absolute compartment pressures from 30 to 45 mmHg (Mubarak et al., 1978;

McQueen, 2006). This threshold for diagnosis is likely too aggressive and subjects a

large number of patients unnecessarily to fasciotomy and the risks associated with it.

McQueen et al. (2006b) have shown that an increase in compartment pressure post tibial

nailing is expected even without the development of a frank compartment syndrome.

Whitesides et al. (1975) astutely suggest that the perfusion of the compartment depends

upon the difference between the patient’s blood pressure as well as the compartment

pressure and recommends fasciotomy when the compartment pressure rises to within 30

mmHg of the diastolic blood pressure, known as the delta p (Δp < 30mmHg). White and

colleagues (2003) have shown that an elevated intramuscular pressure alone is not

38

diagnostic of a compartment syndrome following tibial intramedullary nailing as long as

the Δp remains greater than 30 mmHg. The use of the Δp has been consistently shown to

be a more reliable indicator of the conditions necessary to produce a compartment

syndrome than the compartment pressure alone (McQueen et al., 1990; 1996b; White et

al., 2003). It also allows a more reflective measure in patients with abnormal physiology,

such as shock or hypertension.

One difficulty encountered in the development of pressure thresholds for the

diagnosis of compartment syndrome is the lack of gold standard diagnostic criteria for the

condition. A collection of clinical symptoms and signs in experienced hands serves as the

diagnostic criteria in most circumstances. Attempts to objectify the diagnosis for

purposes of validation of measurement techniques have been made but are not universally

accepted. These include the bulging of muscle compartments on fasciotomy, and

secondly, clinical follow-up looking for the sequelae of the syndrome (Jangzing & Broos,

2001; Ulmer, 2002; McQueen et al., 1996a). The first of these is often dismissed as non-

specific, and the latter does not allow the identification of successfully treated cases.

Some authors have argued that since a rise in compartment pressure must occur

prior to the development of a compartment syndrome, an objective pressure measurement

can diagnose a compartment syndrome prior to the onset of symptoms and prior to the

development of irreversible sequelae of the syndrome. Thus, fasciotomy based upon

continuous pressure measurements for the “impending” compartment syndrome should

be the most appropriate treatment so long as the measurements are adequately sensitive

and specific to the development of a full compartment syndrome. This approach has been

shown to be effective in clinical practice by McQueen and colleagues (1996b) who

39

reported that no compartment syndromes were missed in a large prospective series using

a Δp of < 30 mmHg as the diagnostic criteria for its presence. Despite the apparent

success reported by McQueen et al. (1996b), continuous pressure monitoring has not

become the standard treatment in most centers (Williams et al., 1998). Recently, a

prospective randomized trial has attempted to evaluate the addition of continuous

pressure monitoring to current clinical diagnostic criteria. The recent development of a

hand-held fibre-optic transducer system that uses a unicrystalline piezoelectric

semiconductor has eliminated some of the practical concerns of calibration and blockage

that occurred frequently with catheter based systems (Harris et al., 2006).

2.2.2 Biomarkers

The traumatic injury associated with tibial fractures and ACS both result in the

early onset of inflammatory markers. Generalized inflammatory biomarkers such as an

elevated white blood cell (WBC) count or a positive erythrocyte sedimentation rate

(ESR) cannot specifically indicate the occurrence of a compartment syndrome

(Vrouenraets et al., 1997). Creatine kinase (CK), myoglobin (Mb) and fatty acid binding

protein (FABP) are low molecular-mass cytoplasmic proteins present in the myocardial

muscles as well as skeletal muscles. These proteins have been introduced as plasma

markers for the early detection of myocardial infarction, but at the same time, each of

them show similar plasma release curves after skeletal muscle injury and necrosis.

Following skeletal muscle ischemic damage, both Mb and FABP concentration

significantly increase after 30 minutes while CK concentration reaches a maximum after

40

2 hours. Both Mb and FABP return to the baseline values at 24 hours after injury whereas

CK remains elevated for at least 48 hours (van Nieuwenhoven et al., 1992; Sorichter et

al., 1998). It has been shown that following intracompartmental ischemia when muscle

necrosis occurs, serum level of CK dramatically increases. It is recommended that CK

values over 2000 units/liter after surgery can be a warning sign of ACS in ventilated and

sedated patients (Lampert et al., 1995). Compared to the myocardium, the Mb content in

skeletal muscle is higher and the FABP content is lower. The ratio of Mb/FABP in the

same blood sample is a useful index to determine the origin of the proteins. Normal

Mb/FABP ratio in myocardial muscles is about 5 and in skeletal muscle is more than 20

(van Nieuwenhoven et al., 1995). Frequent measurements of these parameters beginning

shortly after tibial fractures could theoretically alert us to the development of a

compartment syndrome, however they may not be specific enough to differentiate

between direct skeletal muscle injury due to trauma, ACS or myocardial injury, when

used clinically.

Anaerobic metabolism of muscle cells within the ischemic compartment in the

early stages of an ACS produces a high amount of lactic acid. This elevated concentration

of lactate results in a reduced serum pH and may be an indicator of ACS. However, it is

not specific. Measurement and comparison of the local lactate concentrations from the

affected and healthy limb muscles may increase the specificity rather than monitoring the

plasma lactate level (Qvarfordt et al., 1983).

Ischemic modified albumin (IMA) is a relatively new marker of myocardial

ischemia, and IMA concentration can also be affected by skeletal muscle ischemia (Roy

41

et al., 2004). An immediate and transient decrease in plasma concentration of IMA is

reported following skeletal muscle ischemia (Zapico-Muniz et al., 2004), which returns

to baseline one hour after the initial decrease. Such a transient decrease in IMA

concentration in patients experiencing angina might be falsely attributed to only

myocardial ischemia rather than potentially arising from a developing ACS. This

measure cannot be a reliable and specific measure for early diagnosis of ACS.

There is no report of a coenzyme or biomarker specific to skeletal muscle

ischemia to date. Detection of a sensitive and specific biomarker for skeletal muscle

ischemia that is not influenced by inflammation or tissue injury from trauma would be a

tremendous accomplishment in diagnosis of ACS. Therefore, more investigation is

required.

2.2.3 Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is able to detect soft tissue edema and

swollen compartments on T1-weighted spin-echo images (Rominger et al., 2004).

However, MRI cannot differentiate the edema of affected muscles in a compartment

syndrome from the edema of soft tissue injury following trauma (Rominger et al., 1995).

MRI can show the tissue changes in an established compartment syndrome in a very late

stage, but fails to identify early changes of an ACS. MRI is a sensitive and non-invasive

diagnostic tool, but the role of this technology in early diagnosis and monitoring of the

ACS is limited.

42

2.2.4 Ultrasound

Ultrasonography is a noninvasive diagnostic intervention, which can visualize and

monitor soft tissue structure and motion. Several investigators have tried to assess the

geometry and echogenicity of the affected muscles for an early diagnosis of compartment

syndrome by standard sonographic methods with no consistent success (Jerosch et al.,

1989). A new ultrasonic intervention called pulsed phase-locked loop (PPLL) may be

useful in the diagnosis of compartment syndrome (Lynch et al., 2004; Wiemann et al.,

2006). This technique was initially developed by Ueno et al. (1998) as a non-invasive

method to monitor intracranial pressure. The PPLL ultrasound is a low-power ultrasonic

device designed to detect sub-micrometer displacements between the ultrasound emitter

on the skin surface and any targeted tissue, which can reflect the ultrasound waves

(Lynch et al., 2004). The device transmits an ultrasonic wave through the tissue via a

small transducer placed on the skin surface. The depth of penetration of the ultrasonic

waves is set to reach a specific tissue. The transmitted waves reflect off the targeted

tissue and are received by the same transducer. The PPLL ultrasound locks on to a

characteristic reflection that comes from a specific tissue. PPLL ultrasound detects the

very subtle movements of fascia that correspond to local arterial pulsation. These

waveforms have a characteristic shape in the normal compartment. The increased ICP

during compartment syndrome causes a reduction of normal fascial displacements in

response to arterial pulsation, which decreases the complexity of the fascial displacement

waveform (Wiemann et al., 2006). In order to refine its interpretation, the limitations of

this method such as the effect of possible variations of fascial movements indicative of

normal physiology and anatomy should be identified. As a noninvasive monitoring

43

device, PPLL may be a promising method for the early diagnosis of ACS, however, it

requires more investigation.

2.2.5 Scintigraphy

Scintigraphy is a radionuclide imaging intervention. A two dimensional image is

obtained after injection of a soluble radioisotope to evaluate regional perfusion.

Scintigraphy differs from most other imaging modalities since it primarily shows the

physiological function of the system being investigated as opposed to its anatomy. It is

mostly used to study myocardial perfusion and peripheral vascular obstructions. Edwards

et al. (1999) have studied the efficacy of scintigraphy in the diagnosis of chronic

exertional compartment syndrome (CECS). They reported a sensitivity of 80% and a

specificity of 97% for detection of CECS by using 99-Technetium (99Tc)-MIBI

scintigraphy. They concluded that 99Tc-MIBI can detect compartment syndromes with

good positive and negative predictive values. A major strength of this method is that all

four compartments of lower leg can be monitored simultaneously. In addition, it is a

simple, cheap and minimally invasive method. The use of scintigraphy in ACS is limited

by the time required to perform this type of investigation, the potential lack of specificity

in the traumatized limb, and the inability to perform repeated or continuous examinations

(Elliott & Johnston, 2003).

44

2.2.6 Laser Doppler Flowmetry

Laser Doppler flowmetry (LDF) is a well-developed technique for the real-time

measurement of microvascular perfusion in tissue. This velocimetry method is based on

the “Doppler effect” which describes the shift in the frequency of a sound or light wave

when the wave source and/or the receiver is moving (Briers, 2001). LDF works by

illuminating the tissue with low power laser light based on local red blood cell

circulation. Light from one optical fiber is scattered by moving red blood cells. Another

optical fiber collects the backscattered light. By analyzing the differences between the

reflected and returned signals, a functional image is obtained. It is a noninvasive and

highly sensitive method for continuous monitoring of local blood perfusion. Despite all

the advantages, limited studies have been performed to assess the capability of this

method in diagnosis of ACS. In one study, Abraham et al. (1998) successfully used LDF

in the diagnosis of chronic exertional compartment syndrome. Although there is no

documented experience of using this method for the diagnosis of ACS, the technique

shows potential and requires further research.


The pathophysiologic mechanism in ACS is an increased ICP compromising

microvascular flow within the affected compartment, which leads to an acute

intracompartmental hypoxia and muscle necrosis. Near-infrared spectroscopy (NIRS) is

an optical technique, which can determine the redox state of various light-absorbing

45

molecules such as hemoglobin. Its function is based on the relative tissue transparency

for light in the near-infrared spectrum and on the absorption changes of hemoglobin and

myoglobin when they are in their oxygenated versus deoxygenated states (van Beekvelt

et al., 2001b). Based on the different light absorption properties, NIRS can measure the

local changes in concentration of oxygenated and deoxygenated hemoglobin and

perfusion in different tissues including muscle (Mancini et al., 1994). NIRS measures the

most direct pathophysiologic consequence of ACS, intracompartmental tissue oxygen

levels, rather than measuring the indirect mechanism of intracompartmental pressure that

may or may not result in muscle ischemia. In 1977, Jobsis used this technology for the

first time to monitor cerebral and myocardial oxygenation. During the last decade NIRS

has been used to measure tissue oxygenation especially during investigations of exercise.

The NIRS instrument consists of a probe and an analyzer chip. The probe contains light

emitting and receiving fibers.

The depth of penetration of NIRS light is about half the distance between emitter

and receiver probes. Specific wavelengths can be selected for transmission and

subsequent absorption of light for measuring oxygenated and deoxygenated hemoglobin.

The difference between emitted and receiver infrared light signals undergo signal

processing by an integrated software program to extract the tissue oxygen saturation

value. NIRS also has the potential to measure the redox state of cytochrome C, which can

provide more specific information regarding intracellular oxygenation and whether the

oxygen supply is sufficient to meet energy demands at the cellular level (Arbabi et al.,

1999). Local ischemia causes an increased extraction of oxygen by muscular tissue,

which reduces the level of local venous oxyhemoglobin (van den Berand et al., 2005).

46

NIRS can measure the changes of tissue hemoglobin saturation and thus has the potential

to provide continuous, noninvasive monitoring of intracompartmental ischemia and

hypoxia (Garr et al., 1999).

A limited number of studies illustrate the utility of NIRS. In a case report, Tobias

and Hoernschemeyer (2007) presented the use of NIRS in monitoring intracompartmental

oxygenation of a one-month-old infant who developed an acute compartment syndrome

of the lower leg after cardiac surgery. Several studies have shown high sensitivity and

specificity of NIRS in the diagnosis of exertional chronic compartment syndrome (van

den Berand et al., 2004) but no clinical trial is available regarding the diagnostic value of

NIRS in a clinical model of ACS. In an animal model of limb ACS, Garr et al. (1999)

demonstrated a strong correlation between the level of oxyhemoglobin measured by

NIRS and perfusion pressure in the affected muscles. Despite the promising advantages

of near-infrared spectroscopy as a diagnostic tool, it still has some practical limitations,

such as low depth of penetration (maximum depth of 30-40 mm) and extraneous

variables that could affect the penetration and reflection of the emitted infrared light

signal. The system needs further technical improvements and more clinical investigations

are currently under way. NIRS may provide the benefit of a rapid, continuous, non-

invasive, sensitive and specific tool for early detection of ACS (Giannotti et al., 2000).

47

2.2.8 Pulse Oximetry

Pulse oximetry is a noninvasive technique that estimates the level of arterial

oxygen saturation using technology based on somewhat similar principles to the NIRS

device. The pulse oximeter emits red and infrared light, which is absorbed by the

different levels of oxy-hemoglobin, and deoxyhemoglobin during pulsatile flow. The

oximeter probe measures the difference between the emitted and received red and

infrared light during the diastolic and systolic phases of pulsatile flow in order to

compute the estimated oxygen saturation. It is mainly used in clinical care units to

monitor the level of hypoxemia (Styf, 2004). It cannot measure intracompartmental

tissue oxygen saturation and is unable to detect intracompartmental hypo-perfusion

because pulse oximetry technology requires adequate pulsatile flow to compute its signal

(David, 1991). Several reports have demonstrated that pulse oximetry is not an

appropriate aid in the detection or monitoring of impaired perfusion (Mars et al., 1994a;

1994b).

2.2.9 Hardness Measurement Techniques

Several authors have described the use of quantitative hardness measurement

techniques for the diagnosis of compartment syndrome. These techniques are based on

the concept that skin surface pressure (SSP) over a compartment can predict

intracompartmental pressure (Uslu & Apan, 2000; Arokoski et al., 2005). A noninvasive

handheld device formulates a quantitative hardness curve of force versus depth of

48

indentation by applying a 5.0 mm diameter probe to a limb muscle compartment to

estimate the ICP (Steinberg, 2005). Although the accuracy of this technique is not yet

confirmed (Dickson et al, 2003), improvements may enhance the usefulness of this

measure as a noninvasive screening tool for ACS (Joseph et al., 2006).

2.2.10 Direct Nerve Stimulation

Two reports (Sheridan et al., 1977; Rorabeck et al., 1981) have suggested using

direct nerve stimulation at the site where a nerve enters the compartment in patients who

are unable to voluntarily contract the muscles of a compartment. This technique can

differentiate a motor dysfunction due to neuropraxia secondary to a high ICP, from a

primary nerve injury proximal to the compartment. The lack of muscular contraction in

response to electrical stimulation of the compartment nerve can indicate the ACS while

the presence of a muscle contraction in response to the stimulation may indicate a

proximal nerve injury (Styf, 2004). However, since paralysis is a late sign of ACS, this

method is not useful for prospective monitoring of high-risk patients.

2.2.11 Vibratory Sensation

Increased ICP alters regional neural function including the perception of vibratory

sensation (Styf, 2004). In a clinical study, Phillips et al. (1987) demonstrated a direct

correlation between impaired perception of vibration and increasing ICP. They suggested

49

that diminished response to vibratory stimuli as measured with a 256 cycle per second

tuning fork may be a useful and very early indication of a developing ACS (Dellon et al.,

1983). However, like other subjective measurements, it is not practical in children,

unconscious patients, or in those unable to cooperate.

2.2.12 Tissue Ultrafiltration

Tissue ultrafiltration is a technique in which small semi-permeable hollow fibers

are inserted into tissues for the removal of local interstitial fluid. It is primarily used as a

laboratory method of assaying the interstitial space. Initially, Odland et al. (2005)

hypothesized that a tissue ultrafiltration device can extract local interstitial fluid directly

from the involved muscles. Extracted fluid, with a higher than expected concentration of

metabolic markers of muscle ischemia may predict ACS. Further refinement of this

technique in addition to identification of a specific biomarker indicative to skeletal

muscle ischemia could be of some benefit to early diagnosis of acute compartment

syndrome.

2.3 Summary

An ACS is diagnosed by the interpretation of a collection of clinical signs and

symptoms in a high-risk patient. This requires a detailed examination, a vigilant examiner

and a cooperative patient. In situations when the examination is equivocal or unreliable,

50

objective tests that include compartment pressure monitoring can add information for

clinical decision-making. The early identification of compartment syndrome can

significantly reduce the physical, financial and vocational disability experienced by the

injured patient. Advances in the methods, technology and application of techniques for

the early diagnosis of a compartment syndrome have renewed interest for their

investigation. Serum markers of muscle damage, the measurement of changes in

intracompartmental pressure and the measurement of compartmental perfusion and

ischemia provide promising opportunities for clinicians and researchers (Table 2.1).

Further efforts in this area are encouraged.

Table 2.1.

Comparison of advantages between diagnostic methods of acute compartment syndrome.

