Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Monitoring of skeletal muscle ischemia using near infrared spectroscopy Shadgan, Babak 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2011_spring_shadgan_babak.pdf [ 8.35MB ]
Metadata
JSON: 24-1.0071639.json
JSON-LD: 24-1.0071639-ld.json
RDF/XML (Pretty): 24-1.0071639-rdf.xml
RDF/JSON: 24-1.0071639-rdf.json
Turtle: 24-1.0071639-turtle.txt
N-Triples: 24-1.0071639-rdf-ntriples.txt
Original Record: 24-1.0071639-source.json
Full Text
24-1.0071639-fulltext.txt
Citation
24-1.0071639.ris

Full Text

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  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 longterm 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.  ii  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. Tourniquetinduced 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.  iii  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 nearinfrared 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.  iv  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).  v  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  vi  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 vii  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  viii  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  ix  CHAPTER 4 – MONITORING OF TOURNIQUET-INDUCED SKELETAL MUSCLE INJURY BY NEAR INFRARED SPECTROSCOPY DURING ORTHOPAEDIC TRAUMA SURGERY...................................................................................................... 67 4.1 Introduction ......................................................................................................... 68 4.2 Materials and Methods ........................................................................................ 71 4.2.1 Subjects ......................................................................................................... 71 4.2.2 Experimental Overview ................................................................................ 71 4.2.3 Near Infrared Spectroscopy .......................................................................... 73 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 4.3.4 Near Infrared Spectroscopy .......................................................................... 76 4.3.5 Muscle Biopsy .............................................................................................. 78 4.3.6 OxyBlot vs. NIRS Regression ...................................................................... 80  x  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 5.2 Materials and Methods ........................................................................................ 95 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  xi  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  xii  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 tourniquetinduced 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  xiii  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  xiv  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  xv  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  xvi  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  xvii  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  xviii  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”.  xix  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.  xx  CHAPTER 1  Introduction  1  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.  2  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  3  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.  4  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  5  & 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  6  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.  7  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.  8  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 preischemic 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).  9  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  10  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 11  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  Intracompartmental pressure  Limb Circulation  Ischemic Biomarkers  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  12  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).  13  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  14  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 15  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  16  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 17  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 tourniquetinduced 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),  18  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.  19  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;  20  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  21  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 22  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.  