51

CHAPTER 3

Wireless Near-Infrared Spectroscopy of Skeletal

Muscle Oxygenation During Exercise and

Ischemia*

* A version of this chapter has been published in a peer-reviewed journal as:

Shadgan B, Reid WD, Gharakhanlou R, Stothers L and Macnab A. Wireless near-infrared spectroscopy of skeletal muscle oxygenation and hemodynamics during exercise and ischemia. Spectroscopy. 23(5):233-241, 2009.

52

3.1 Introduction

Near-infrared spectroscopy (NIRS) is a well-established optical technique that

monitors change in concentration of the chromophores oxygenated (O2Hb), deoxygenated

(HHb) and total hemoglobin (tHb) in a variety of tissues (Wolf et al., 2007; Hamaoka et

al., 2007). In the study of muscle, the application of NIRS utilizes the relative

transparency of tissue to photons in the near-infrared (NIR) spectrum, and the oxygen-

dependent absorption changes of these photons by hemoglobin (Hb) and myoglobin

(Mb). Spectrum overlap prevents distinction between Hb and Mb, but with continuous

wave (CW) NIRS instruments using multiple NIR wavelengths, it is possible to use

software algorithms to derive chromophore concentrations from raw optical data, and

distinguish between oxy and deoxy hemoglobin/myoglobin (O2Hb/O2Mb and

HHb/HMb).

The feasibility of conventional CW NIRS in monitoring the patterns of skeletal

muscle chromophore changes during rest, isometric exercise and ischemia are reported by

different investigators (De Blasi et al., 1993; van Beekvelt et al., 2002a; Kime et al.,

2003; Usaj et al., 2007). NIRS investigations have contributed new knowledge related to

muscle physiology at a basic science level, as a measure of performance in exercise

science (Quaresima et al., 2003; Pereira et al., 2007) and also to monitor muscle hypoxia

and ischemia in sports medicine (van den Brand et al., 2004). The non-invasive nature of

the transcutaneous NIRS interface, and ability to monitor continuously even during

physical movement and active exercise enables measurement of oxygenation and

hemodynamics in muscle tissue in health and disease.

53

The majority of commercially available NIRS instruments are continuous wave

spectrophotometers, and have proven reliability in the measurement of changes in O2Hb

and HHb. Multi-channel CW instruments can monitor more than one site simultaneously,

and when configured with a grid capable of holding multiple source-detectors and

appropriate software, are used for topographic mapping (fNIRS). The fNIRS technique

does not require strict motion restriction so it is well suited for monitoring during normal

activities including exercise (Hoshi, 2007). CW instruments can use a variety of light

sources including laser diodes with discrete wavelengths, a white light source combined

with a charge-coupled-device (CCD) array and a grating to discriminate for wavelength

(Cope et al., 1989), or light emitting diodes (LED). In spite of having a broader emission

spectrum, LEDs have several advantages when compared to lasers, particularly their low

cost and ability to be applied directly to the skin without the need for optical cables or

lenses (Muehlemann et al., 2008). CW instruments detect light returning from tissue

using photon counting hardware, usually consisting of a photodiode, photo multiplier

tube or CCD. With CW technology, the assumption is made that photon scatter in tissue

is constant and a tissue specific differential path length factor (DPF) is used to calculate

the optical path length from the inter optode distance (Delpy et al., 1988).

The basic components of a CW NIRS instrument are: a) a pulsed light source for

each chromophore being sampled emitting light at a specific wavelength in the 729 to

920 nanometers (nm) NIR range; b) fibreoptic bundles that transmit light from the source

to a tissue interface (probe or patch) and back to the instrument’s photon counting

hardware; c) an emitter and receiver in the tissue interface that introduces light into the

tissue and receives the photons returning, respectively; d) photon counting hardware; d)

54

computer with software containing algorithms for converting raw optical data into

chromophore concentrations, storing and displaying data; e) a visual display where NIRS

data are displayed numerically and/or graphically against time.

Recent refinements to this basic monitoring methodology have broadened the

research potential of NIRS. Some instruments provide the option to select wavelengths

from multiple options; the ability to use more than one data channel to allow comparison

between monitoring sites and/or tissue (e.g. opposite limbs, or to monitor muscle and

brain simultaneously); or a signal weighted towards brain or muscle tissue (the former is

achieved by subtracting a superficial signal from a deeper signal) (McCormic et al.,

1991; van der Sluijs et al., 1997). Research instruments incorporate other technology

such as phase modulation, or time resolved spectroscopy. In addition, a range of

instruments are now configured for spatially resolved spectroscopy (SRS). SRS

incorporates multiple sensors at different distances from the emitter, which enables the

ratio of oxygenated to total tissue hemoglobin to be measured and a quantitative measure

of tissue oxygenation to be derived (Rolfe, 2000; Boushel et al., 2001; Ferrari et al.,

2004). Current technology affords the opportunity for successfully monitoring during

exercise or participation in a range of sports (Neary, 2004).

CW instruments of small size, particularly those with telemetric capacity,

represent an important advance in sports medicine and exercise physiology studies (Wolf

et al., 2007). Instruments with wireless capability include: a single-channel wearable

NIRS system capable of monitoring brain activity in freely moving subjects (Shiga et al.,

1997; Hoshi et al., 2006); a commercially available miniaturized device (Arquatis GmbH,

Switzerland) that combines spatially resolved NIR spectroscopy with a 3D-accelerometer

55

for muscle measurement during field training; a NIR imaging sensor incorporating 4 light

sources and 4 detectors capable of a sampling rate of 100 Hz (Muehlemann et al., 2008);

and a 3 wavelength single channel CW reflectance instrument used to study muscle

oxygenation in athletes (Liu et al., 2003).

CW instruments have demonstrated feasilibility in clinical applications. The NIR

imaging sensor was tested prior to brain study using a conventional arterial occlusion

experiment to validate the functionality of the system. Using CW NIRS, changes in

chromophore concentration in the brachioradialis muscle of a male volunteer was

comparable to data obtained with conventional NIRS in response to 5 consecutive periods

of ischemia. The feasibility of assessing hemodynamic changes in the cerebral cortex

during thumb and index finger tapping was then demonstrated (Muehlemann et al.,

2008). Another study used 3-wavelength single channel CW NIRS to study oxygenation

in the quadriceps muscle of 10 athletes using 2 experimental protocols; a maximal output

power experiment and three-step incremental load exercise (Liu et al., 2003).

We report the use of a CW wireless NIRS instrument in a spatially resolved

configuration to monitor forearm muscle oxygenation and hemodynamics in healthy

subjects during isometric exercise and tourniquet-induced ischemia. The ease of use of

this instrument and internal consistency of data obtained confirm the feasibility of

applying miniaturized NIRS technology with telemetry for clinical conditions that require

continuous monitoring of skeletal muscle oxygenation and hemodynamics.

56

3.2 Materials and Methods

3.2.1 Instrumentation

The model used is a compact, self-contained unit (PortaMon® developed by

Artinis Medical Systems, BV, the Netherlands) that incorporates the following features.

The unit measures 83 X 52 X 20 millimeters and weighs 84 grams, and uses paired light

emitting diodes with wavelengths of 760 and 850 nm as the NIR light source. Three pairs

of these LEDs are mounted in a spatially resolved configuration so that the three light

sources and one receiver provide 3 source-detector separation distances (30, 35 and 40

millimeters). The sensor is an avalanche photodiode with ambient light protection.

Power is supplied by a rechargeable lithium polymer battery with a capacity of about 6

hours of continues monitoring. The unit has an internal memory with a capacity of 2

megabytes to store data during ambulatory measurement, and incorporates Bluetooth®

technology with broadcast range of 30 meters to transfer data to a laptop computer for

data analysis, graphic display and storage. The spatially resolved configuration of the

emitters to the sensors provides pathlength geometry, which makes it possible to also

derive a measure of tissue oxygen saturation (TSI%) (Suzuki et al., 1999). The

instrument is attached to a subject by means of an adjustable strap or tape.

Software controlling the device allows data interpretation at each source-detector

distance, and derives changes in the concentration of the chromophores, oxygenated

(O2Hb) and deoxygenated (HHb), from the raw optical data and total hemoglobin (tHb)

as the sum of O2Hb and HHb. Spatially resolved configuration of this NIRS equipment

enables real time calculation of tissue oxygen saturation.

57

3.2.2 Subjects

Ten volunteer male athletes were recruited following interview to exclude any

major co-morbidity and/or significant acute or chronic injury that could affect their

ability to perform the forearm muscle contraction protocol required for the study (see

Table 3.1 for demographic data). The study received ethical approval and all subjects

provided informed consent.

3.2.3 Protocol

The equipment was set up was standardized for all subjects (Figure 3.1). Each

subject sat in a chair with his right arm placed on a table with the elbow extended at the

level of the heart, and the forearm was positioned in an upward angle of 30 degrees with

the hand resting on a handgrip dynamometer. Support was provided to maintain a stable

position and an unrestricted circulation while leaving the forearm free.

58

Figure 3.1. The wireless instrument positioned over the flexor digitorum superficialis

muscle for study of muscle oxygenation and hemodynamics.

The mid point of the belly of the flexor digitorum superficialis muscle was identified and

marked. The skin-fold thickness at this location was measured using a skin-fold caliper,

and the wireless NIRS device placed over the marked point and fixed by taping. A

sphygmomanometer cuff was placed loosely around the upper arm in order for arterial

occlusion to be applied as part of the experiment. Although the sensor in the instrument

incorporates an ambient light filter the forearm was covered by an opaque cloth to avoid

any possibility of signal contamination by ambient light.

59

Maximum voluntary contraction force (MVC) was measured three times with 1-

minute periods of rest in between. The highest of the three measures was defined as the

MVC. Values for 10, 30 and 50% of the final MVC were calculated and marked on the

dynamometer display (Smedley Hand Dynamometer) so as to be visible to the subject.

After a 10-minute rest period monitoring began with one minute of baseline

measurement and continued throughout the remainder of the protocol at 10 Hz. Each

subject was asked to perform a 30 seconds sustained isometric handgrip contractions at

defined values of MVC followed by 3 minutes periods of rest; first at 10% of MVC, then

at 30% MVC, and finally at 50% MVC. After 10 minutes of rest, the sphygmomanometer

cuff was inflated to a pressure of 250 mmHg and held at this pressure for 30 seconds to

sustain forearm ischemia, and then deflated. NIRS monitoring was continued for another

10 minutes. At this point, the NIRS monitoring and the experimental protocol was

complete.

3.2.4 Data and Statistical Analysis

Chromophore concentrations of O2Hb and HHb and their variable tHb and TSI%

were monitored in real time, sampled at 10 Hz, filtered (Moving Gaussian) and stored on

hard disk for further off-line analysis using dedicated software (Oxysoft, Artinis Medical

Systems, BV, The Netherlands). All NIRS values were zeroed at the start point of the

experiment. Changes of chromophore concentrations and variables were calculated

during each muscle contraction and ischemia interval. Changes in NIRS values at

different MVC% and ischemia period were compared using two-tailed Student’s paired t-

60

test. Data are presented as means ± SD. The level of significance was set at P<0.05 for all

statistical comparisons.

3.3 Results

Ten healthy male athletes participated. All subjects were right-handed. Table 3.1

shows their mean age, height and weight.

Table 3.1.

Subjects Mean SD

Age 30.7 6.2

Weight (kg) 79.1 6.7

Height (cm) 177.0 2.7

Skinfold thickness (mm) 6.8 1.8

Physical characteristics of subjects. Mean (±SD)

Identical patterns of change in tHb, O2Hb, HHb and TSI% were seen in all 10

subjects during the 3 sets of isometric voluntary forearm muscle contraction at 10, 30 and

50% of MVC, and during the subsequent period of ischemia. Figure 3.2 is a

representative graph of data from a typical subject that shows the pattern of change in

61

chromophore concentration (O2Hb and HHb) and tHb observed over the course of the

protocol; TSI% is included below on the same time scale.

Figure 3.2. The pattern of change in chromophore concentration (O2Hb and HHb) and

tHb and TSI% over the experimental protocol in a representative subject.

Some variation in the magnitude of change was observed between subjects. Mean values

(±SD) for all 10 subjects of changes in tHb, O2Hb, HHb and TSI% during 10, 30 and

50% of MVC and the subsequent period of ischemia are shown in Table 3.2.

62

Table 3.2.

NIRS variable or Chromophore

MVC Ischemia

10% 30% 50%

Δ O2Hb -8+5.3 -25.7+13.5* -35.9+11.6* -5.7+3.6

Δ HHb 6.4+4.8* 15.5+5.6* 16.7+5.1 6+2.2

Δ tHb -1+4.2 -9.7+9.5* -19.2+7.2* 0.3+2.5

Δ TSI% -12.2+6.6* -37+13.1* -50.3+7.9* -8.4+1.9*

Mean (±SD) changes of O2Hb, HHb and tHb along with TSI% during 10, 30 and 50% of

MVC and ischemia. *Different than baseline measure (P<0.05).

During isometric contraction of forearm muscles at three different intensities, an

overall decrease in O2Hb (-25.7 ± 13.5 µM & -35.9 ± 11.6 µM, P<0.05) and tHb (-9.7 ±

9.5 µM & -19.2 ± 7.2 µM, P<0.05) were observed at 30 and 50% of MVC respectively

compared to baseline. The level of HHb increased only during muscle contraction at 10

and 30% of MVC compared to baseline (6.4 ± 4.8 µM, 15.5 ± 5.6 µM, P<0.05). The

changes observed in the value of muscle TSI% also differed in response to 10, 30 and 50

% of MVC and during induced ischemia compared to baseline. Tissue muscle oxygen

saturation declined most during 30 and 50% of MVC. This was a consistent finding in all

subjects. Variation in the values obtained was greater during isometric contractions at 30

and 50% MVC than during ischemia (P<0.05).

An example of a typical NIRS tracing is shown in Figure 3.3. Of note,

microvascular arterial pulsation is obvious before tourniquet inflation obstructs arterial

63

flow and immediately after tourniquet deflation. During the ischemia phase of the

protocol, an initial increase in tHb was observed during the inflation of the pneumatic

cuff, consistent with blood flow congestion in the forearm. Once cuff pressure completely

obstructed forearm blood flow, a sharp decrease in O2Hb (-5.7 ± 3.6 µM) and an increase

in HHb (6 ± 2.2 µM) were observed, while tHb tended to remain stable or slightly

increase (0.3 ± 2.5 µM).

Figure 3.3. An example of a typical pattern of tourniquet induced muscle ischemia, which

indicates microvascular arterial pulsation before tourniquet inflation and immediately

after tourniquet deflation.

64

The technical features and software provided with the instrument made it

straightforward to use. Reliable attachment to the subject was readily achieved and data

transferred to the laptop without issue. Clear signals, free of noise or movement artifact

were obtained in all subjects studied. Subjects found the device comfortable to wear

during the period of study; and the compact nature of the instrument and freedom from

optical cables facilitated the set up and conduct of each element of the study protocol.

3.4 Discussion

We have demonstrated the feasibility of monitoring changes in chromophore

concentration in forearm muscle of healthy male subjects during sequential isometric

contractions and a period of induced ischemia using a wireless NIRS instrument. The

data collected show a similarity in the patterns and some variation of the magnitude of

change seen in the NIRS chromophore concentration data.

Each isometric contraction decreased local muscle blood volume by increasing

intramuscular pressure and compressing the small intramuscular blood vessels; tHb fell

while O2Hb decreased and HHb increased. A significant decrease in O2Hb and tHb

occurred in each subject during isometric contraction, and the magnitude of change

became larger at 30 and 50% of MVC as the percentage of maximum voluntary

contraction increased.

During the period of tourniquet-induced forearm ischemia all subjects showed a

similar pattern of increase in HHb and decrease in O2Hb (Table 3.2). The alteration

65

pattern of muscle oxygen saturation in response to three isometric contractions and

subsequent arterial obstruction was consistent and similar in all subjects. The effects of

ischemia on the patterns of change in tissue O2Hb and HHb in our subjects are also

consistent with those observed in other tissues such as the brain and spinal cord (Macnab

et al., 2003).

The changes we observed reflect what would be expected to occur physiologically

in healthy subjects in response to the experimental protocol employed. Our results also

match those observed in prior studies involving muscle using conventional CW NIRS

instruments by van Beekvelt et al. (2003) and Kahn et al. (1998). Even the initial rise in

tHb during the first minute following cuff inflation, due to tourniquet-induced venous

congestion, has been observed in other studies (van Beekvelt et al., 2003).

The changes observed in the value of muscle TSI% differed in response to 10, 30

and 50 % of MVC and during induced ischemia, declining during 30 and 50% of MVC.

This finding is in accordance with the previous NIRS experiments, which demonstrated

that sustained muscle contraction above 30% MVC induces a complete local ischemia

(Kahn et al., 1998; van Beekvelt et al., 2003). The NIRS data we obtained, confirms the

precision of the LED light source and integrity of the technical design of the wireless

instrument, and the accuracy of the algorithm it incorporates.

We recognize limitations in our study including our data being limited to

individual measurements in ten male subjects. However, our experience and results from

this feasibility study advance the potential for wireless NIRS technology to contribute to

studies of basic muscle physiology and performance assessment in sports medicine. This

66

is the first report of a prospective series of NIRS measurements of muscle oxygen

saturation using a wireless device with dual wavelengths in multiple subjects, and adds to

prior reports of a single forearm measurement (Muehlemann et al., 2008) and to the

observations of Liu and colleagues (2003) with a single channel prototype instrument in

10 athletes.

3.5 Conclusions

We have described our successful use of this commercially available wireless

NIRS instrument in the belief that other investigators will be able to design experiments

that benefit from this technology. Wireless monitoring is a significant advance that will

contribute to multiple future applications of NIRS in a range of tissues where changes in

oxygenation and hemodynamics are of relevance. Independent trials are required to

validate our findings. However, we believe that investigators conducting such trials will

rapidly expand the use of this device and the technology it incorporates to other studies,

particularly when their research questions relate to the study of muscle, sports medicine,

and exercise physiology.

67

CHAPTER 4

Monitoring of Tourniquet-Induced Skeletal

Muscle Injury by Near Infrared Spectroscopy

During Orthopaedic Trauma Surgery *

* This chapter has been submitted to a peer-reviewed journal for publication:

Shadgan B, Harris RL, Reid WD, Jafari S, Powers SK, and O’Brien PJ. Monitoring of tourniquet-induced skeletal muscle injury by near infrared spectroscopy during orthopaedic trauma surgery.

68

4.1 Introduction

Clinical studies have reported evidence of tourniquet-induced post-surgical

complications such as muscle paresis, impaired wound healing, skin blistering, infection,

compartment syndrome, deep vein thrombosis and higher levels of limb pain and

swelling (Shenton et al., 1990; Mohler et al., 1999; Wakai et al., 2001; Murphy et al.,

2005; Oddinson & Finsen, 2006; Konrad et al. 2005). Moreover, many studies have

demonstrated a range of neuromuscular functional impairments following application of

pneumatic tourniquet, such as muscle weakness, fatigue and impaired postoperative

recovery (Saunders et al., 1979; Dobner & Nitz, 1982; Shenton et al., 1990; Mafulli et

al., 1993; Daniel et al., 1995; Abdel-Salam & Eyres, 1995; Mohler et al., 1999; Wakai et

al., 2001; Murphy et al., 2005; Oddinson & Finsen, 2006).

The main causes of tourniquet-induced neuromuscular dysfunction appear to be

direct pressure on the nerve and skeletal muscle directly underlying the tourniquet,

ischemic oxidative injury of muscle and microvasculature (neural and muscular) distal to

the tourniquet, and reperfusion-associated oxidative and inflammatory injury distal and

proximal to the tourniquet. The neurological complications of pneumatic tourniquets

have been reported by others (Shenton et al., 1990; Mohler et al., 1999; Wakai et al.,

2001; Murphy et al., 2005; Oddinson & Finsen, 2006), however, the mechanism

underlying deficits in muscle function after tourniquet use remains uncertain. Although

several variables related to tourniquet cuff inflation contribute to the development of

ischemic muscle injury, the duration of ischemia has been identified as the primary factor

(Artacho-Perula et al., 1991; Odinsson & Finsen, 2006).

69

Several studies have reported that skeletal muscle escapes irreversible damage

due to ischemia and the reperfusion that subsequently occurs upon tourniquet release, as

long as the ischemic period does not exceed 3 hours (Harris et al., 1986; Idstrom et al.,

1990; Blebea et al. 1987; Pasupathy & Homer-Vanniasinkam, 2005a; 2005b; Pedowitz et

al. 1992). However, the safe time limit for tourniquet use in humans remains

controversial (Flatt, 1972; Wakai et al., 2001; Chiu et al. 1976; Pedowitz et al. 1992;

Appell et al., 1993). The current clinical standard for the maximum continuous tourniquet

time during orthopedic surgery of the lower extremity is 90 minutes (Noordin et al.,

2009). The primary evidence for this benchmark appears to be based on animal studies,

but adopting the results of such animal studies for the clinical setting is limited by the fact

that, compared to human skeletal muscles, animal limb muscles differ in their metabolic

and muscle fiber types, their structure, and their biomechanics (Sapega et al., 1985;

Heppenstal et al., 1986; Pedowitz et al., 1992; Mohler et al., 1999; Coirault et al, 2007).

A practical and noninvasive method that assists surgeons to monitor the safe

tourniquet time and pressure during surgery would enable them to find a balance between

the advantages of a bloodless operative field and the risk of ischemic muscle injury,

which could prolong muscle recovery and rehabilitation. A potential method to facilitate

this process is near infrared spectroscopy (NIRS), a non-invasive optical method to

monitor tissue oxygenation and hemodynamics in real time (Terakadu et al. 1999; Moalla

et al. 2005). In recent years, NIRS has been validated and used by many investigators to

monitor regional tissue oxygenation, hemodynamics and metabolism in health and

disease (Mancini et al., 1994; Boushel et al., 2001; van Beekvelt et al., 2003). However,

the application of NIRS in monitoring limb muscle ischemia is limited to a number of

70

studies regarding limb muscle oxygenation during short-duration venous and arterial

occlusion (Gentillelo et al., 2001; Yu et al., 2005; Vo et al., 2007; Gomez et al., 2008;

Shadgan et al., 2009).

Accordingly, the purpose of the present study was to examine the feasibility and

convergent validity of NIRS for continuous monitoring of skeletal muscle oxygenation

and hemodynamics during tourniquet-induced ischemia. We also aimed to investigate the

predictive value of NIRS-derived variables for evaluation of limb muscle oxidative

changes during tourniquet-induced ischemia. Specifically, we used NIRS to monitor

skeletal muscle oxygenation and hemodynamics distal to the tourniquet during surgery,

and examined ischemia-related muscle protein degradation using biochemical analyses of

surgical muscle biopsies.

We hypothesized that NIRS would be proved to be a feasible method for the continuous

monitoring of tourniquet-induced limb muscle ischemia. We also hypothesized that

protein oxidative changes in muscles distal to the tourniquet would be correlated

positively to tourniquet time and changes in HHb and reoxygenation rate, and inversely

to changes in O2Hb and tHb as monitored by NIRS.

71


4.2.1 Subjects

A convenience sample of patients, admitted to a level 1 trauma hospital with

closed ankle fractures requiring emergency surgery, were recruited. Inclusion criteria

were: adults with unilateral ankle fractures, no major comorbidity (Charlson et al., 1987)

and no current or previous contralateral injuries that might affect the reliability of NIRS

measurements on either limb. Twenty-four subjects entered the study, of whom 17

patients had a complete data set of outcomes including biopsy and NIRS data. The study

received institutional research ethics board approval and informed written consent was

obtained from all volunteers before participating. All procedures complied with the

Declaration of Helsinki.

4.2.2 Experimental Overview

All patients underwent a standardized general anesthetic. After surgical

preparation and positioning of the lower limbs, a pair of NIRS probes (Oxymon III,

Artinis) were placed and fixed over the proximal third of the tibialis anterior (TA)

muscles bilaterally. A thigh tourniquet (Zimmer ATS 2000, IN, USA) was applied to the

injured leg, and was inflated to 300 mm Hg after elevating the limb. Using the NIRS

apparatus, chromophore concentrations of oxygenated (O2Hb) and deoxygenated

hemoglobin (HHb) were measured bilaterally in the TA muscles before and during

tourniquet inflation, and after tourniquet release until O2Hb returned to the baseline

72

value. Mean systemic arterial pressure, heart rate and arterial oxygen saturation were

obtained from the upper extremity using an automated blood pressure cuff and a pulse

oximeter (AS3000™ Anesthesia Delivery System, Mahwah, NJ). Muscle biopsies were

collected from the peroneus tertius (PT) distal to the tourniquet: 1) immediately after

tourniquet inflation (Pre-biopsy), and 2) towards the end of the surgical procedure

immediately before tourniquet deflation (Post-biopsy). The tourniquet was released when

the surgeon decided that arterial obstruction was no longer required. The TA muscle was

chosen for NIRS monitoring because it is the most superficial muscle within the anterior

compartment of the leg with a thin layer of subcotanous fat and a mixed fibre type

composition. The proximal third of the TA was selected for NIRS monitoring since at

that point NIRS probes could be placed over the skin outside of the sterile field necessary

for ankle surgery. Rather than the TA, the PT was selected for Pre and Post surgical

biopsies because during ankle surgery there is direct surgical access through the surgical

incision to the PT but not to the TA. In addition to their common anatomical location in

the anterior compartment, these muscles are comparable in their distribution of

functionally-classified motor unit types (Dum & Kennedy, 1980; Jami et al., 1982), so it

was assumed that biochemical assays from the PT would be an appropriate comparison to

NIRS measurements from the TA. The experimental protocol is summarized in Figure

4.1.

73

Figure 4.1. A summarized overview of the experimental protocol.


Oxygenation and hemodynamics were continuously monitored bilaterally, in both

TA muscles, under-tourniquet and the contralateral side as a control, using a four-channel

continuous-wave near-infrared spectroscope (Oxymon M III, Artinis Medical Systems,

BV, the Netherlands). The principle of NIRS and the calculation of NIRS-derived

parameters have both been described elsewhere (van der Sluijs et al., 1997; Boushel et

al., 2001; Tachtsidis et al., 2007; Wolf et al., 2007; Shadgan et al., 2009). In this study

we measured changes in chromophore concentrations of O2Hb and HHb in the TA

muscles throughout the surgical procedure from at least 10 minutes before tourniquet

inflation until O2Hb returned to the baseline value following tourniquet deflation.

Tourniquet time (i.e., duration of tourniquet inflation) and several NIRS variables were

calculated for each subject. These variables included total hemoglobin (tHb) (van

Beekvelt et al., 2002a), Hbdiff (Grassi et al., 1999), hyperemia (see Chapter 1), recovery

74

time (Chance et al., 1992), and reoxygenation rate (van Beekvelt et al., 2002b). Data

were monitored in real time, sampled at 10 Hz, filtered (Moving Gaussian with filter

width of 1 second) and recorded by the NIRS instrument for further off-line analysis

using dedicated software (Oxysoft, Artinis Medical Systems, BV, The Netherlands).

Changes in tissue oxygenation, deoxygenation and local blood volume were estimated

from changes in O2Hb, HHb, Hbdiff and tHb. Figure 4.2. shows NIRS data acquisition

during the surgery.

In addition to the NIRS monitoring, any tourniquet adjustment or changes in

surgical setup, such as limb repositioning, that might alter the NIRS reading during the

experiments were recorded. Furthermore, to detect and record any considerable blood

loss, surgical fields were visually inspected during the operations.

Figure 4.2. NIRS data acquisition during surgery in operation room.

75

4.2.4 Biopsy Collection and OxyBlot Analysis

Immediately upon collection, fat and connective tissue were removed from the

biopsy, it was blotted gently by saline-soaked gauze to remove blood, and flash frozen in

liquid nitrogen. Following flash freezing, biopsies were maintained frozen on dry ice and

then stored in a -70ºC freezer until processing. Protein oxidation was measured using

Western blot technique for reactive carbonyl derivatives using the commercially available

OxyBlot protein oxidation detection kit, according to the manufacturer’s instructions

(Millipore, Billerica, MA) as previously described (Kavazis et al. 2009; Zergeroglu et al.

2003). Degree of oxidation was analyzed by comparing signal intensities of the Pre

versus the Post samples for the whole lane (total protein oxidation), according to the

protocol by Zergeroglu et al. (2003).

4.2.5 Statistical Methods

Descriptive statistics were used to summarize subject characteristics. A two-tailed

Student’s paired t-test was used to assess statistical differences in average protein

oxidation between the Pre and Post biopsies, and a Student’s unpaired t-test to assess

statistical differences in the average protein oxidation increase observed between male

and female subjects. We also used a linear regression model to investigate the effect of

each of the following variables on Pre-Post biopsy results: age, gender, body mass index

(BMI), tourniquet time, O2Hb, HHb, tHb, Hbdiff, hyperemia interval, recovery time, and

reoxygenation rate. Each of these independent variables was entered into the linear

regression model one at a time. Data were analyzed using SPSS software (SPSS for

76

Windows, Rel. 11.0.1. 2001. Chicago: SPSS Inc.). Values are presented as means ±

standard deviation. Statistical significance was accepted at P<0.05.

4.3 Results

4.3.1 Descriptive characteristics

A total of 17 patients (13 females and 4 males) with unilateral ankle fractures

were included in this study. The mean age of participants was 49 ±15 with a range of 19-

69 years. The mean participant BMI was 25.9 ± 4.4 kg·m−2.

4.3.2 Tourniquet

A tourniquet application pressure of 300 mmHg was maintained in all subjects. A

bloodless surgical field was obtained in all subjects. The average duration of the

tourniquet time was 43.2 ± 14.6 minutes with a range of 20.7 – 73.8 minutes.

4.3.3 Cardiovascular

Mean arterial pressure during the tourniquet application increased (7.5 ± 4.9

mmHg) while pulse rate and SpO2 showed no significant change from resting values.


Figure 4.3. shows changes of HHb, O2Hb and tHb in the TA muscle in a single

representative subject before, during and after thigh tourniquet inflation.

77

Following tourniquet inflation a progressive increase in HHb (23.7 ± 8.2µM) and

a progressive decrease in O2Hb (-23.4 ± 8.2 µM) in the under-tourniquet TA muscles

were both consistent across subjects. These changes in HHb and O2Hb began to reverse

immediately after tourniquet deflation. Of interest, tHb, a measure of blood volume,

increased in 8 subjects (7.8 ± 5.2 µM) and decreased in 9 subjects (-8.2 ± 5.6 µM) during

tourniquet time. As an index of tissue oxygenation, Hbdiff showed a significant decrease

(-47.1 ± 13.4 µM) during tourniquet application. The rate of Hbdiff decrease was higher

among males and this difference was statistically significant (P = 0.007). Recovery time

and re-oxygenation rate of hemoglobin following tourniquet release were 138.7 ± 63.7

seconds and 5.8 ± 4.1 µM/seconds respectively.

The pattern of changes in muscle oxygenation and blood volume for the TA

muscle following tourniquet inflation and release was significantly different (P < 0.05) in

the experimental leg compared to the control leg, and demonstrated a consistent pattern

across the subjects. In control TA muscles, no significant changes in NIRS variables were

observed throughout the duration of the tourniquet application.

78

Figure 4.3. Chromophore concentration changes for O2Hb and HHb, and NIRS variables

of tHb and Hbdiff, shown in a representative tibialis anterior muscle before, during and

after thigh tourniquet inflation. Changes in tHb (the sum of O2Hb and HHb

concentrations) reflect changes in local blood volume. Changes in Hbdiff (the difference

between changes of O2Hb and HHb concentrations) indicate local tissue oxygenation in

the tibialis anterior muscle. A: tourniquet inflation time; B: tourniquet release time; C:

point of maximum hyperemia; D: point of maximum O2Hb recovery.

4.3.5 Muscle Biopsy

Based on the OxyBlot analysis of protein oxidation in the Pre and Post PT

biopsies from 17 patients, Pre-Post differences in reactive carbonyl derivatives were

compared using a Student’s paired t-test. An average of 43.2 (±14.6) minutes of

tourniquet-induced ischemia resulted in a large increase in total protein oxidation of

174.3 ± 128% (P < 0.0005) (Figure 4.4). There was no significant association between

tourniquet duration and Pre-Post biopsy changes (P = 0.49, R2 = 0.031).

79

Figure 4.4. Raw oxidized protein volume in peroneus tertius samples at the beginning

(Pre) and end (Post) of tourniquet inflation. The raw contents of carbonylated proteins in

peroneus tertius muscle biopsy homogenates were determined by integrated densitometry

of Western blots prepared using the commercially available OxyBlot method. The mean

values for Pre (white bar) and Post (grey bar) samples were calculated after correcting

the raw values to the loading control sample that was run on all gels. The percent

difference between Pre and Post (black bar) indicates the increase in protein oxidation

that occurred during the ischemic period. As indicated, the Pre-Post difference was

significant at P<0.00005.

In the examination of the influence of gender, in Post samples compared to Pre

samples, protein oxidation increased 227.7 ± 97% in men and 129.2 ± 136.8% in women.

That is, the average increase in protein carbonylation as determined using the OxyBlot

method was 51% higher in male subjects than in female subjects (P = 0.022, R2 = 0.30)

(Fig 4.4). No significant correlation was found between Pre-Post biopsy analysis and

subject age (P = 0.48, R2 = 0.03), or BMI (P = 0.8, R2 = 0.04).

80

Figure 4.5. Percent increase in peroneus tertius (PT) protein oxidation in men and

women. These average percent differences were recalculated separately (cf. Figure 4.3)

for the two gender subgroups of male (white bar) and female (black bar) subjects. As

indicated (*), the gender difference in PT protein oxidation during ischemia was

significant at P <0.05.

4.3.6 OxyBlot vs. NIRS Regression

Linear regression was conducted in order to assess the relationship between each

of several NIRS variables and the degree of Pre-Post change in total protein oxidation.

We found that changes in both O2Hb and tHb were significantly negatively correlated

with the average increase in protein oxidation for all subjects. Specifically, 1 µM

decrease in O2Hb resulted in 6.1% increase in protein oxidation (P = 0.040, R2 = 0.25)

n = 4

n = 13

81

(Figure 4.6), and 1 µM increase in tHb resulted in 11.8% decrease in protein oxidation (P

= 0.003, R2 = 0.58) (Figure 4.7).

Figure 4.6. Scatter plot and regression line showing the correlation between ΔO2Hb and

Pre-Post changes in muscle protein oxidation.

82

Figure 4.7. Scatter plot and regression line showing the correlation between ΔtHb and

Pre-Post changes in muscle protein oxidation.

In contrast, we found that reoxygenation rate was significantly positively

correlated with the average increase in protein oxidation for all subjects. In fact, for each

unit increase in reoxygenation rate the increase in protein oxidation was 18.1% higher (P

= 0.041, R2 = 0.57) (Figure 4.8). No significant correlation between the average increase

in total protein oxidation and changes in NIRS-derived hyperemia interval (P = 0.65, R2

= 0.02) and also the recovery time of oxygenated hemoglobin (P = 0.42, R2 = 0.03) was

found. Additionally, we did not observe correlations between HHb or Hbdiff with protein

oxidation (P = 0.63 & P = 0.31 respectively).

83

Figure 4.8. Scatter plot and regression line showing the correlation between

reoxygenation rate and Pre-Post changes in muscle protein oxidation.