23  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.  24  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.  25  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 tourniquetinduced limb muscle ischemia during ankle surgery without being affected by EMI of medical devices commonly used in orthopaedic operation room.  26  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 NIRSderived 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).  27  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).  28  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.  29  Table 1.2.  Overview of overall objectives and hypotheses of the studies in this thesis.  30  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.  31  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  32  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 33  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.  34  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).  35  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  36  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  37  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 nonspecific, 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 38  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  39  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  40  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.  41  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  42  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).  43  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.  2.2.7  Near Infrared Spectroscopy 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  44  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).  45  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, noninvasive, sensitive and specific tool for early detection of ACS (Giannotti et al., 2000).  46  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  47  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  48  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,  49  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. 50  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 nearinfrared spectroscopy of skeletal muscle oxygenation and hemodynamics during exercise and ischemia. Spectroscopy. 23(5):233-241, 2009. 51  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 oxygendependent 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.  52  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)  53  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 54  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.  55  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.  56  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.  57  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.  58  Maximum voluntary contraction force (MVC) was measured three times with 1minute 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 t59  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  60  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. 61  Table 3.2. NIRS variable or Chromophore  MVC 10%  30%  Ischemia 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  62  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.  63  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  64  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  65  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.  66  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.  67  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).  68  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  69  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.  70  4.2 4.2.1  Materials and Methods 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  71  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.  72  Figure 4.1. A summarized overview of the experimental protocol.  4.2.3  Near Infrared Spectroscopy 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  73  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.  74  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 75  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 1969 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.  4.3.4  Near Infrared Spectroscopy 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.  76  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.  77  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).  78  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).  79  n=4  n = 13  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)  80  (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.  81  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).  