4.4 Discussion

The present study showed that NIRS is a feasible and efficient method for non-

invasive monitoring of skeletal muscle ischemia. We have also demonstrated that an

average of 43 minutes of tourniquet-induced ischemia, without reperfusion, resulted in

significant limb muscle protein oxidation. The observed muscle protein oxidation was

greater in men than in women. We also demonstrated predictive criterion validity of

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NIRS by the significant negative association between the extent of tourniquet-induced

muscle protein oxidation and changes of local muscle blood volume measured by NIRS.

Since 1904, when Harvey Cushing introduced the first pneumatic tourniquet,

many investigations have explored the relationship among tourniquet-induced ischemia,

reperfusion and skeletal muscle injury. Recent investigations of tourniquet use in human

patients have emphasized the impact of the reperfusion period that necessarily follows

ischemia (Haljamae & Enger, 1975; Huda et al., 2004; Mathru et al., 2007; Barreiro &

Hussain, 2010). In this context, it has been observed that about 2 hours of tourniquet-

induced ischemia for elective knee surgery followed by 2 hours of reperfusion leads to

increases in pro-inflammatory markers and adhesion molecules both locally, in the

affected muscle, and systemically, in the plasma (Huda et al., 2004; Mathru et al., 2007).

In contrast to previous studies that report no protein carbonylation after ischemia

without reperfusion (Hammerson et al., 1989; Barreiro & Hussain, 2010; Grisotto et al.,

2000, Ozyurt et al., 2006), we found large and significant increases in the oxidation of

muscle proteins after 43 minutes (on average) of tourniquet-induced ischemia per se. Our

data are somewhat consistent with findings of Appell et al., (1993), which demonstrated

that in healthy young subjects undergoing ACL reconstruction even as little as 15

minutes of tourniquet-induced ischemia alone, without reperfusion, results in muscle

damage characterized by myofiber edema in combination with thickening of capillary

basement membranes. It has been purported, however, that intramuscular oxidative stress

is directly linked to increases in myofiber edema and ruptures of capillary endothelia,

leading to increased capillary permeability and exudation of myofiber contents (e.g.,

85

proteins) into the vasculature (Appell et al. 1997; Duarte et al., 1997; Nylander et al.

1989).

It is well documented that tourniquet duration for more than 90 minutes is

positively associated with an increased rate of tourniquet-induced complications (Sapega

et al., 1985; Artacho-Perula et al., 1991; Odinsson & Finsen, 2006, Bollman & Flock,

1944). In our study although we observed that tourniquet-induced ischemia was

associated with a significant increase in the oxidative modification of muscle proteins, it

may be important for clinical practice that we also observed no significant correlation

between the increase in muscle protein oxidation and the average tourniquet time interval

of 43 minutes (range: 21-74 minutes). This indicates that for tourniquet applications up to

approximately 1¼ hours, the degree of tourniquet-induced muscle damage is not

determined solely by the ischemia interval.

We observed a significantly greater increase in skeletal muscle protein oxidation

in the peroneus tertius of male subjects than in the peroneus tertius of female subjects.

This gender difference in oxidative muscle damage may contribute to the tourniquet-

related post-surgical pain management that is more difficult to manage in men than in

women (Omeroglu et al., 1997). Likewise, exercise-induced oxidative muscle damage

and associated soreness are considerably more severe in men than in women, perhaps

because women exhibit significantly more robust endogenous cellular antioxidant

mechanisms (Kerksick et al., 2006). The apparent protective effect of estrogen on cell

membrane stability might be another explanation for these findings (Tiidus, 2000;

Sugiura et al., 2006). Mounting evidence from recent animal studies strongly indicates

that ageing-related oxidative modification of proteins, lipids, and DNA in skeletal and

86

cardiac muscle, blood plasma, and brain tissue are greater in males than in females

(Cakatay et al., 2010; Fano et al., 2001; Kayali et al., 2007a; 2007b; Uzun et al., 2010),

and thus the possibility remains that the gender difference we observed in the current

study was due in part to a gender-age interaction, although this interaction was not a

statistically significant finding in our study.

Theoretically, muscle oxidative damage should have a dramatic impact on muscle

recovery and post-surgical functional rehabilitation. Tourniquet-induced oxidation of

limb muscles, and the subsequent inflammatory response, may represent the initial step

towards muscle atrophy (Huda et al., 2004; Appell et al., 1993), especially in those cases

that require a period of immobilization post-surgery. Importantly, simply unloading or

immobilizing a muscle, or reducing neuromuscular activation, results in much slower

atrophy than what is observed when oxidative stress and inflammation participate in the

signaling of more dramatic myonuclear apoptosis (Powers et al., 2007). Moreover,

oxidative (and inflammatory) damage renders myofibrillar proteins more vulnerable to

proteolysis, likely because oxidative modification leads to protein unfolding and hence

lends better proteolytic access to degradative enzymes such as calpains and caspase-3

(Smuder et al., 2010). In other words, tourniquet use during orthopaedic surgery is likely

to exacerbate functional deficits and prolong post-surgical recovery if the deleterious

effects of oxidative stress on myofibrillar protein integrity are not somehow mitigated

(Powers et al., 2007; Smuder et al., 2010). It is not surprising, then, that tourniquet-

induced muscle protein oxidative changes are also associated with increased incidence of

post-surgical complications (Konrad et al., 2005). In addition, the inflammatory

component of this relationship also appears crucial to patient recovery since it has been

87

shown that ankle fracture fixation leads to considerably higher levels of pain and swelling

when tourniquets are used compared to when they are not used, and this pain and

swelling persist for at least 6 weeks (Konrad et al., 2005). From the clinical perspective,

therefore, it is ultimately important to consider what the effects are of tourniquet-induced

muscle ischemia on patient outcomes, and in this context it may be crucial to monitor

muscle ischemia during surgery to find the most safe tourniquet time for each individual

patient. Furthermore, predicting the degree of tourniquet-induced muscle damage at the

end of surgery could help surgeons to consider the necessary therapeutic measures for

limiting the muscle damage.

Reactive hyperemia following tourniquet release is well documented. Reactive

hyperemia refers to the post-ischemia increase in limb muscle blood perfusion (i.e.,

reperfusion) that, using NIRS, can be measured as an increase in tHb (Fahmy & Patel,

1981). In contrast to the early reperfusion period, however, increases in tHb during

tourniquet inflation should not be expected. Gradual increases in tHb in muscles distal to

the tourniquet indicate that the tourniquet pressure is insufficient to induce a complete

limb arterial occlusion, and gradual decreases in tHb in muscles distal to the tourniquet

indicate that there is blood loss from the surgical field. In this study, tHb decreased in 8

patients and increased in 9 patients during tourniquet usage. Regression analysis of NIRS

variables demonstrated a significant negative relationship between the degree of

tourniquet-induced muscle protein oxidation and both ΔtHb and ΔO2Hb.

Taken together, our findings that protein oxidation is not related to tourniquet

time, up to 74 minutes in the lower extremity, but is negatively correlated with both ΔtHb

and ΔO2Hb suggest that muscle oxidative injury distal to the tourniquet is closely

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associated with changes in oxygenated blood volume. In other words, it appears that

muscles are in fact protected against ischemic injury (i.e., protein oxidation) when there

is arterial leakage at the tourniquet site, but that oxidative injury is intensified when blood

loss distal to the tourniquet leads to decreased limb muscle blood volume.

Previous studies have shown that the actual amount of tourniquet pressure for a

complete arterial occlusion varies across individuals, and must account for several

variables including tourniquet cuff design, limb circumference, limb morphology and fat

distribution, and systolic blood pressure (Noordin et al., 2009; McLaren & Rorabeck,

1985; Shaw et al., 1982). In fact, for a given pressure of a tourniquet cuff, patients with

higher circumference of the limb, higher BMI, or uncontrolled hypertension may show a

small amount of blood leakage at the tourniquet site (Ishii et al., 2008). Obviously,

reducing the pressure of the tourniquet cuff would further increase the risk of blood

leakage.

Several investigations have also shown that lowering the cuff pressure to a safe

limit above the systolic blood pressure during the limb surgery minimizes the rate and

severity of the tourniquet-induced complications by reducing the external compression on

limb neurovasculature (Ishii et al., 2005; Shenton et al., 1990; Wakai et al., 2001;

Murphy et al., 2005; Oddinson & Finsen, 2006). Our data from ∆tHb and ∆muscle

protein oxidation relationship suggest that using a pneumatic tourniquet that permits a

very small arterial leak rather than completely occluding arterial flow may protect the

muscle tissue distal to the tourniquet against ischemia-associated oxidative injury.

Further investigations are required to confirm this observation.

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It has been reported previously that the ischemia and the subsequent reperfusion

are characterized by endothelial damage, increased leukocyte-endothelial cell adhesion,

inflammation, increased microvascular permeability, increased numbers of no-flow

capillaries, and erythrocyte aggregation (Appell et al., 1993; Durán & Dillon 1989;

Schoen et al., 2007; Tamas et al., 2010). In this context, we found that in muscles distal

to the tourniquet the degree of tourniquet-induced muscle protein oxidation was

positively correlated with changes in reoxygenation rate. This implies that the oxidative

damage that occurs during tourniquet use contributes to faster reoxygenation during

reperfusion. The tourniquet-induced muscle protein damage that occurs during ischemia

may also be caused in part by the endothelial damage and inflammatory response that

worsen progressively throughout ischemia (Appell et al., 1993; Schoen et al., 2007;

Tamas et al., 2010), which should theoretically explain how the degree of protein

oxidation could be linked to the rate of reoxygenation observed during reperfusion. Thus,

measuring this NIRS-derived index upon tourniquet release may help surgeons to

estimate the degree of tourniquet-induced muscle protein oxidation that developed during

the surgery. In fact, it may be possible to use reoxygenation rate as a predictive index of

the degree of muscle protein damage for planning more effective postoperative care.

This study showed that CW NIRS is a feasible noninvasive method for the

continouous monitoring of tourniquet-induced limb muscle ischemia during lower limb

operations. We found that instrument set-up and sensor placement do not limit the

operation of NIRS inside the orthopaedic OR. All patients tolerated the NIRS sensors and

no operational limitation was observed in patients with different age, gender, BMI and

skin colour.

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There are limitations to this study. Although NIRS data have been extensively

used for studying muscle oxygenation and blood flow, this technique should be

considered as a non-invasive measure for estimating changes in muscle oxygenation and

blood volume (Boushel et al., 2000b; 2002). Examining only the tibialis anterior for

NIRS analysis and the peroneus tertius for muscle biochemical analysis may not

represent other muscles outside the anterior compartment. These two muscles were

selected due to the ease of NIRS monitoring and surgical biopsy access, respectively.

Both muscles are of mixed fiber type, however, so they are likely to provide more

broadly representative measures than leg muscles that are predominantly fast or slow

twitch.

It should also be recognized that the small sample size in the current study might

have a negative effect on finding a statistically significant relationship between muscle

protein changes and some of the other study variables such as age, BMI, and recovery

time. Additionally, we limited our subjects to those people without major comorbidities.

Finally, we did not follow up the patients to assess their postoperative condition and the

functional rehabilitation of their leg muscles, so we cannot directly compare the extent of

tourniquet-associated protein oxidation to longer-term functional outcomes.

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4.5 Conclusions

This study demonstrates that assuming a constant safe tourniquet time for all

lower limb surgeries may put some patients at risk for oxidative changes in skeletal

muscle proteins, which could potentially damage the affected muscle tissue and delay

post-surgical rehabilitation. We showed that tourniquet-induced muscle ischemia for 21

to 74 minutes during lower extremity surgery leads to considerable ischemic muscle

injury as measured according to myofibrillar protein oxidation. Importantly, this injury

occurs even without reperfusion. Our data indicate that the extent of skeletal muscle

oxidative injury is greater in men than in women, but is not related to age or of ischemia

interval for tourniquet applications up to ~75 minutes in our sample.

Using NIRS, we observed that leakage of a small amount of oxygenated blood

volume from the tourniquet site could effectively protect skeletal muscles distal to

tourniquet against oxidative damage during surgery. Further studies with larger sample

size, and preferably control trial designs, are required to prove this protective concept of

tourniquet leakage.

Our findings also indicate that NIRS is a feasible and efficient technique for non-

invasive monitoring of muscle oxygenation and hemodynamics in the clinical setting.

The findings of this study provide a foundation for future studies regarding the use of

NIRS for continuous monitoring of skeletal muscle oxygenation, as well as regarding the

possible use of NIRS for early diagnosis of critical muscle ischemic conditions such as

acute compartment syndrome in high-risk trauma patients.

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CHAPTER 5

Do Radio Frequencies of Medical Instruments

Common in the Operating Room Interfere with

Near-Infrared Spectroscopy Signals? *

* A version of this chapter has been published in a peer-reviewed journal as:

Shadgan B, Molavi B, Reid WD, Dumont G, Macnab AJ. Do radio frequencies of medical instruments common in the operating room interfere with near-infrared spectroscopy signals? Proc. SPIE, Vol. 7555, 755512;doi:10.1117/12.842712, 2010.

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5.1 Introduction

All electric devices including monitoring equipment used in the intensive care

unit (ICU) and the operating room (OR) have the potential to produce radiofrequency

electromagnetic radiation, which may affect the functioning of other medical devices in

use (Silberberg, 1993; Segal et al., 1995). Electromagnetic interference (EMI) may

corrupt and alter digital data and analog signals of essential medical instruments.

Although it is difficult to predict all the factors that can contribute to the generation of

significant EMI, it is known that electric devices with electromagnetic frequencies

between 10 kHz and 1GHz at distances closer than 1 meter have the potential to be

significant sources of EMI (Lapinsky & Easty, 2006). Older instruments with less

electromagnetic compatibility are also reported to be more susceptible to EMI (IEC,

2002). It is important to evaluate the potential of any new electric medical device that is

being considered for use in the ICU or OR for its potential to generate significant EMI

and also be affected by other devices.

Near infrared spectroscopy (NIRS) is an optical technology, which uses the near

infrared region (NIR) of the electromagnetic spectrum (700 - 1000 nm) to monitor

changes in local blood volume and tissue oxygenation (Ferrari et al., 2004). NIRS is

based on two main physics principles: 1) the transparency of tissue to NIR photons which

scatter widely and are variably absorbed by naturally occurring chromophores, with

oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb) being the two chromophores of

principal interest; and 2) the ability to measure changes in local concentrations of

chromophores O2Hb and HHb by comparing the photons transmitted through and

returned from the tissue (Hamaoka et al., 2007). This technique has been widely used in

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research, and applied in medicine as a non-invasive tool for clinical diagnosis of ischemic

and hypoxic conditions, and continuous monitoring of local tissue oxygenation and

hemodynamics (Jobsis, 1977; Gagnon & Macnab, 2005a; Hamaoka et al., 2007; Wolf et

al., 2007; Stothers et al., 2008).

NIRS has been applied in the OR for different clinical purposes such as:

evaluating cerebral blood flow and oxygenation during cardiovascular surgeries

(Steinbrink et al., 2006), monitoring the severity of shock in trauma surgeries (Beilman et

al., 1999), and diagnosis of acute compartment syndrome and limb ischemia during limb

surgeries (Tobias & Hoernschemeyer, 2007). There is a progressive trend of using NIRS

in OR and critical care units as a research tool and also a monitoring method.

The objective of this study was to address the question of whether EMI generated

by three devices commonly used in orthopedic surgery have any effect on NIRS signals.

The category of devices into which NIRS optodes fall is “Optical Fiber Sensors”;

these are made of dielectric material and are immune to any form of EMI and can be used

in environments in which conventional electronic sensors are unsuitable (López-Higuera,

2002). NIRS monitoring is based on attenuation of an optical signal during passage

through tissue, and therefore operates in a specific region of the electromagnetic

spectrum where signals will not be affected by radio frequency interference (RFI) from

other devices. Noise generated by light can cause interference, for example fluorescent

lights with certain wavelengths, OR lights of high intensity, or even bright daylight.

However, none of the three devices we evaluated emit light in the wavelengths close to

the NIR region. The electronic components of NIRS instruments that generate and

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control the lasers, however, are susceptible to RFI and EMI. These components record

the signal from the source/detector interface, and transmit data to a computer. To ensure

reliable clinical use of NIRS devices in the OR or ICU environments, it is necessary to

study how the instrument performs in proximity to other equipment in these locations.

Hence, the potential of three devices commonly used by orthopaedic surgeons in the OR

to generate EMI capable of affecting simultaneous monitored NIRS signals was

investigated in this study. Our hypothesis was that medical devices commonly used

during orthopedic surgery would prove not to generate interference that compromises

NIRS intra-operative monitoring.


NIRS data were collected from patients with an ankle fracture who required

orthopaedic surgery at a university hospital. All patients provided informed consent. A 4-

channel continuous wavelength NIRS instrument (Oxymon III, Artinis Medical

Technologies, the Netherlands) with lasers of 760 and 864 nm and a sampling rate of 10

Hz was used to monitor changes in O2Hb, HHb, and total hemoglobin (tHb) in leg muscle

on the fractured and non-fractured limbs. The full specifications and performance of the

instrument have been described in the context of muscle studies (van Beekvelt et al.,

2002a). Data was obtained in each subject throughout the operation for every occasion

each device under evaluation was used, and included the start and end times and duration

of use. The devices were: an electrocautery (The System 5000TM, ConMed), a battery-

powered orthopaedic drill (Hall PowerPro®, ConMed Linvatec) and a portable imaging

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system (OEC MiniView 6800, GE Medical System). The distances between the NIRS

instrument and the three devices were about 1 to 1.5 meters.

Signal analysis involved comparison of the distribution of the signal amplitude

while the devices were running with a reference set. For the reference set we chose the 1

second-long portion of the signal immediately before the operation of each device. We

expected to see a change in the distribution of the signal amplitude if interference

occurred. Prior to signal analysis it was necessary to remove the effect of physiological

changes in the signal in order for these not to contribute to signal variations.

Hemodynamic changes were quite slow, and study of the frequency spectrum of the

signals showed most of the energy of hemodynamic changes was concentrated in the

lower frequency band, so a high-pass “finite impulse response” (FIR) filter of order 20

with corner frequency of 0.8Hz was used to remove physiological signal components.