82  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  83  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 tourniquetinduced 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.,  84  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 tourniquetrelated 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 85  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 tourniquetinduced 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  86  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  87  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.  88  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.  89  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.  90  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 noninvasive 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.  91  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.  92  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 93  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  94  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.  5.2  Materials and Methods NIRS data were collected from patients with an ankle fracture who required  orthopaedic surgery at a university hospital. All patients provided informed consent. A 4channel 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 batterypowered orthopaedic drill (Hall PowerPro®, ConMed Linvatec) and a portable imaging  95  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.  96  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 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20  2 3 0 4 0 0 2 0 0 3 0 0 0 0 5 0 1 1 3 0  7 4 5 0 5 7 5 5 6 7 0 1 5 5 6 5 5 4 2 5  2 8 6 10 7 13 8 5 11 6 3 5 5 2 5 5 5 1 4 4  Total  24  89  124  97  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 98  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.  99  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.  100  CHAPTER 6  Conclusions  101  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).  102  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,  103  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 74 104  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  105  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.  106  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 60 107  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.  108  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.  109  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 nonlinear 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  110  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  111  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 112  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.  113  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  114  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 115  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 116  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.  117  REFERENCES Abdel-Salam A, Eyres KS: Effects of tourniquet during total knee arthroplasty. A prospective ran- domized study. J Bone Joint Surg. 1995;77B:250-3. Abe T, Kearns CF, Sato Y. Muscle size and strength are increased following walk training with restricted venous blood flow from the leg muscle, Kaatsu-walk training. J. Appl. Physiol. 2006; 100:1460Y6. Abraham P, Leftheriotis G, Saumet JL. Laser Doppler flowmetry in the diagnosis of chronic compartment syndrome. J Bone Joint Surg Br. 1998;80:365-9. Allen DG, Westerblad H, Lee JA, Lannergren J. Role of excitation-contraction coupling in muscle Fatigue. Sports Med 1992; 13:116-76. Al-Rawi PG. Near infrared spectroscopy in brain injury: today’s perspective. Acta Neurochir. 2005[Suppl];95:453-7. Amendola A, Twaddel BC. Compartment Syndromes. In: Browner BD, Jupiter JB, Levine AM, Trafton PG, eds. Skeletal Trauma; Basic Science, Management, and Reconstruction. Philadelphia: Saunders; 2003:268-92. Appell HJ, Glöser S, Duarte JA, Zellner A, Soares JM. Skeletal muscle damage during tourniquet-induced ischaemia. The initial step towards atrophy after orthopaedic surgery? Eur J Appl Physiol Occup Physiol. 1993;67:342-7. Appell HJ, Duarte JA, Gloser S, Remiao F, Carvalho F, Bastos ML, and Soares JM. Administration of tourniquet. II. Prevention of postischemic oxidative stress can reduce muscle edema. Arch Orthop Trauma Surg. 1997;116: 101-5. Arbabi S, Brundage SI, Gentilello LM. Near-infrared spectroscopy: a potential method for continuous, transcutaneous monitoring for compartmental syndrome in critically injured patients. J Trauma Inj Infec Critic Care. 1999;47:829-33.  118  Arokoski JP, Surakka J, Ojala T, et al. Feasibility of the use of a novel soft tissue stiffness meter. Physiol Meas. 2005;26:215-28. Artacho-Pdrula E, Rolan-Villalobos R, Vaamonde-Lemos R. Capillary and fibre size interrelationships in regenerating rat soleus muscle after ischemia: a quantitative study. Acta Anat. 1991;142:70-6. Arthur PG, Hogan MC, Bebout DE, Wagner PD, Hochachka PW. Modeling the effects of hypoxia on ATP turnover in exercising muscle. J. Appl. Physiol. 1992; 73:737Y42. Artinis Medical Systems B.V. The Oxymon Mk III System. http://www.artinis.com/ accessed on July 20, 2010. Asgari S, Rohrborn HJ, Engelhorn T, Stolke D. Intra-operative characterization of gliomas by near-infrared spectroscopy: possible association with prognosis. Acta Neurochir (Wien). 2003;145:453-9. Barreiro E, and Hussain S. Protein carbonylation in skeletal muscles: impact on function. Antioxid Redox Signal. 2010;12:417-29. Bayliss WM. General discussion on shock. Proc R Soc Med. 1919;12:33-34. Beilman GJ, Myers D, Cerra RFB. Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia. Shock. 2001;15:392-7. Benaron DA, Parachikov IH, Friedland S, Soetikno R, Brock-Utne J, van der Starre PJ, Nezhat C, Terris MK, Maxim PG, Carson JJ, Razavi MK, Gladstone HB, Fincher EF, Hsu CP, Clark FL, Cheong WF, Duckworth JL, Stevenson DK. Continuous, noninvasive, and localized microvascular tissue oximetry using visible light spectroscopy. Anesthesiology 2004;100:1469-75. Bhambhani Y, Buckley S, Susaki T. Muscle oxygenation trends during constant work rate cycle exercise in men and women. Med Sci Sports Exerc. 1999;31:90-8.  119  Bhattacharyya T, Vrahas MS. The medical-legal aspects of compartment syndrome. J Bone Joint Surg Am. 2004;86-A:864-8. Blaisdell FW. The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome: a review. Cardiovasc Surg. 2002;10:620–630. Blebea J, Kerr JC, Shumko JZ, Feinberg RN, Hobson RW. Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury. J Surg Res. 1987;43: 311-21. Block F, Detko G. Minimizing interference and false alarms from electrocautery in the Nellcor N-100 pulse oximeters. J Clin Monitoring 1986;2:203-5. Boldt J. Clinical review: hemodynamic monitoring in the intensive care unit. Critical Care (London). 2002;6:52-9. Bollman JL, and Flock EV. Changes in phosphate of muscle during tourniquet shock. Am J Physiol. 1944;142:290-2. Boros P, Bromberg JS. New cellular and molecular immune pathways in ischemiareperfusion injury. Am J Transplant. 2006;6:652-8. Boushel R, Piantadosi CA. Near-infrared spectroscopy for monitoring muscle oxygenation. Acta Physiologica Scandinavica. 2000a;168:615-22. Boushel R, Langberg H, Olesen J, Nowak M, Simonsen L, Bulow J, Kjaer M. Regional blood flow during exercise in humans measured by near-infrared spectroscopy and indocyanine green. J Appl Physiol. 2000b;89:1868–1878. Boushel R, Langberg H, Olesen J, Gonzales-Alonzo J, Bulow J, Kjaer M. Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease. Scand J Med Sci Sports. 2001;11:213-22.  120  Boushel R, Langberg H, Gemmer C, Olesen J, Crameri R, Scheede C, Sander M, Kjaer M. Combined inhibition of nitric oxide and prostaglandins reduces human skeletal muscle blood flow during exercise. J Physiol. 2002;543:691-8. Brazy JE, Lewis DV, Mitnisk MH, Jobsis-van der Vliet FF. Noninvasive monitoring of cerebral oxygenation in preterm infants: preliminary observation. Pediatrics. 1985;75:217-25. Briers JD. Laser Doppler, speckle and related techniques for blood perfusion mapping and imaging. Physiol Meas. 2001;22:R35-66. Buunk G, van der Hoeven JG, Meinders AE. A comparison of near-infrared spectroscopy and jugular bulb oximetry in comatose patients resuscitated from a cardiac arrest. Anaesthesia. 1998;53:13-9. Cakatay U, Kayali R, Uzun H. Relation of plasma protein oxidation parameters and paraoxonase activity in the ageing population. Clin Exp Med. 2008;8:51-7. Cakatay U, Aydin S, Yanar K, Uzun H. Gender-dependent variations in systemic biomarkers of oxidative protein, DNA, and lipid damage in aged rats. Aging Male. 2010;13:51-8. Carini R, de Cesaris MG, Splendore R, Bagnati M, Albano E. Ischemic preconditioning reduces Na(C) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia. Hepatology 2000;31:166–172. Casavola C, Paunescu LA, Fantini S, Gratton E. Blood flow and oxygen consumption with near-infrared spectroscopy and venous occlusion: spatial maps and the effect of time and pressure of inflation. J Biomed Opt. 2000;5:269-76. Chance B, Dait MT, Zhang C, Hamaoka T Hagerman F. Recovery from exercise-induced desaturation in the quadriceps muscles of elite competitive rowers. Am J Physiol. 1992; 262: C766–C775.  121  Chang YH, Tu YK, Yeh WL. Tibial platue fracture with compartment syndrome: a complication of higher incidence in Taiwan. Chang Gung Med J. 2000;23:149-55. Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987;40:373-83. Chiu D, Wang HH, Blumenthal MR. Creatine phosphokinase release as a measure of tourniquet effect on skeletal muscle. Arch Surg. 1976;111:71-4. Choudhary RK, Waseem M, Thalva R, Dunlop DG. Acute compartment syndrome: diagnosis and immediate care. Hosp Med 2003;64:296-8. Christodoulou AG, Ploumis AL, Terzidis IP, Chantzidis P, Metsovitis SR, Nikiforos DG. The role of timing of tourniquet release and cementing on perioperative blood loss in total knee replacement. Knee. 2004;11:313-7. Clanton TL. Hypoxia-induced reactive oxygen species formation in skeletal muscle. J. Appl. Physiol. 