The Kolmogorov-Smirnov test was used to compare the distribution of signal

amplitudes before and during operations of the devices (Massey, 1951). This test

essentially measures the difference between two distributions that is more discriminating

than simply comparing the mean and variance of the two sets, as it compares the entire

distributions of the data sets. If this test shows the distributions are identical, this

indicates that variances and means are identical too.

A secondary method of analysis compared the frequency spectrum of the NIRS

signal before and after starting the devices to identify if the frequency content changed.

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5.3 Results

Data from 20 patients were studied. The NIRS instrument monitored data

successfully in real time in all trials. The number of time points for each subject was

different and is summarized in Table 5.1. Visual inspection of the recorded NIRS signals

showed no visible interference at the times use of any of the three devices began. The

results of the mathematical analysis also showed no evidence of significant change in the

signal at significance level 0.01 for any of the devices.

Table 5.1. The frequency of use of each device evaluated in each of the operative cases

studied.

Subjects Total Time Points Cautery Drill X-ray

1 2 7 2 2 3 4 8 3 0 5 6 4 4 0 10 5 0 5 7 6 0 7 13 7 2 5 8 8 0 5 5 9 0 6 11

10 3 7 6 11 0 0 3 12 0 1 5 13 0 5 5 14 0 5 2 15 5 6 5 16 0 5 5 17 1 5 5 18 1 4 1 19 3 2 4 20 0 5 4

Total

24

89

124

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Comparison analysis of the frequency spectrum of the NIRS signals before and

after starting the three devices could not be used. The sampling frequency of the devices

were not high enough, and the duration of devices activity were also limited in the range

of 1 to a few seconds, consequently the number of available signal samples were

insufficient for meaningful frequency spectrum analysis.

5.4 Discussion

We report an analysis for confirming the absence of EMI in NIRS data sets. In the

OR environment tested, the three devices evaluated did not contribute significant

interference. This implies that NIRS monitoring can be conducted effectively in similar

OR environments. However, each situation, combination of instruments and specific

environment has the unique potential to generate problematic EMI such that further

testing in other invirenments might be required. In our study, the distances between the

devices and the NIRS instrument was not fixed in all 20 trials, but was never less than 1

meter. Previous studies indicate the potential for clinically significant EMI is very low

with inter-instrument distances greater than 1m. However, in some instances interference

at distances up to 3m have been reported (van der Toght et al., 2008).

Some earlier studies have identified significant interference of optical devices

from specific items of medical equipment. Pulse oximetry is another optical technology

that uses many of the physics principles employed by NIRS, although the depth of

penetration is greater in the latter. Pulse oximetry is widely used in a number of clinical

settings and both optical and electrical interferences from other common devices have

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been reported. In one study, a Stealth Station image guidance system, which is a

frameless stereotactic surgical positioning system, was shown to interfere optically with

oximeter readings (van Ooostrom et al., 2005). This system uses IR light to detect the

position of fixed fiducial markers. The interference at approximately 4 Hz caused the

pulse oximeter to display saturations erroneously if the actual oxygen saturation was

below 80%. Interference of oximeters by electrocautery in the OR has also been reported

to cause inappropriately low oxygen saturation readings and generate false alarms (Block

& Detko, 1986). In a similar study, effect of EMI on NIRS data from a NIRO 500 device

(Hamamatsu, Japan) was studied during neonatal data collection and the results showed

the possibility of errors occurring during transmission of NIRS-derived data from the

instrument to a computer (Macnab et al., 1994).

Modern medical instruments have to comply with IEC Standard 60601-1-2 that

requires equipment not intended for life support to have radiated RF immunity for fields

of up to 3 V/m. The fact that no interference comparable to that detected in earlier studies

was evident in our study can be attributed in large part to an improved level of EMI

immunity in modern medical equipment including the cautery, x-ray, drill and NIRS

device used in this study. Another contributing factor might be the adequate spacing

between the devices, which is known to greatly reduce signal distortion by EMI.

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5.5 Conclusions

The current findings confirmed the absence in NIRS monitoring data of

significant EMI originating from other devices. The three devices we evaluated, which

are commonly used during orthopedic surgery, did not generate interference that

compromised NIRS intra-operative monitoring in 20 patients. The results show no

significant impact from the devices on NIRS recording at P < 0.01 in the configuration

and OR setting we used. The results indicate that NIRS monitoring free of EMI can be

achieved in the OR.

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CHAPTER 6

Conclusions

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6.1 Overview

The main purposes of this thesis were to: 1) examine the feasibility and

convergent validity of CW NIRS for continuous monitoring of skeletal muscle

oxygenation and hemodynamics during transient and long-term tourniquet-induced LMI.

2) investigate the predictive value of NIRS-derived data for evaluation of limb muscle

oxidative changes during tourniquet-induced LMI. These data have provided a strong

initial basis to further explore the feasibility of using NIRS to facilitate the early

diagnosis of acute limb muscle ischemia in high-risk patients (i.e. patients at risk for ACS

and those that undergo tourniquet-induced ischemia during surgical procedures).

The studies presented within this thesis highlight five main findings. First, CW

NIRS is a feasible method for monitoring skeletal muscle oxygenation and

hemodynamics during isometric muscle contraction and tourniquet-induced ischemia

(Chapters 3 and 4). Second, sustained muscle contraction at intensities above 30% MVC

induces a complete local ischemia in forearm muscles, as measured by NIRS (Chapter 3).

Third, tourniquet-induced muscle ischemia for 21 to 74 minutes, without reperfusion,

leads to oxidative muscle damage (Chapter 4). Fourth, the extent of tourniquet-induced

oxidative muscle damage is negatively associated with changes in local muscle

oxygenated blood volume (O2Hb / tHb) as measured by NIRS and is greater in men than

in women, but is not associated to age or an ischemia interval up to ~75 minutes (Chapter

4). Fifth, EMI from the operation of three medical devices commonly used in the

orthopaedic operation room (surgical drill, surgical cutter and portable X-ray) does not

affect NIRS signals (Chapter 5).

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6.2 Conclusions addressing Thesis Hypotheses

Hypothesis 1: “Conventional and wireless NIRS will prove to be feasible methods

for the continuous monitoring of transient and long-term tourniquet-induced limb muscle

ischemia”. The findings discussed in Chapter 3 and 4 of this thesis support this

hypothesis.

In recent years, conventional NIRS instruments have been validated and used by

many investigators to monitor tissue oxygenation, hemodynamics and metabolism in

health and disease (Mancini et al., 1994; van der Sluijs et al., 1997; Boushel et al., 2001;

van Beekvelt et al. 2003; Ferrari et al., 2004). However, the application of NIRS in

monitoring limb muscle ischemia is limited to a handful of animal studies (Sapega, 1985;

Heppenstal, 1986; Pedowitz, 1992; Mohler, 1999; Benaron et al., 2004; Kim et al., 2009)

and clinical studies that investigated limb muscle oxygenation during only short-duration

venous and arterial occlusion at workplace or during muscle contraction (Garr et al.,

1999; Casavola, 2000; Gentillelo, 2001; Kragelj et al., 2001; van Beekvelt et al., 2001b;

Tobias & Hoernschemeyer, 2007; Yu, 2005; Vo, 2007; Gomez, 2008; Shadgan et al.,

2009).

The findings of Chapter 4 demonstrate that CW NIRS is able to continuously

monitor limb muscle ischemia at 10 Hz for at least 74 minutes and can sensitively detect

the inflection points of muscle ischemia and reperfusion. This is the longest NIRS

monitoring of ischemic limb muscles in people that can be found in the literature to the

best of our knowledge. The results of this study provide important information in support

of the feasibility of NIRS for the continuous monitoring of limb muscle ischemia,

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clinically.

The use of lasers in CW NIRS instruments provides high spectral resolution, the

ability to detect very small changes in the propagation medium (coherence), and high

sensitivity when used with ultra fast photo detectors (Ferrari et al., 2004). Chapter 3 of

this thesis meanwhile demonstrates that the pattern of changes in chromophore

concentrations during muscle ischemia and reperfusion as measured by a wireless NIRS

instrument are highly comparable with previous reports using conventional NIRS

instruments, highlighting wireless NIRS as a possible substitute for conventional NIRS in

certain settings.

The studies of Chapters 3 and 4 further confirm that, with our study samples and

protocols, ambient light, and instrument set up and placement do not confound

experimental and intraoperative NIRS measurements, and that subjects can comfortably

tolerate NIRS sensor placement. Together, all of these findings confirm that conventional

and wireless NIRS are both feasible technologies to monitor limb muscle ischemia over

extended intervals.

Hypothesis 2 “Changes in muscle protein oxidation state in muscles distal to the

tourniquet during tourniquet-induced ischemia will correlate positively to tourniquet time

and changes in HHb and reoxygenation rate, and inversely to changes of O2Hb and tHb

as monitored using NIRS.” This hypothesis was partially supported.

Based on previous studies, we expected to observe a direct relationship between

ischemia time and measures of muscle tissue deoxygenation and muscle protein

oxidation. Our data from Chapter 4 reveals that limb muscle ischemia for 21 to 74

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minutes without perfusion, during lower extremity surgery, leads to considerable

ischemic muscle injury as measured by myofibrillar protein oxidation, however, we

found no significant correlation between the extent of muscle oxidative changes and

ischemia time interval. This finding suggests that the degree to which limb muscle

undergoes oxidative damage is determined not only by the ischemic interval, but also by

other factors such as muscle fibre type composition and metabolic characteristics

(Yamada et al., 2006), gender (Tiidus, 2000; Kayalia et al., 2007; Chapter 4), as well as

patient age (Cakatay et al., 2008). From a clinical perspective, this is a notable

observation as it highlights the somewhat arbitrary guideline of a fixed tourniquet time of

90 minutes for all limb surgeries rather than tailoring tourniquet time to suit individual

differences of patients. A better understanding of the patient-related factors that

contribute to oxidative injury facilitate titration of optimal tourniquet protocols.

In addition, we found that protein oxidation of ischemic limb muscles correlates

positively with re-oxygenation rate and negatively with changes in both tHb and O2Hb,

suggesting that muscle oxidative injury is closely associated with changes in oxygenated

blood volume. All together, our data from Chapter 4 suggest that continuous monitoring

of the hemodynamics and oxygenation status of muscles distal to the tourniquet by CW

NIRS may facilitate decision making by surgeons to determine the time of intermittent

tourniquet deflation rather than relying solely on a fixed tourniquet time. Our data from

Chapter 4 also indicates that the extent of skeletal muscle oxidative injury is greater in

men than in women. While data from animal and human studies are consistent with this

finding (Tiidus, 2000; Fano et al., 2005; Kayalia et al., 2007; Sanz et al., 2007; Gomez et

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al., 2008), further clinical investigation is warranted to explore the implications in this

clinical scenario.

Hypothesis 3: “NIRS signals will not be affected by EMI of medical devices that

are commonly used in the orthopedic operating room” (Chapter 5). Mathematical signal

analysis of intraoperative NIRS data in Chapter 5 confirmed that EMI derived from

common electrical devices used in the OR does not disrupt NIRS data being

simultaneously recorded. This unique observation reported in the thesis, helps validate

NIRS as a useful monitoring technology during limb muscle surgery.

In conclusion, the results of this thesis support potential application of CW NIRS

as a feasible, practical and cost-effective technology for routing monitoring of limb

muscle oxygenation and hemodynamics during limb surgery, in high-risk patients for

early detection of LMI when it is still possible to prevent irreversible complications and

to study muscle dysfunctions in workplaces or during exercise when a sustained isometric

muscle contraction is required.

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6.3 Significance

Recent reviews (Katzen, 2002; Henke, 2002; Kasirajan & Ouriel, 2002;

Choudhary et al., 2003) including our review reported in Chapter 2 support the need for

access to a reliable clinical method for continuous monitoring and early diagnosis of limb

muscle ischemia. Indeed, a large gap exists in the literature on strategies for obtaining

early diagnosis of this serious condition. The unique clinical data presented in this thesis

informs the existing literature of the role NIRS may be able to play in filling this gap. To

date, the majority of studies on NIRS monitoring of limb muscle oxygenation and

hemodynamics have either investigated short-duration limb muscle ischemia in humans

or long-duration ischemia in animal models. To the best of our knowledge, the findings

of this thesis are the first to demonstrate the feasibility and efficacy of CW NIRS in

monitoring tourniquet-induced limb muscle ischemia during long duration limb surgery

(Chapter 4). We have shown that CW NIRS can not only monitor and detect inflection

points of human limb muscle oxygenation and hemodynamics but can also be used as a

proxy to predict the extent of ischemia-induced muscle oxidative damage. Our data have

also indicated that, for ischemic durations of up to approximately 1¼ hours, the degree of

muscle damage is not determined solely by the ischemic interval. These findings strongly

support previous studies that suggest NIRS is a valuable method for the diagnosis and

monitoring of acute limb muscle ischemia including ACS (Garr et al., 1999; Gentilello et

al., 2001; Shuler et al., 2010).

There exists, to the best of our knowledge, no clinical study that investigates

skeletal muscle protein oxidation over a range of ischemic time intervals during leg

surgery. We have shown that limb muscle ischemia for durations of even less than 60

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minutes before reperfusion can cause muscle damage (Chapter 4). This finding is

particularly important for surgeons that rely solely on the “90-minute maximum” as a

safe tourniquet time during limb surgeries; this thesis provides evidence that this

guideline requires further investigation.

Moreover, this research is the first report of a limb muscle study using a wireless

CW NIRS device (Chapter 3). Wireless monitoring greatly broadens the clinical

scenarios in which NIRS monitoring can be conveniently and practically used and marks

a significant accomplishment that will contribute to a continued increase in the

applications of NIRS for non-invasive monitoring of limb muscles in high-risk

individuals. Lastly, this research presents the first statistical approach to investigate the

effects of EMI from other electrical devices on NIRS signals and demonstrates that NIRS

can be used in conjunction with several other OR electrical devices without interference

of the signal (Chapter 5); a finding that confirms NIRS as a useful and safe tool for use in

the OR and other clinical settings.

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6.4 Study Strengths and Limitations

The primary strengths of this thesis relate to the study design of the clinical trial

in Chapter 4 that coupled NIRS-derived data to changes in muscle protein in a cohort of

surgical patients, and the unique mathematical analysis of NIRS signals presented in

Chapter 5. The methodological approaches adopted in these studies allowed us to

examine our hypotheses by way of objective, non-invasive and previously validated

methods.

Specific limitations for each of the studies in this thesis are described in Chapters

3, 4 and 5, as appropriate. Common to many clinical studies, small sample size was a

general limitation. While all of the sample sizes used in this thesis were of a power

sufficient to address our main questions, they nevertheless precluded the possibility of

more powerful statistical analyses and greater generalizability. Furthermore, in all

studies, recruited subjects were not completely homogenous in their characteristics and

were also limited to those without major comorbidities, which could be viewed as another

limitation to this thesis.

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6.5 Future Directions

This thesis provides novel information regarding skeletal muscle ischemia,

current limitations of its diagnosis, and the potential for clinical application of NIRS for

continuous monitoring and early diagnosis of acute ischemic conditions of limb muscles,

including ACS. Despite the many advances of NIRS realized through this thesis, there

remain a number of relevant issues that will require further investigation.

Future clinical trials should utilize longer ischemia intervals and include larger

sample sizes with equal gender distribution and inclusion of patients with common

comorbidities, complemented by long term follow up of patients. Such trials will inform

the association between ischemia interval and post-operative muscle recovery in male

and female subjects and further provide insight into the patient-related factors that might

accentuate oxidative injury during the previously accepted 90 minutes tourniquet time.

Larger samples size will also allow for more powerful statistical analyses including non-

linear regression analysis of involved variables, which could further enhance our

understanding of limb muscle ischemia, its risk factors, and its diagnosis and prevention.

6.5.1 Effect of Ischemia on Skeletal Muscle Lipid Peroxidation

In this thesis, the effect of ischemia without reperfusion on muscle protein

oxidation is investigated however, little is known about the impact of ischemia on

skeletal muscle lipid peroxidation; an indication of cell membrane damage (Mathru et al.,

2007). Investigations of this nature could increase our understanding of IR-injury and

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help to delineate the individual impact of each component of ischemia and reperfusion on

skeletal muscles.

6.5.2 Advancing the Clinical Use of NIRS

Wireless CW NIRS devices are now available with spatially resolved

configuration; an option that enables measurement of absolute TSI% (see Chapter 3).

Increasing the use of this NIRS prototype, in conjunction with the use of conventional

NIRS devices in future clinical trials, will allow investigation into its ability to provide

more rapid and accurate real-time monitoring of limb muscle oxygenation and

hemodynamics. Figure 6.1 shows a wireless NIRS instrument, fixed over the vastus

lateralis muscle of a patient during lower limb trauma surgery. This compact, portable

and wireless device stands to be a useful, non-invasive, real-time tool that can provide

information on oxygenation and hemodynamics during a wide range of in-field exercise

occupational, or dynamic clinical examinations. It therefore promises a significant,

positive impact on the promotion of functional rehabilitation in sports medicine as well as

on the promotion of improved skeletal muscle function and neuromuscular control in

both the general population as well as athletes.

Even further advancements in this use of NIRS technology involve the

investigation of internal organs. Indeed, several studies have reported the use of NIRS

optodes incorporated into surgical catheters allowing for the study of deeper organs and

tissues, albeit using a more invasive approach than the surface monitoring approach

112

applied in the studies of this thesis (Macnab et al., 2003; Asgari et al., 2003; Mitsuta et

al., 2006).

From here, a possible next step in the clinical evaluation and validation of NIRS

monitoring of limb muscles could be to use NIRS in parallel with other standards of

practice for the study of different groups of patients at risk of acute limb muscle ischemia

such as patients predisposed to vascular obstruction or ACS.