2007; 102:2379Y88. Coirault C, Guellich A, Barbry T, Samuel JL, Riou B, and Lecarpentier Y. Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure. Am J Physiol Heart Circ Physiol. 2007;292: H1009-1017. Cooper CE, Springett R. Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy. Philos Trans R Soc Lond B Biol Sci. 1997;352:669-76. Cope M, Delpy DT, Wray S, J.Wyatt JS, Reynolds EOR. A CCD spectrometer to quantitate the concentration of chromophores in living tissue utilizing the absorption peak of water at 975 nm. Adv Exp Med Biol. 1989;248:33-40. Costes F, Barthelemy JC, Feasson L, Busso T, Geyssant A, Deni C. Comparison of muscle near-infrared spectroscopy and femoral blood gases during steady-state exercise in humans. J Appl Physiol. 1996;80:1345–50.  122  Court-Brown CM, McBirnie J. The epidemiology of tibial fractures. J Bone Joint Surg Br. 1995;77:417-21. Court-Brown CM. Fractures of the Tibia and Fibula. In: Buchholz RW, Heckman JD, Court-Brown CM, eds. Rockwood and Green's Fractures in Adults. Philadelphia: Lippincott, Williams & Wilkins; 2006:2079-146. Court-Brown CM, Koval KJ. The Epidemiology of Fractures. In: Buchholz RW, Heckman JD, Court-Brown CM, eds. Rockwood and Green's Fractures in Adults. Philadelphia: Lippincott Williams & Wilkins, 2006:96-143. Cross KM, Leonardi L, Payette JR et al. Clinical utilization of near-infrared spectroscopy devices for burn depth assessment. Wo™und Repair Regen. 2007;15:332-40. Cui W, Kumar C, Chance B. Experimental study of the migration depth for the photons measured at sample surface. Time resolved spectroscopy and imaging. Proc Int Soc Opt Eng. 1991;1431:180-91. Daniel DM, Lumkong G, Stone ML, Pedowitz RA: Effects of tourniquet use in anterior cruciate ligament reconstruction. Arthroscopy. 1995;11:307-11. Davey PR, Thorpe RD, Williams C. Fatigue decreases skilled tennis performance. J Sports Sci. 2002;20:311-8. David HG. Pulse oximetry in closed limb fractures. Ann R Coll Surg Engl.1991;73:2834. De Blasi RA, Cope M, Elwell C, Safoue F, Ferrari M. Noninvasive measurement of human forearm oxygen consumption by near infrared spectroscopy, Eur J Appl Physiol. 1993;67:20-5. De Blasi RA, Ferrari M, Natali A, Conti G, Mega A, Gasparetto A. Noninvasive measurement of forearm blood flow and oxygen consumption by near-infrared spectroscopy. J Appl Physiol. 1994;76:1388-93.  123  Dellon AL, Schneider RJ, Burke R. Effect of acute compartmental pressure change on response to vibratory stimuli in primates. Plast Reconstr Surg. 1983;72:208-16. Delpy DT, Cope M, Zee P van der, Arridge S, Wray S, Wyatt J. Estimation of optical pathlength through tissue from direct time of flight measurements. Phys med Biol. 1988;33:1433-42. Dickson KF, Sullivan MJ, Steinberg B. Noninvasive measurement of compartment syndrome. Orthopedics. 2003;26:1215-8. Dobner JJ, Nitz AJ: Postmeniscectomy tourniquet palsy and functional sequelae. Am J Sports Med. 1982;101:211-4. Duarte JA, Gloser S, Remiao F, Carvalho F, Bastos ML, Soares JM, Appell HJ. Administration of tourniquet. I. Are edema and oxidative stress related to each other and to the duration of ischemia in reperfused skeletal muscle? Arch Orthop Trauma Surg. 1997;116:97-100. Dum RP, Kennedy TT. Physiological and histochemical characteristics of motor units in cat tibialis anterior and extensor digitorum longus muscles. J NeurophysioL. 1980;43:1615-30. Duncan A, Meek JH, Clemence M, Elwell CE, Tyszczuk L, Cope M, Delpy DT. Optical pathlength measurements on adult head, calf and forearm and the head of the newborn infant using phase resolved optical spectroscopy. Phys Med Biol. 1995;40:295-304. Dunn R. Plastic Surgery Education Foundations: Essay. Plastic Surgery Edu. Foundation, 1990. Chicago, IL. Durán WN, Dillon PK. Effects of ischemia-reperfusion injury on microvascular permeability in skeletal muscle. Microcirc Endothelium Lymphatics. 1989;5:22339. Eberlin KR, McCormack MC, Nguyen JT, Tatlidede HS, Randolph MA, Austen WG Jr.  124  Ischemic preconditioning of skeletal muscle mitigates remote injury and mortality. J Surg Res. 2008;148:24-30. Edwards PD, Miles KA, Owens SJ. A new non-invasive test for the detection of compartment syndromes. Nucl Med Commun. 1999;20:215-8. Eliason JL, Wakefield TW. Metabolic consequences of acute limb ischemia and their clinical implications. Semin Vasc Surg. 2009;22:29-33. Elliott KG, Johnstone AJ. Diagnosing acute compartment syndrome. J Bone Joint Surg Br. 2003;85:625-32. Fahmy NR, Patel DG. Hemostatic changes and postoperative deep-vein thrombosis associated with use of a pneumatic tourniquet. J Bone Joint Surg Am. 1981;63:461-5. Fanò G, Mecocci P, Vecchiet J, Belia S, Fulle S, Polidori MC, Felzani G, Senin U, Vecchiet L, Beal MF. Age and sex influence on oxidative damage and functional status in human skeletal muscle. J Muscle Res Cell Motil. 2001;22:345-51. Ferrari M, Binzoni T, Quaresima V. Oxidative metabolism in muscle. Philos Trans R Soc Lond B Biol Sci. 1997;352(1354):677-83. Ferrari M, Mottola L, Quaresima V. Principles, techniques and limitations of near infrared spectroscopy. Can J Appl Physiol. 2004;29:463-87. Finkelstein Ja, Hunter GA, Hu RW. Lower limb compartment syndrome: course after delayed fasciotomy. J Trauma. 1996;40:342-4. Fitzgerald AM, Gaston P, Wilson Y, Quaba A, McQueen MM. Long-term sequelae of fasciotomy wounds. Brit J Plast Surg. 2000;53:690-3. Flatt AE. Tourniquet time in hand surgery. Arch Surg. 1972;104:190-2.  125  Gagnon RE, Macnab AJ. Near Infrared spectroscopy (NIRS) in the clinical setting – An adjunct to monitoring during diagnosis and treatment. Spectroscopy 2005a;19:221-33. Gagnon R, Macnab AJ, Gagnon F. Brain, spine and muscle Cu-A redox patterns of change during hypothermic circulatory arrest in swine. Comp Biochem Physiol. 2005b;141:264-70. Garr JL, Gentilello LM, Cole PA. Monitoring for compartmental syndrome using nearinfrared spectroscopy: a noninvasive continuous transcutaneous monitoring technique. J Trauma. 1999;46:613-8. Gentilello LM, Sanzone A, Wang L, Liu PY, Robinson L. Near-infrared spectroscopy versus compartment pressure for the diagnosis of lower extremity compartmental syndrome using electromyography-determined measurements of neuromuscular function. J Trauma. 2001;51:1-8. Giannotti G, Cohn SM, Brown M. Utility of near-infrared spectroscopy in the diagnosis of lower extremity compartment syndrome. J Trauma Inj Infec Critic Care. 2000;48:396-401. Giannoudis PV, Nicolopoulos C, Dinopoulos H. The impact of lower leg compartment syndrome on health related quality of life. Injury. 2002;33:117-21. Gómez H, Torres A, Polanco P, Kim HK, Zenker S, Puyana JC, Pinsky MR. Use of noninvasive NIRS during a vascular occlusion test to assess dynamic tissue O2 saturation response. Intensive Care Med. 2008;34:1600-7. Grassi B, Quaresima V, Marconi C, Ferrari M, Cerretelli P. Blood lactate accumulation and muscle deoxygenation during incremental exercise. J Appl Physiol. 1999;87: 348-55. Gravvanis AI, Tsoutsos DA, Karakitsos D, Panayotou P, Iconomou T, Zografos G, Karabinis A, Papadopoulos O. Application of the pedicled anterolateral thigh flap to defects from the pelvis to the knee. Microsurgery. 2006;26:432-8. 126  Grisotto PC, dos Santos AC, Coutinho-Netto J, Cherri J, Piccinato CE. Indicators of oxidative injury and alterations of the cell membrane in the skeletal muscle of rats submitted to ischemia and reperfusion. J Surg Res. 2000;92:1-6. Grubhofer G, Plöchl W, Skolka M, Czerny M, Ehrlich M, Lassnigg A. Comparing Doppler ultrasonography and cerebral oximetry as indicators for shunting in carotid endarterectomy. Anesth Analg. 2000;91:1339-44. Haljamae H, and Enger E. Human skeletal muscle energy metabolism during and after complete tourniquet ischemia. Ann Surg. 1975;182:9-14. Hamaoka T, Iwane H, Shimomitsu T, et al. Noninvasive mea- sures of oxidative metabolism on working human muscles by near-infrared spectroscopy. J Appl Physiol 1996; 81: 1410-7. Hamaoka T, McCully KK, Quaresima V, Yamamoto Y, Chance B. Near-infrared spectroscopy imaging for monitoring muscle oxygenation and oxidative metabolism in healthy and diseased humans. J Biomed Optics. 2007;12:062105. Hammersen F, Barker JH, Gidlöf MD, Menger MD, Hammersen E, Messmer K. The ultrastructure of microvessels and their contents following ischemia and reperfusion. Prog Appl Microcirc. 1989;13:1-26. Harel F, Olamaei N, Ngo Q, Dupuis J, Khairy P. Arterial flow measurements during reactive hyperemia using NIRS Physiol Meas. 2008;29:033–40. Harris K, Walker PM, Mickle DA, Harding R, Gatley R, Wilson GJ, Kuzon B, McKee N, Romaschin AD. Metabolic response of skeletal muscle to ischemia. Am J Physiol. 1986;250:213-20. Harris IA, Kadir A, Donald G. Continuous compartment pressure monitoring for tibial fractures: does it influence outcome? J Trauma Inj Infec Critic Care. 2006;60:1330-5.  127  Henke PK. Approach to the patient with acute limb ischemia: diagnosis and therapeutic modalities. Cardiol Clin 2002;20:513-20. Heppenstall RB, Scott R, Sapega A, Park YS, Chance B. A comparative study of the tolerance of skeletal muscle to ischemia. Tourniquet application compared with acute compartment syndrome. J Bone Joint Surg Am. 1986;68:820–8. Hicks A, McGill S, Hughson RL. Tissue oxygenation by near-infrared spectroscopy and muscle blood flow during isometric contractions of the forearm. Can J Appl Physiol. 1999;24:216-30. Homma S, Eda H, Ogasawara S, Kagaya A. Near-infrared estimation of O2 supply and consumption in forearm muscles working at varying intensity. J Appl Physiol. 1996; 80:1279 -84. Hope MJ, McQueen MM. Acute compartment syndrome in the absence of fracture. J Orthop Trauma. 