Figure 6.1. A wireless NIRS instrument (PortaMon, Artinis, the Netherlands) monitors

vastus lateralis muscle oxygenation and hemodynamics during lower limb trauma

surgery.

6.5.3 Early Diagnosis of ACS in High-Risk Patients Using NIRS

The literature review (Chapter 2) of the current diagnostic strategies for ACS

highlights notable gaps in the methods available for the diagnosis of this critical

condition that warrants further investigation. As discussed in Chapter 2, the

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pathophysiology of ACS involves an insult to muscle perfusion within a compartment in

conjunction with increased tissue pressure due to trauma, bone fracture or other

etiologies. The clinical studies of Chapters 3 and 4 show that CW NIRS can sensitively

detect upper and lower limb muscle ischemia and deoxygenation immediately after blood

flow cessation. We therefore postulated that NIRS might be a more valuable method for

the early diagnosis of ACS as compared to intracompartmental pressure measurement,

the current standard of practice. A recent clinical study by Shuler et al. (2010) has

underscored a significant negative correlation between NIRS-measured muscle

oxygenation and the intracompartmental pressure in legs affected by ACS. Further

prospective clinical trials are needed to examine the value of monitoring limb

hemodynamic using CW NIRS for evidence of the critical threshold of ischemia and

muscle deoxygenation and, as such, an early diagnosis of ACS in high-risk individuals,

such as those with tibial fractures. Figure 6.2 illustrates a hypothetical example of a NIRS

set up for monitoring and diagnosis of ACS in a high-risk patient.

Moreover, NIRS may prove to be a useful method for diagnosis of other clinical

conditions that cause skeletal muscle ischemia. For instance, acute arterial obstruction

may cause acute limb muscle ischemia in the absence of local tissue pressure changes; a

condition for which NIRS based diagnostics may be extremely useful.

114

Figure 6.2. Schematic presentation of a hypothetical NIRS set-up for monitoring anterior

compartment of a fractured leg.

6.5.4 Monitoring of Limb Muscle Oxygenation and Hemodynamics During

Tourniquet-Induced Ischemia

In Chapter 4 we demonstrate that CW NIRS is capable of monitoring muscle

oxygenation and hemodynamics in muscles distal to a pneumatic tourniquet during lower

limb surgery. Our data demonstrate predictive criterion validity of NIRS indexes as

shown by the corralations of ∆O2Hb, ∆tHb and re-oxygenation rate to changes in protein

oxidation in ischemic limb muscles. This finding highlights the potential clinical

application of CW NIRS during limb surgeries in which a tourniquet is used, so as to

allow surgical staff to remain aware of the extent of tourniquet-induced muscle oxidative

damage. This finding warrants further investigations with more subjects, longer ischemia

115

intervals and more in depth histochemical and biochemical analyses of limb muscles.

Examining the reliability of NIRS in the investigation of limb muscle ischemia and

deoxygenation in other cohorts of patients such as people with cardiorespiratory disorders

or myopathies also warrant further investigations.

6.5.5 Developing Safer Tourniquet Systems Using NIRS

In Chapter 4 we report a significantly negative correlation between muscle

oxidative stress and the volume of oxygenated hemoglobin within limb muscles distal to

pneumatic tourniquets during limb surgeries. While this finding remains to be confirmed

by further studies, it may provide evidence in support of setting an optimal tourniquet

pressure to allow a minimum volume of oxygenated blood in under-tourniquet muscles to

be maintained. In other words, safer tourniquet systems that use a lower dynamic

tourniquet pressure intraoperatively could perhaps be developed by way of continuous

monitoring of the level of NIRS-derived blood volume in muscles distal to the tourniquet.

Developing a NIRS-integrated tourniquet of this sort would mark an important

step for reducing tourniquet-induced muscle oxidative damage and related complications

during limb surgery. Moreover, minimizing the tourniquet cuff pressure and adapting it to

the lowest pressure required by each individual patient could potentially reduce the risk

of tourniquet-induced nerve injuries and other tourniquet pressure related complications,

especially in under-tourniquet tissue. Such a tourniquet system may also enable surgeons

to increase tourniquet time without increasing the risk of muscle damage. This possibility

invites additional animal studies to explore and quantify the relationship between changes

116

in leaking blood volume and muscle oxidation. Clinical trials should then investigate the

feasibility, sensitivity and specificity of the method in clinical settings.

6.5.6 Monitoring the Effects of Ischemic Preconditioning by NIRS

A growing body of evidence indicates that various hypoxia-sensitive tissue cells,

including skeletal muscles, can be protected from IR-injury by prior exposure to a short

period of ischemia. This method, known as ischemic preconditioning (IPC) (Murry et al.,

1986; Jerome et al., 1995; Pang et al., 1995; Papanastasiou et al., 1999), induces

ischemia tolerance in striated muscles and protects skeletal muscles during the

reperfusion component of IR by way of various mechanisms (Jerom et al., 1995; Yadav

et al., 1999; Papanastisious et al., 1999; Carini et al., 2000; Jennings et al., 2001; Pucar et

al., 2001; Kharbanda et al., 2001; Kim et al., 2003), the overall effect of which decreases

inflammation, interstitial edema and pressure on the capillaries after reperfusion of

ischemic muscle (Duarte et al, 1997).

Validation of this method in different animal studies resulted in its introduction as

a therapeutic adjunct for prevention and attenuation of the deleterious effects of muscle

ischemia (Schoen et al., 2007; Eberline et al., 2008). Recent clinical studies have shown

that IPC can reduce the severity of tourniquet-induced IR-injury to limb muscles in

healthy individuals (Kharbanda et al., 2001), however, assessing the effect of IPC and

standardizing the best practice of this method in clinical settings is still under study.

NIRS may prove useful, both in a research and clinical context, as a noninvasive method

for the evaluation and quantification of the effects of IPC on IR-injury of limb muscles in

117

high-risk individuals. Further studies using NIRS to monitor and compare oxygenation

and hemodynamic response of skeletal muscles to IR with and without IPC may increase

our knowledge of the mechanism governing this phenomenon and the best therapeutic

approach for the use of IPC for protecting skeletal muscles against IR-injury.

As a final mention, improvements of study design in order to minimize the

limitations outlined in the studies presented in this thesis will likely further enhance the

quality of future relevant research projects that will focus on clinical applications of

NIRS to optimize tissue oxygenation and minimize risk of tissue oxidative injuries.

118

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APPENDIX I

Informed Consent Forms

150

THE UNIVERSITY OF BRITISH COLUMBIA and VANCOUVER GENERAL HOSPITAL

DEPARTMENT OF ORTHOPAEDICS

# 110 - 828 West 10th Avenue

Vancouver, BC, V5Z 1L8 CANADA

Tel: (604) 875-5239 Fax: (604) 875-4438

Consent Form

Skeletal Muscle Deoxygenation and Degradation Induced by

Tourniquet

Principal Investigator: Dr. P. J. O’Brien, MD, FRCSC Department of Orthopaedics

Vancouver General Hospital, (604) 875-5239

Co-Investigators: Dr. W.D. Reid, PhD, Professor, Department of Physical Therapy, Dr.

B Shadgan MD, MSc, PhD student, Faculty of Medicine, Dr P. Blachut, MD, FRCSC, Dr

H. Broekhuyse, MD, FRCSC, Dr P. Guy, MD, FRCSC, Dr K. Lefaivre, MD, FRCSC,

Orthopaedic Trauma, Vancouver General Hospital.

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Background:

We are inviting subjects such as yourself, who have a current or previously injured lower

leg or ankle that requires surgery, to participate in a research study.

In lower leg and ankle surgeries a routine part of the surgical procedure is to block blood

flow to the area being surgically treated for a short period of time (the time varies

depending on the type of injury and surgery) by using a pneumatic tourniquet (a device

that operates by compressed air) in order to prevent excessive bleeding. Although the

established rule is to only apply the tourniquet for a maximum of 90 minutes there is not

to date, scientific data that can show whether this is in fact the maximum or if the

maximum time should be shorter.

This study has been designed to assist in gaining a better understanding of what happens

to muscle tissue during surgery in these types of cases. The study will use the Near-

Infrared Spectroscopy (NIRS) to measure the level of oxygen that is reaching the

surrounding muscle tissue as well as measure the level of blood flow occurring during the

surgical procedure.

Purpose:

The purpose of this study is to examine the effects on muscle tissue (specifically, the

changes in oxygen supply to the muscle) during operative treatment of the lower limb.

Alternatives:

If you choose not to participate in this study, your surgical treatment will be carried out

as it normally does in these cases. If you choose not to participate it will simply mean

that the NIRS device will not be placed to measure oxygen or blood flow to the muscle

tissue of the lower limb and a tissue sample will not be retrieved.

Who May Participate

§ 16 to 70 years of age

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§ Ankle or foot injury requiring surgery

§ Subjects who are able to understand the consent form and provide consent on

their own behalf

Who May Not Participate

Factors which will exclude you from participation in this study include:

§ Less than 16 or more than 70 years of age

§ Lower limb injuries on both sides

§ Severe leg injury in the past on either side

§ Subjects with a diagnosis of Congestive Heart Failure

§ Subjects with any disease/condition that affects the arteries or veins (organs for

carrying blood throughout the body)

§ Hemiplegia (paralysis of one side of body)

§ Acquired Immune Deficiency Syndrome (AIDS)

§ Subjects who are not able to understand the consent form on their own and

provide consent on their own behalf.

Study Procedures:

Regardless of whether you choose to participate or not in the study you will have surgery

as you normally would for the injury you have experienced. If you choose to participate

you may consent to having the NIRS device placed but elect not to provide a tissue

sample.

NIRS:

If you enter the study, after you have been prepared in the usual manner for surgery NIRS

probes will be placed on the skin of both the lower legs and of both thighs. The probes

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work with optical fibers that give off and receive light. The probes measure oxygen in

the tissue with wavelengths of light. The probes are connected to a computer which

processes the signals as a result of the wavelengths and it calculates and displays the

measurements of oxygen and blood flow in real-time. The probes will be left on for the

duration of your surgery and removed once the surgery is completed.

Tissue Sample:

You may elect to undergo NIRS monitoring without providing a tissue sample. Please

indicate your choice at the end of this document by either checking the box to consent to

providing a tissue sample or leave it unchecked to decline providing a tissue sample.

During your surgical procedure a small tissue sample (0.7 x 1.0 cm in total size which is

about the size of an eraser head on a pencil) will be taken from the same area that will be

accessed for your surgical treatment. Because the muscle sample will be taken from the

same area to be accessed for your operation, it will be easily accessible during your

operation. The taking of the sample will not lengthen the time needed to perform your

surgery. There is no evidence of an increased risk as a result of providing a small tissue

sample at the time of surgery for the type of injury you have. You will not be aware of

any effect from the retrieval of the tissue such as pain or functional loss. The follow-up is

the same whether you choose to participate in the study or not. The sample retrieved, will

be taken to the Muscle Research Lab at Vancouver General Hospital where it will be

tested immediately with various chemicals and equipment to assess its composition at the

cellular level. Portions of the same tissue sample will be sent to the University of Florida

research facility where more analysis using very specialized equipment will take place.

The tissue will not be used for any commercial purposes and will not be accessible to

anyone outside of this study. The sample will be labeled with a unique study identifier,

which is not linked in any way to information that could identify you. This de-indentified

sample will be kept in a locked storage facility, in a locked laboratory space within

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Vancouver General Hospital excluding the de-identified portions to be transferred to the

University of Florida.

Reimbursement for Participation:

You will not receive payment for your participation.

Benefits:

You will not benefit from the study. However, this study may provide information about

what happens to muscle tissue during surgical repair of these injuries.

Risks:

There are general risks associated with having surgery, including the standard treatment

for your injury. These risks are not decreased or increased by your participation in this

study. There have been no reports of risks associated with placing the probes on to the

skin to the best of the study team’s knowledge and there is no evidence of an increased

risk as a result of providing a small tissue sample at the time of surgery in these types of

cases.

Although we employ all of the measures available to us in order to protect the

confidentiality of hospital and research records we cannot offer a 100% guarantee that the

methods are 100% foolproof.

Study Withdrawal:

Your participation in this study is entirely voluntary. You may withdraw from this study

at any time without providing any reasons. If you decide to enter the study and to

withdraw at any time in the future there will be no penalty or loss of benefits to which

you are otherwise entitled, and your future medical care will not be affected.

If you choose to enter the study and then decide to withdraw at a later time, all data

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collected about you and analyzed during your enrollment in the study will be retained.

By law, this data cannot be destroyed. If you decide to withdraw before your information

has been analyzed or is being stored for analysis at a later date, the data will be destroyed

and discarded in accordance with established protocols to complete your requested

withdrawal from the study.

Confidentiality:

Your confidentiality will be respected. No information that discloses your identity will

be released or published without your specific consent to the disclosure. However,

research records and medical records identifying you may be inspected in the presence of

the investigator or his designate by representatives of Health Canada, and the UBC

Research Ethics Board for the purpose of monitoring research. However, no records

which identify you by name or initials will be allowed to leave the Investigators’ offices.

Contact:

If you have any questions or desire further information with respect to this study, please

contact the research office at (604) 875-5239. (24 hours).

If there are any concerns about your treatment or rights as a research subject you may

contact the Research Subject Information Line at the University of British Columbia,

Office of Research Services, at (604) 822-8598. Signing this consent form in no way

limits your legal rights against the sponsor, investigators, or anyone else.

Subject Consent:

I understand that participation in this study is entirely voluntary and that I may refuse to

participate or I may withdraw from the study at any time without any consequences to my

continuing medical care.

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I have received a signed and dated copy of this consent form for my own records.

By signing below you consent to participate in this study.

Subject Name (please Print) Subject Signature Date

Witness Name (please Print) Witness Signature Date

Investigator Name (please Print) Investigator Signature Date

If you also consent to providing a Tissue Sample please check

this box:

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SUBJECT INFORMATION AND CONSENT FORM

Evaluation of muscle oxygenation in primary and accessory respiratory muscles during tidal and

incremental threshold loaded breathing using near infrared spectroscopy

Principal Investigator: Dr. W. Darlene Reid, PhD Department of Physical Therapy

University of British Columbia Vancouver Coastal Health Research Institute

Co-Investigator(s): Dr. Babak Shadgan, MD, MSc Muscle Biophysics Laboratory

University of British Columbia Vancouver Coastal Health Research Institute

Dr. Bill Sheel, PhD School of Human Kinetics University of British Columbia

Jordan A. Guenette, BHK, MSc Health and Integrative Physiology Laboratory University of British Columbia

1. INTRODUCTION

You are being invited to take part in this research study because you are a healthy person

with no breathing problems. We are interested in examining respiratory muscle function

in healthy people.

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2. YOUR PARTICIPATION IS VOLUNTARY

Your participation is entirely voluntary, so it is up to you to decide whether or not to take

part in this study. Before you decide, it is important for you to understand what the

research involves. This consent form will tell you about the study, why the research is

being done, what will happen to you during the study and the possible benefits, risks and

discomforts.

If you wish to participate, you will be asked to sign this form. If you do decide to take

part in this study, you are still free to withdraw at any time and without giving any

reasons for your decision.

If you do not wish to participate, you do not have to provide any reason for your decision

not to participate nor will you lose the benefit of any medical care to which you are

entitled or are presently receiving.

Please take time to read the following information carefully and to discuss it with your

family, friends, and doctor before you decide.

3. WHO IS CONDUCTING THE STUDY?

This study is being conducted by the Muscle Biophysics Laboratory at VCHRI,

University of British Columbia.

4. BACKGROUND

This study is being conducted to examine whether muscle fatigue occurs after exercise in

the muscles that we use for breathing (the respiratory muscles). This study is of interest

because people with chronic obstructive pulmonary disease (COPD) show signs of

muscle damage and fatigue in their respiratory muscles (the major muscles we use for

breathing). The muscle fatigue can affect their ability to breathe but it is difficult to

measure whether muscle fatigue is present in these people and if so, which respiratory

muscles (primary or accessory) are more vulnerable to fatigue and weakness. This study

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will help to find ways to measure muscle fatigue using clinical non-invasive

measurement tools that can then be used in people with COPD.

5. WHAT IS THE PURPOSE OF THE STUDY?

The purpose of this study is to look for signs of muscle fatigue following a bout of

incremental inspiratory threshold loading breathing in healthy subjects using Near

Infrared Spectroscopy (NIRS) Device. An exercise bout of resistive breathing will be

used to induce fatigue to the muscles of breathing. This muscle fatigue might result in

muscle force loss (muscle weakness). If this occurs, the muscle fatigue is temporary and

it is reversible.

6. WHO CAN PARTICIPATE IN THE STUDY?

Healthy people who are 20-50 years old can participate in this study.

7. WHO SHOULD NOT PARTICIPATE IN THE STUDY?

People who currently smoking or are ex-smokers should not participate in this study.

People with asthma or other lung diseases or breathing problems should not participate in

this study.

8. WHAT DOES THE STUDY INVOLVE?

This study will be conducted at the University of British Columbia. Twenty subjects will

be enrolled to take part in the study.

Overview of the Study

The study involves participation in one baseline and testing session. The total time

requirement is 1-2 hours. All subjects will undergo the same testing procedures.

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If You Decide to Join This Study

If you agree to take part in this study, the procedures you can expect will include the

following:

On your visit, we will take the following steps:

1- You will be asked to complete a screening questionnaire to ensure that you do not

have any health problems that would prevent you from participating in the study.

2- You will be asked to complete a respiratory Muscle Force Test – the amount of

force that you can produce using your breathing muscles will be measured. You

will put a mouthpiece in your mouth and nose clips on your nose. You will be

asked to breathe in through the mouthpiece as hard you can for a period of 3

seconds and the force you produce will be recorded. You will be asked to repeat

this test up to 10 times until the forces you produce are within a small range. You

will get a one-minute break between trials. Then you will do a similar test with a

small probe that will be placed in one nostril. You will be asked to “sniff as hard

as possible” and the force produced will be measured. You will be asked to repeat

this test 3- 10 times until the forces you produce are within a small range. You

will get a one-minute break between trials.

3- You will be asked to perform a respiratory muscle exercise that will take 10-30

minutes to complete. You will breathe through a mouthpiece and a one-valve that

is attached to a weight. In order for you to get air to flow into the mouthpiece, you

will have to breathe in hard enough to lift the weighted valve that will be set at 30

% of your maximal inspiratory muscle force. You will be asked to breathe against

the increasing wieght until you are unable to meet the force. Your heart rate and

oxygen saturation will be monitored throughout the test using a clip on your index

finger. The exercise will last between 10 to 30 minutes.

4- During the exercise we put a simple plastic probe on your neck (over the

sternocleidomastoid muscle) and another similar probe over your chest. These

probes are connected to a non-invasive diagnostic system, which is called Near-

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Infrared Spectroscopy (NIRS). Using this system we try to monitor the level of

oxygen and carbon dioxide in your respiratory muscles, which are in the neck and

chest. An adhesive tape would be placed around the probe to protect it from sweat

and moisture and fix it over the skin. In addition, a black soft cervical collar will

be used around the neck to avoid movement of the probe. Monitoring the changes

of O2 and CO2 of respiratory muscles during exercise will provide us with useful

information about physiological aspects of respiratory muscle fatigues.

9. WHAT ARE MY RESPONSIBILITIES?

During the study, you will asked to refrain from consuming caffeine or alcohol and from

engaging in heavy physical activity for at least 24 hours prior to testing.

10. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF

PARTICIPATING?

There are only minimal risks involved with this study. During the exercise bout, you

may feel uncomfortable, anxious or breathless when you are breathing against the

weighted valve (about 20% of people experience these symptoms).

In rare instances (less than 2% of people), you may experience lightheadedness, dizziness

or fainting. Your heart rate, oxygen level and your carbon dioxide level will be

monitored throughout the test to ensure your safety. The test would be stopped by the

investigator if your values exceed normal values or if you are unable to continue for any

reason.

You may experience muscle soreness in the muscles of your neck, shoulders, chest and/or

abdomen following the exercise bout, which is temporary and reversible in a short time.

11. WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY?

There are no direct benefits to you for participating in this study except you will find out

some information about your respiratory muscle forces. We hope that the information

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learned from this study can be used in the future to benefit people with chronic lung

disease.

You will be provided with a copy of the results of your respiratory muscle force tests, if

requested.

12. WHAT ARE THE ALTERNATIVES TO THE STUDY TREATMENT?

There are no alternatives to the study as it does not provide a treatment.

13. WHAT IF NEW INFORMATION BECOMES AVAILABLE THAT MAY

AFFECT MY DECISION TO PARTICIPATE?

If new information arises during the research study that may affect your willingness to

remain in the study, you will be advised of this information.

14. WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO

PARTICIPATE?

Your participation in this research is entirely voluntary. You may withdraw from this

study at any time. If you decide to enter the study and to withdraw at any time in the

future, there will be no penalty or loss of benefits to which you are otherwise entitled, and

your future medical care will not be affected.

The study investigators may decide to discontinue the study at any time, or withdraw you

from the study at any time, if they feel that it is in your best interests.

If you choose to enter the study and then decide to withdraw at a later time, all data

collected about you during your enrolment in the study will be retained for analysis. By

law, this data cannot be destroyed.

15. WHAT HAPPENS IF SOMETHING GOES WRONG?

Signing this consent form in no way limits your legal rights against the investigators, or

anyone else.

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16. CAN I BE ASKED TO LEAVE THE STUDY?

If you are not complying with the requirements of the study or for any other reason, the

study investigator may withdraw you from the study.

17. AFTER THE STUDY IS FINISHED

You will receive a copy of your results within one month of completing the study, if

requested.

18. WHAT WILL THE STUDY COST ME?

You will be responsible for your transportation and/or parking costs to and from UBC

campus to participate in the study. There will be no reimbursement for these expenses.

No honorarium will be provided for participating in the study.

19. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?

Your confidentiality will be respected. No information that discloses your identity will

be released or published without your specific consent to the disclosure. However,

research records and medical records identifying you may be inspected in the presence of

the Investigator or his or her designate by representatives of Health Canada and the UBC

Research Ethics Board for the purpose of monitoring the research. However, no records

which identify you by name or initials will be allowed to leave the Investigators' offices.

20. WHO DO I CONTACT IF I HAVE QUESTIONS ABOUT THE STUDY

DURING MY PARTICIPATION?

If you have any questions or desire further information about this study before or during

participation, you can contact Dr. Darlene Reid [Principal Investigator].

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21. WHO DO I CONTACT IF I HAVE ANY QUESTIONS OR CONCERNS

ABOUT MY RIGHTS AS A SUBJECT DURING THE STUDY?

If you have any concerns about your rights as a research subject and/or your experiences

while participating in this study, contact the Research Subject Information Line in the

University of British Columbia, Office of Research Services.

SUBJECT CONSENT TO PARTICIPATE

• I have read and understood the subject information and consent form.

• I have had sufficient time to consider the information provided and to ask for

advice if necessary.

• I have had the opportunity to ask questions and have had satisfactory responses to

my questions.

• I understand that all of the information collected will be kept confidential and that

the result will only be used for scientific objectives.

• I understand that my participation in this study is voluntary and that I am

completely free to refuse to participate or to withdraw from this study at any time

without changing in any way the quality of care that I receive.

• I understand that I am not waiving any of my legal rights as a result of signing

this consent form.

• I understand that there is no guarantee that this study will provide any benefits to

me.

• I have read this form and I freely consent to participate in this study.

• I have been told that I will receive a dated and signed copy of this form.

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SIGNATURES

________________________________________________________________________ Printed name of subject Signature Date ______________________________________________________________________ Printed name of witness Signature Date ________________________________________________________________________ Printed name of principal investigator/ Signature Date designated representative ________________________________________________________________________ Printed name of translator (if applicable) Signature Date

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APPENDIX II

Motion artifact removal from muscle NIR spectroscopy measurements *

* A version of this appendix is in press as:

Molavi B., Dumont G., Shadgan B., Motion artifact removal from muscle NIR spectroscopy measurements, IEEE Canadian Conference on Electrical and Computer Engineering 2010.

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APPENDIX III

Sternocelidomastoid Muscle Oxygenation and Hemodynamics Response to Incremental

Inspiratory Threshold Loading Measured by Near Infrared Spectroscopy. *

* This appendix has been submitted for publication in a peer-reviewed journal as:

Shadgan B., Guenette J., Sheel B., Reid D., Sternocleidomastoid muscle oxygenation and hemodynamic response to incremental inspiratory threshold loadeing measured by near infrared spectroscopy.

172

ABSTRACT

This study investigated the pattern of muscle oxygenation and hemodynamic responses in

the sternocleidomastoid (SCM) in comparison with the parasternal (PS) and intercostal

(IC) muscles during a bout of incremental inspiratory threshold loading (ITL) in healthy

subjects using near-infrared spectroscopy. As loading started the PS and IC showed a

significant increase in oxygenated hemoglobin (5.9 ± 2.3 & 6.8 ± 2.4 µM, P<0.05) and

the SCM showed an increase in deoxygenated hemoglobin (17.3 ± 3.8 µM, P < 0.05).

Total hemoglobin also steadily increased in the SCM whereas it decreased in the

quiescent vastus lateralis muscle (20.7 ± 6.1 vs. -6.6 ± 2.4 µM, P<0.05), which was used

as the control muscle during the ITL. Our data suggests that although the SCM is

recruited progressively during progressive ITL, the metabolic demand exceeds the

aerobic potential of this muscle. Our findings suggest that blood redistribution from limb

muscles is a mechanism for maintaining inspiratory muscle oxygenation during high

respiratory motor output.

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1. Introduction

The sternocleidomastoid muscle (SCM), responsible for the majority of head

movements (de Mayo et al., 2005), is also an important accessory muscle of inspiration

(Epstein, 1994; Yokoba et al., 1999; Masubunchi et al., 2001). The SCM becomes active

during ventilation at high lung volumes (Hudson et al., 2007) and elevated levels of

inspiratory work (Mananas et al., 2000). However, the uniformity of SCM recruitment

during high ventilatory demands is equivocal, which may relate to its differing role in

animal models (De Troyer et al., 2005), humans (Hudson et al., 2007), and in chronic

respiratory diseases such as chronic obstructive pulmonary disease (Tobin et al. 2009).

In healthy humans, SCM activation occurs in accordance with “neuromechanical

matching” based on their mechanical advantage (Butler, 2007). Accordingly, the SCM is

progressively activated during incremental static and dynamic maneuvers to maximal

inspiratory pressure in healthy individuals, although at a later onset than the obligatory

inspiratory muscle, the scalene (Hudson et al., 2007). Because of their hypertrophy and

prominent appearance in stable COPD, the SCM was postulated to be active at rest (Aora

& Rochester, 1984). Evidence of their activation during tidal ventilation, however, was

not found (De Troyer et.al., 1994; Laghi et al., 1998). The clinical importance of

monitoring SCM in COPD patients may be most obvious in those who undergo weaning

from mechanical ventilation after suffering from respiratory failure. COPD patients

demonstrated greatly elevated electromyography (EMG) activation of SCM in all “failed

weaning” cases, and also showed SCM activity during the majority of breaths during the

spontaneous breathing trials (83 versus 19% of breaths during spontaneous breathing)

compared to those who successfully weaned (Parthasarathy et al., 2007).

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Near infrared spectroscopy (NIRS) is a noninvasive, continuous, and direct

method to study oxygenation and hemodynamics of living tissue in real time (Delpy &

Cope, 1997; Ferrari et al., 2004). In recent years, NIRS has been widely used to monitor

muscle oxygenation, hemodynamics and metabolism in health and disease (Boushel et

al., 2001; Hamaoka et al., 2007; Nielsen et al., 2001, van Beekvelt et al., 2003). Few

studies have examined the oxygenation and hemodynamic responses of respiratory

muscles using NIRS. For example, serratus anterior muscle deoxygenation has been

studied in patients with chronic heart failure (Terakadu et al., 1999) and in children

during incremental cardiopulmonary exercise testing (Moalla et al., 2005). In 2001,

Nielson et al. monitored intercostal muscle oxygenation during resistive breathing and

simultaneous submaximal cycling using NIRS in healthy males and in 2007, Cannon et

al. utilized simultaneous EMG and NIRS to determine the recruitment and oxygenation

of the serratus anterior during graded upright cycling. In spite of studies examining EMG

activity of the SCM (Campbell, 1970; De Troyer et al., 2005; Butler et al., 2007), the

oxygenation and hemodynamic response of the SCM and its coordination with the

primary inspiratory muscles during loaded breathing has not been extensively studied.

Determining the amount of loading that results in significant changes of muscle

oxygenation in different inspiratory muscles may provide insight into their respective

contribution to function, and force loss in healthy people as well as those with chronic

respiratory disease. Despite the few studies that have examined ventilatory muscle

strength during exercise, there have been no studies to systematically assess oxygenation

and hemodynamics of ventilatory muscle endurance during incremental inspiratory

threshold loading (ITL). Also there have been no studies on SCM muscle activation,

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oxygenation and hemodynamics during respiration. Accordingly, the purpose of the

present study was to determine the pattern of muscle oxygenation changes in the SCM

using NIRS during ITL in healthy subjects and to compare SCM oxygenation with 1) the

respiratory muscles including the parasternal (PS) and intercostal muscles (IC) and; 2)

vastus lateralis (VL) as a quiescent control muscle. We hypothesized that total and

deoxygenated hemoglobin will increase during ITL whereas the PS and IC will show

more stable responses during ITL. We hypothesized that muscle deoxygenation of SCM

during ITL in healthy subjects will more closely correspond to the intensity of loading

than the PS and IC.

2. Methods

2.1 Subjects. Subjects were included if they were: 1) male; 2) healthy; 3) non-smokers,

with no history of asthma, COPD or any other respiratory condition; 4) able to provide

informed consent; and if they 5) had adipose tissue thickness of less than 10 mm at the

site of NIRS monitoring. This adipose tissue thickness was selected based on the depth

of NIRS penetration and interoptode distance available on the muscle of interest (Homa

et al., 1996). The study received institutional ethical approval and informed written

consent was obtained from all volunteers before participating. All procedures complied

with the Declaration of Helsinki.

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2.2 Experimental Overview. Subjects refrained from caffeine, exercise and alcohol for

12 hours prior to testing. A screening questionnaire was completed by each subject prior

to testing to ensure inclusion criteria were met and to identify any health conditions that

might interfere with testing. In a seated position, forced expiratory volume in 1 second

(FEV1), the forced vital capacity (FVC) and FEV1/FVC ratio were obtained. Before

starting the ITL exercise, all subjects were instrumented with surface electromyography

(EMG) electrodes and transcutaneous NIRS optodes on the SCM, PS, IC (eight

intercostal space) and VL. Mouth pressure (Pm), partial pressure of end-tidal CO2

(PETCO2), oxygen saturation (SpO2), heart rate (HR), and mean arterial blood pressure

(MAP) were monitored continuously and are described in detail under the measurements

section. Maximal inspiratory pressure (MIP) was measured using an inspiratory muscle

force meter. Subjects performed an ITL test against progressive loading that was

increased every 2 minutes until task failure. Monitoring of all variables continued for a 5

minutes recovery period. MIP was repeated 5 minutes after ITL. The experimental set-up

is illustrated in Fig. 1.

2.3 Incremental Inspiratory Threshold Loading Test. The participant was seated

comfortably in an upright position with his arms supported on a table at the level of the

heart and his feet on the floor. While wearing nose clips, the subject inspired against a

weighted plunger through a fitted one-way valve attached to a mouthpiece using a

threshold loading apparatus identical to that described previously (Mathur et al., 2010).

With this apparatus, the weighted plunger lifts once the threshold inspiratory pressure is

achieved and allows air to flow so long as this threshold pressure is maintained.

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Fig. 1: Experimental setup (panel A), placement of NIRS probe over the left SCM

muscle (panel B) and over VL muscle (panel C).

Each trial began with the subject breathing in and out through the apparatus with

no load for 10 minutes to collect baseline cardiorespiratory, EMG and NIRS measures.

For the first stage of the ITL test, the subject breathed against an initial threshold load of

100 grams while an additional 50 grams of weight was added at each 2-minute interval.

Subjects were prompted by listening to a computer-generated audio signal with distinct

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inspiratory and expiratory tones to target breathing pattern at 10 breaths per minute with a

33% duty cycle (2 seconds inspiration and 4 seconds expiration in each breath).

Breathing continued without interruption until the point of task failure, defined as the

point when the subject could no longer lift the inspiratory threshold valve after at least

two attempts to breathe in.

2.4 Measurements

2.4.1 Spirometry and Maximal inspiratory pressure (MIP). In a seated position,

forced expiratory volume in 1 second (FEV1), the forced vital capacity (FVC), and the

FEV1/FVC ratio were obtained using a portable spirometer (Spirolab II, Medical

International Research, Vancouver, BC) and expressed using prediction equations (ATS,

2005). MIP was determined before and 5 minutes after the ITL test according to

standardized procedures (ATS, 2002) using an inspiratory muscle force meter

(MicroRPM, Vacumed). To ensure good reliability, the procedure was repeated until

three values within 5% were obtained.

2.4.2 Mouth pressure and CO2. Pm was continuously measured via a port located in the

mouthpiece that was connected to a calibrated pressure transducer (Model MP45–36-871,

Validyne, Northridge CA, USA) by polyethylene tubing. PETCO2 was continuously

monitored using a gas analyzer (CD-3A, AEI Technologies, Pittsburgh, PA, USA)

connected to the mouthpiece through a three-way stopcock and was maintained at

eucapnoeic levels by titrating 100% CO2 into the inspiratory circuit as needed.

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Monitoring continued for 5 minutes during the recovery period immediately after

cessation of ITL.

2.4.3 Cardiovascular variables. HR and mean arterial pressure (MAP) were monitored

continuously on a beat-by-beat basis with the use of a finger arterial plethysmograph

(Finometer, FMS, Finapres Medical Systems BV, Arnhem, the Netherlands). SpO2 was

measured using a finger pulse oximeter (Nonin 8600, Nonin Medical Inc., Plymouth,

MN). EMG and NIRS of the SCM, PS, IC and VL muscles were measured before,

during and after the ITL until the end of the 5-minute recovery period.

2.4.4 Surface EMG. EMG was obtained using surface electrodes (Soft-E H59P:

Kendall-LTP, Chicopee, MA, USA) with a 1 cm inter-electrode distance. The SCM

electrodes were placed midway between the mastoid process and the medial end of the

clavicle. The PS EMG electrodes were placed over the right second intercostal space just

lateral to the sternum and IC EMG electrodes were placed on the right eighth intercostal

space at the anterior axillary line. EMG electrodes for the VL were placed along the

vertical axis of this muscle approximately 12 to 15 cm above the knee joint. The

common electrode was placed and fixed over the coracoid process of the scapula. The

skin overlying each muscle was shaved and cleaned with alcohol prior to placing the

EMG electrodes. To minimize the interaction of electrocardiogram signals, SCM, PS and

IC EMG monitoring was measured on the right side of the body (Cram et al., 1998).

2.4.5 NIRS. Oxygenation and hemodynamics of the left SCM, PS, IC and VL muscles

were monitored continuously using a four-channel continuous-wave near-infrared

spectroscope (Oxymon M III, Artinis Medical Systems, BV, the Netherlands). The

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principle of NIRS and calculation of NIRS-derived parameters have been described

elsewhere (van der Sluijs et al., 1997; Grassi et al., 1999; Kirkpatrick et al., 1997;

Tachtsidis et al., 2007). In this study we determined changes in chromophore

concentrations of the oxygenated hemoglobin (O2Hb), deoxygenated hemoglobin (HHb),

total hemoglobin (tHb) and the difference between changes of O2Hb and HHb

concentrations (Hbdiff) in the tissue of interest.

Four NIRS optodes, attached to the NIRS instrument via fiber optic cables were

placed directly on the skin on top of the left SCM, PS, IC and VL muscles, at the similar

sites defined for EMG electrode placement. Skinfold thickness at the four points of

NIRS monitoring were measured using a skinfold caliper (Jamar 2058, Sammons Preston

Rolyan, USA) prior to placement of the NIRS optodes in order to determine the adipose

tissue thickness covering the muscles. A thickness of more than 10 mm was considered

as exclusion criteria to minimize the confounding effect of local adipose layer on in vivo

NIRS measurements (Homma et al., 1996; van Beekvelt, 2001). Interoptode distance of

the optodes was set at 25 mm for SCM and 30 mm for PS, IC and VL muscles, giving the

depth of penetration equal to half the interoptode distance in each site (Homma et al.,

1996). Differential path-length factor (DPF) of the NIRS instrument was set at 4 (van

Beekvelt, 2001) and calibration was repeated before each test. For each muscle, changes

in tissue oxygenation and local blood volume were estimated from changes in

chromophore concentrations of O2Hb and HHb and their variables tHb and Hbdiff

measured by NIRS.

2.5 Data and statistical analysis. All raw data including Pm, PETCO2, SpO2, HR, MAP

and EMG were recorded continuously at 1000 Hz using an analog-to-digital converter

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(PowerLab / 16SP model ML 795, ADI, Colorado Springs, CO, USA) and stored in a

computer for subsequent analysis.

Raw EMG signals were amplified, rectified, low-pass filtered (between 10 and

100 Hz) and smoothed using a 50-ms triangular (Bartlett) window (LabChart 6,

ADInstruments, Sydney, Australia). Integrated EMG signals were normalized to the

maximal EMG measured during the MIP maneuvers for each muscle across all subjects.

Time synchronized chromophore concentrations of O2Hb and HHb and their

variables tHb and Hbdiff were monitored in real time, sampled at 10 Hz, filtered (Moving

Gaussian) and stored on hard disk for further off-line analysis using dedicated software

(Oxysoft, Artinis Medical Systems, BV, The Netherlands). All NIRS values were zeroed

at the start point of the ITL test. Changes of chromophore concentrations and variables

were calculated for the entire duration and during each loading interval of the ITL test.

The ITL test duration was divided into ten equal intervals for each subject, in order to

calculate mean changes of chromophore concentrations of SCM, PS, IC and VL muscles

during each tenth percentile of the ITL.

Descriptive characteristics and spirometry values were used to summarize subject

characteristics. MIP values, obtained pre- and post-ITL from each subject, were

compared using Student’s paired t-test. NIRS values were tested for normality using the

Shapiro-Walk test. Analysis of variance (ANOVA) with Tukey’s post-hoc test was

performed to examine differences of ∆O2Hb, ∆HHb, ∆tHb, and ∆Hbdiff for the entire

duration of ITL in the four muscles (SCM, PS, IC, VL). Two-way repeated-measure

ANOVA was performed to examine differences in the pattern of changes of NIRS

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variables (O2Hb, HHb, tHb, and Hbdiff) between four muscles during ITL. When a

significant difference was detected, a single factor ANOVA with Tukey’s post-hoc test

was performed to locate statistically significant changes between baseline and the 10th

decile time intervals in each muscle. Data are presented as means ± SE. The level of

significance was set at P<0.05 for all statistical comparisons.

3. Results

3.1 Descriptive characteristics. Subjects (n=10) were male, young adults (27.5 ± 0.7

years), of normal body mass index (24 ± 0.7 kg·m−2) and had normal spirometry (Table

1).

Table 1. Pulmonary function data

n = 10

FVC, liters 6.0 ± 0.4 (4.2-7.9) % predicted 109 ± 6 (88-147)

FEV1, liters 4.7 ± 0.3 (3.4-6.36) % predicted 101 ± 5 (74-133)

FEV1/FVC 0.79 ± 0.01 (0.70-0.82) % predicted 93 ± 2 (74-98)

FVC, forced vital capacity; FEV1, forced expired volume in 1 second. Values are mean ±

SE. Ranges are presented in parentheses.

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3.2 Incremental Inspiratory Threshold loading test. Subjects began ITL exercise with

an initial resistive load of 100 grams (Pm = 20.8 ± 0.7 cmH2O) and ended at 635 ± 55.7

grams (Pm = 108.2 ± 9 cmH2O). The average duration of the ITL test was 22.4 ± 2.2

(11.3-32.1) minutes. As a group, there was a modest but non-significant increase in

PETCO2 from rest to the end of the ITL test (41.2 ± 1.3 vs. 44.2 ± 0.7 mmHg: P = 0.09).

The PETCO2 response was variable between subjects with some subjects demonstrating

mild CO2 retention (1-3 mmHg) whereas others were kept isocapnic by the administration

of supplemental CO2 during the course of the ITL.

3.3 Cardiorespiratory measures. MAP and HR increased significantly from the ITL start

point to task failure (Fig. 2). SpO2 was maintained ≥ 96% and no significant change was

observed across all subjects during the trials. There was no significant change in MIP

measured pre- and post-ITL (148.6 ± 10.7 vs. 152.9 ± 11.1 cmH2O, P > 0.05).

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Fig. 2. Blood volume redistribution during ITL. Changes in blood volume (ΔtHb) of the

SCM and VL and changes in heart rate and mean arterial pressure in 10 subjects during

ITL. Values are mean ± SE. * indicates significantly different from baseline (P < 0.05), †

indicates significantly different from the first point of significance (P < 0.05).

185

3.4 Near infrared spectroscopy. The rate and pattern of changes in muscle oxygenation

and blood volume for the SCM muscle differed from the other 3 muscles during the

entire duration of ITL. Fig. 3 shows the overall changes of NIRS variables in the four

muscles during ITL. While the PS and IC muscles showed an overall significant increase

in O2Hb (PS: 5.9 ± 2.3 µM, P<0.05 & IC: 6.8 ± 2.4 µM, P<0.05), a significant increase in

HHb was observed only in the SCM (17.3 ± 3.8 µM, P < 0.05). The level of tHb

increased in the SCM muscle during the ITL (20.7 ± 6.1 µM, P < 0.05) and decreased

significantly in the quiescent VL (-6.6 ± 2.4 µM, P < 0.05).

Fig. 3. Mean changes in NIRS variables of SCM, PS, IC and VL muscles for entire

duration of ITL. The mean changes of O2Hb, HHb, tHb and Hbdiff in the SCM, PS, IC

and VL muscles during the ITL test are shown indicating comparisons between muscles

and comparisons from baseline versus task failure within the same muscle. Brackets

show comparisons between muscles. * indicates statistical significance for mean

changes of NIRS variables between muscles (P < 0.05), † indicates statistical

significance in each muscle (baseline vs. task failure) (P < 0.05).

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Mean changes of O2Hb, HHb and tHb in the SCM, PS, IC and VL muscles across

ITL time (normalized to maximal duration) are depicted in Fig. 4. As loading started, PS

and IC showed a significant increase in O2Hb at 10% of ITL compared to baseline and

then at 80% (PS) and 70% (IC) compared to the first decile, which continued until task

failure. The SCM showed consistent increases in HHB throughout ITL, which was

significantly higher than baseline or from the previously significant time point at 30, 50,

80, 90 and 100% of the ITL duration. Progressive increase in tHb until task failure was

only observed in the SCM muscle. HHb tended to decrease and tHb decreased in VL

during the ITL. The significant decreases in tHb of the VL was detected at 60% and

progressed to the 90% of the ITL test duration. Chromophore concentrations changed

immediately towards baseline values after task failure when ITL was discontinued and

recovery began (Fig. 4).

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Fig. 4. Changes in chromophore concentration of SCM, PS, IC and VL muscles during

deciles of ITL. Mean changes of O2Hb, HHb and tHb in the SCM, PS, IC and VL muscles

during each decile of the ITL test (solid line) and recovery (dashed line) as measured by

NIRS. Values are mean ± SE. Closed markers indicate significant differences from

baseline and then from the last closed marker (P<0.05). For example, changes of PS

O2Hb were significant at the first decile (10% of ITL vs. baseline) and at the eighth decile

(80% vs. 10% of ITL duration).

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Changes in Hbdiff in the four muscles during and after the ITL test are shown in

Fig. 5. Only the SCM showed significant muscle deoxygenation as determined by a

steady decrease in Hbdiff (-13.8 ± 5.3 µM, P < 0.05) during the ITL test, which was

significantly lower at 40% and 70% of the ITL duration. No significant change in Hbdiff

was observed in the PS, IC and VL. The sudden increase in the SCM Hbdiff following

task failure reflects a rapid decrease in HHb and concomitant increases in O2Hb

concentrations (Fig. 4).

3.5 EMG. Timing of initial activation and progressive changes of EMG magnitude

differed among the 4 muscles throughout the duration of the ITL. No EMG activity was

detected in the SCM and IC during quiet breathing whereas the PS was active during

every breath. As soon as loading started, the SCM EMG showed a progressive

activation, which significantly increased at 70% of ITL time (vs. baseline, P<0.05) and

continued to the point of task failure. Compared to baseline, the EMG activity of the PS

increased significantly at 30% of ITL (P<0.05). Unlike PS and SCM, EMG activation of

IC was delayed until 30% of ITL duration (P<0.05) with no significant increase in

magnitude as loading continued to task failure. EMG data from the VL confirmed

muscle quiescence during all trials.

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Fig. 5. Changes in Hbdiff of SCM, PS, IC and VL muscles during deciles of ITL.

Changes of mean difference between O2Hb and HHb concentrations (Δ Hbdiff) in the

SCM, PS, IC and VL muscles during ITL test (solid line) and recovery (dashed line).

Values are mean ± SE. * indicates the first significant inflection point of Δ Hbdiff from

baseline (P < 0.05). † indicates significantly different from the first point of significance

(P < 0.05).

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Fig. 6 shows the raw EMG signals and concurrent changes of HHb, O2Hb and tHb

in the SCM, PS, IC and VL muscles in a single representative subject during ITL.

Changes of NIRS variables and EMG activities represented in this figure are consistent

with group mean observations from figures 3, 4, and 5.

Fig. 6. Pattern of EMG and NIRS changes. Raw EMG signals and changes of

chromophore concentrations in the SCM, PS, IC and VL muscles during the ITL test in a

representative subject. Note that the NIRS and EMG signals were related temporally and

in magnitude as they increased. For example, tHb and EMG both increased

progressively in SCM and PS throughout the duration of the ITL. Other important issues

191

were shown for the VL. Firstly, observable EMG in the VL was absent throughout ITL

verifying its quiescence and secondly, tHb was not only flat but actually decreased at

approximately the midpoint of the ITL duration.

4. Discussion

The unique finding of this study is that loaded inspiration causes the greatest

increases of muscle deoxygenation in the SCM in healthy men. We have also shown that

blood volume decreased in quiescent lower limb muscle and increased in working

respiratory muscles during ITL, which may be an indication of preferential blood flow

redistribution to the respiratory muscles. To our knowledge, this is the first study that has

used non-invasive NIRS for monitoring respiratory muscle oxygenation and

hemodynamic responses during ITL.

4.1 Physiological responses to ITL. We observed a significant increase in MAP and HR

in our subjects during ITL indicating that intensive respiratory workloads affected

systemic hemodynamics. Inspiratory threshold loading is considered a valid method for

assessing respiratory muscle endurance and function (Martyn et al., 1987) as well as for

improving respiratory muscle strength (Clanton et al., 1985; Turner & Jackson 2002) in

healthy individuals and those with respiratory disease. In this method, endurance of

respiratory muscles is measured as the maximum inspiratory load achieved during an

incremental loading regimen.

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We did not observe a significant decrease in MIP, measured 5 minutes after

cessation of ITL. This observation is consistent with previous studies that questioned

MIP as an index of force deficits in inspiratory muscles following the ITL and a reliable

measure of inspiratory muscle fatigue particularly for research purposes when accurate

and reproducible measurements are necessary (Eastwood et al., 1994; Aldrich & Spiro,

1995; Mathur et al., 2010).

4.2 Respiratory muscle activation. As respiratory loading begins and progresses,

accessory muscles of respiration in the neck and chest wall are recruited to expand the

ribcage in order to increase lung volumes. However, they represent different patterns of

activation (Campbell, 1970). Several investigators have quantified activation of

accessory muscles based on their mechanical advantages. By definition, a respiratory

muscle with a high mechanical advantage appears to be recruited earlier and contracts

stronger than a muscle with low mechanical advantage (De Troyer et al., 2005). Based

on this “neuromechanical matching” principle, it is postulated that the SCM, due to its

lower mechanical advantage, will contract later and with less force than PS and IC

muscles (Butler et al., 2007). Several studies concluded that, as an accessory muscle of

inspiration, the SCM does not activate during quiet breathing and is recruited after an

inspiratory load equal to 35% of MIP during static inspiration and 20% of MIP during

dynamic inspiration (De Troyer et al., 1994; Yokoba et al., 2003; Hudson et al., 2007).

We observed no active contraction of the SCM before and after the ITL test. Significant

inspiratory activation of SCM, as shown by EMG, was detected at 70% of the ITL

duration, which increased progressively to the point of task failure. In contrast with

193

delayed activation of IC, PS was active during every breath before, during and after ITL

with a continued increase in activity in response to loading, indicating its role as an

obligatory muscle of inspiration. Progressive activation of the SCM during ITL implies

its important function under conditions of increased ventilatory demand.

4.3 SCM oxygenation and hemodynamics during ITL. One of the key findings of this

study was that the concentration of oxygenated hemoglobin increased only in the PS and

IC during high intensity ITL. During breathing at rest, no significant difference or

specific pattern was observed for SCM oxygenation, which is in agreement with the work

of Hudson et al., (2007). SCM blood volume increased early upon loading which later

accelerated after 50% of ITL. The observed pattern was associated with an increase in

SCM HHb after 50% of ITL, which steadily continued until task failure (Fig. 4).

Significant increases in O2Hb concentrations of PS and IC muscles in the presence of a

constant blood volume, exhibited a significantly enhanced oxygenation in the PS and IC

muscles during ITL (Grassi et al., 1999). Our data showed that the ability of the PS and

IC muscles to be supplied by oxygenated hemoglobin throughout the entire ITL was well

preserved whereas the SCM failed to maintain adequate oxygenation. This confirms the

fact that primary muscles of respiration are well suited for working during strenuous

loaded breathing.

4.5 Blood flow redistribution from quiescent limb muscle during ITL. Another finding

of the present study was that ITL to the point of task failure elicited a reduction in blood

volume of the non-working quadriceps muscle. There is evidence to suggest that high

194

intensity respiratory muscle loading activates a metaboreflex that originates from fatigued

inspiratory muscles. This sympathetically mediated reflex reduces limb blood flow and

increases respiratory muscle circulation (Harms et al., 1997; Sheel et al., 2002). Some

studies have suggested that activation of the respiratory muscle metaboreflex starts at a

critical threshold of inspiratory muscle workload and does not occur during submaximal

efforts (Wetter et al., 1999; St Croix et al., 2000; Sheel et al., 2001 & 2002).

In the current study, a decrease in blood volume was detected in the VL after 60%

of ITL. A concurrent increase in SCM blood volume implied a redistribution of blood

volume from a quiescent limb muscle to an over loaded respiratory muscle which was

deoxygenated during ITL. Simultaneous time-dependent increases in HR and MAP

during ITL indicating an increase in cardiac output supports the occurrence of the blood

flow redistribution phenomenon in this experiment (Fig. 2).

4.6 Clinical implications. In healthy men SCM activates very early after loading

and deoxygenates prior to task failure. Further investigations may indicate whether this

phenomenon is accentuated in patients with imminent ventilatory failure such as those

with chronic obstructive pulmonary disease. NIRS has potential to advance our

understanding of human respiratory muscle oxygenation and hemodynamics during

loading in healthy individuals and those with respiratory disease.

4.7 Limitations of the study. In our experiment, while overall increases in PETCO2 over

the course of ITL was not statistically significant, there were four subjects that retained

modest levels of CO2 during the experiment. Changes of oxy- and deoxygenated

195

hemoglobin concentrations in working muscles are mainly affected by changes in

systemic and local oxygen delivery, extraction and consumption. Exhaustive respiratory

workloads that result in CO2 retention can influence muscle perfusion and therefore alter

muscle oxygenation and hemodynamic responses measured by NIRS. Nielsen et al.

(2001) demonstrated that hypercapnia increases O2Hb concentration and blood flow in

skeletal muscles while HHb concentration remained stable and unchanged. Although

CO2 levels were not significantly different from rest, we cannot rule out the possibility

that modest levels of CO2 retention may have influenced our results.

The results obtained in this study were limited to ten healthy male subjects.

Therefore, we cannot extend these observations to clinical populations that may have

compromised respiratory muscle oxygenation responses. Accordingly, future studies

should be conducted to assess respiratory muscle oxygenation under different

physiological conditions in different patient populations.

5. Conclusions

We found that during incremental loading in healthy young men, NIRS-derived

blood volume maintaind and oxygenated hemoglobin increased in the primary inspiratory

muscles. We demonstrated that compared to the SCM, primary respiratory muscles

including PS and IC are better able to be supplied by oxygenated hemoglobin throughout

progressive resistive breathing and therefore are more efficient to work under respiratory

loading conditions. Our findings also demonstrate that redistribution of blood flow from

quiescent limb muscles is a mechanism for maintaining inspiratory muscle oxygenation

196

during high respiratory motor output. We have also shown that NIRS is a useful

technique for non-invasive monitoring of respiratory muscle oxygenation and

hemodynamic responses during ITL. Additional studies on SCM oxygenation,

hemodynamics and activation may provide insight into the pathophysiology of chronic

obstructive pulmonary disease.

197

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