2004;18:220-4. Hoshi Y, Chen SJ. New dimensions of cognitive neuroscience research with near-infrared spectroscopy: Free motion neuroimaging studies In: Progress in Brain Mapping Research. Ed FJ Chen Nova Science, New York 2006:205-229. Hoshi Y. Functional near-infrared spectroscopy: current status and future prospects. J Biomed Optics. 2007;12:062106. Huda R, Solanki DR, and Mathru M. Inflammatory and redox responses to ischaemia/reperfusion in human skeletal muscle. Clin Sci (Lond). 2004;107:497503. Humphreys PW, Lind AR. The blood flow through the active and inactive muscles of the forearm during sustained handgrip contractions. J Physiol. 1963;166:120–35. Idstrom JP, Soussi B, Elander A, Bylund-Fellenius AC. Purine metabolism after in vivo ischemia and reperfusion in rat skeletal muscle. Am J Physiol. 1990;258:1668-73.  128  International Electrotechnical Commission (IEC) Standard 60601-1-2. 2nd edition, Part 1-2: General requirements for safety—collateral standard: Electromagnetic compatibility—requirements and tests, 2002. Ishii Y, Matsuda Y. Effect of tourniquet pressure on perioperative blood loss associated with cementless total knee arthroplasty: a prospective, randomized study. J Arthroplasty. 2005;20:325–30. Ishii Y, Noguchi H, Matsuda Y. A new tourniquet system that determines pressure in synchtony with systolic blood pressure. Arch Orthop Trauma Surg. 2008;128:297-300. Jami L, Murthy KSK, Petit J, Zytnicki D. Distribution of physiological types of motor units in the cat peroneus tertius muscle. Exp Brain Res. 1982;48:177-84. Janzing HMZ, Broos PLO. Routine monitoring of compartment pressure in patients with tibial fractures: beware of overtreatment! Injury. 2001;32:415-21. Jennings RB, Sebbag L, Schwartz LM, Crago MS, Reimer KA. Metabolism of preconditioned myocardium: effect of loss and reinstatement of cardioprotection. J Mol Cell Cardiol 2001;33:1571–1588. Jerome SN, Akimitsu T, Gute DC, Korthuis RJ. Ischemic preconditioning attenuates capillary no-reflow induced by prolonged ischemia and reperfusion. Am J Physiol 1995;268:H2063–2067. Jerosch J, Geske B, Sons HU. The value of sonography in assessing intracompartmental pressure in the anterior tibial compartment. Ultraschall Med. 1989;10:206-10. Jobsis FF. Noninvasive, infrared monitoring of cerebral and myocardial oxygen sufficiency and circulatory parameters. Science. 1977;198:1264-7. Joseph B, Varghese RA, Mulpuri K. Measurement of tissue hardness: can this be a method of diagnosing compartment syndrome noninvasively in children? J Pediatr Orthop. 2006;Part B.15:443-8.  129  Kagaya A, Homma S. Brachial arterial blood flow during static handgrip exercise of short duration at varying intensities studied by a Doppler ultrasound method. Acta Physiol Scand. 1997;160: 257–65. Kahn JF, Jouanin JC, Bussiere JL, Tinet E, Avrillier S, Ollivier JP, Monod H. The isometric force that induces maximal surface muscle deoxygenation. Eur J Appl Physiol. 1998; 78: 183–7. Kahraman S, Kayali H, Atabey C, Acar F, Gocmen S. The accuracy of near-infrared spectroscopy in detection of subdural and epidural hematomas. J Trauma. 2006;61:1480–3. Kasirajan K, Ouriel K. Current options in the diagnosis and management of acute limb ischemia. Prog Cardiovasc Nurs 2002;17:26-34. Katzen BT. Clinical diagnosis and prognosis of acute limb ischemia. Rev Cardiovasc Med 2002;3:S2-6. Kavazis AN, Talbert EE, Smuder AJ, Hudson MB, Nelson WB, and Powers SK. Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production. Free Radic Biol Med. 2009;46:842-50. Kayali R, Cakatay U, Uzun H, Genç H. Gender difference as regards myocardial protein oxidation in aged rats: male rats have increased oxidative protein damage. Biogerontology. 2007a;8:653-61. Kayali R, Cakatay U, Tekeli F. Male rats exhibit higher oxidative protein damage than females of the same chronological age. Mech Ageing Dev. 2007b;128:365-9. Kell RT, Bhambhani Y. Relationship between erector spinae muscle oxygenation via in vivo near infrared spectroscopy and static endurance time in healthy males. Eur J Appl Physiol. 2008;102:243-50. Kent M. Oxford Dictionary of Sports Science and Medicine. New York: Oxford;2007.  130  Kerksick C, Taylor LT, Harvey A, Willoughby D. Genderrelated differences in muscle injury, oxidative stress, and apoptosis. Med Sci Sports Exerc. 2006;40:1772–80. Kharbanda RK, Peters M, Walton B, Kattenhorn M, Mullen M, Klein N et al. Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans in vivo. Circulation 2001;103:1624–30. Kim JG, Lee J, Roe J, Tromberg BJ, Brenner M, Walters TJ. Hemodynamic changes in rat leg muscles during tourniquet-induced ischemia-reperfusion injury observed by near-infrared spectroscopy. Physiol Meas 2009;30:529-40. Kim GT, Chun YS, Park JW, Kim MS. Role of apoptosisinducing factor in myocardial cell  death  by  ischemia-reperfusion.  Biochem  Biophys  Res  Commun  2003;309:619–624. Kime R, Hamaoka T, Sako T, Murakami M, Homma T, Katsumura T,Chance B. Delayed reoxygenation after maximal isometric handgrip exercise in high oxidative capacity muscle. Eur J Appl Physiol. 2003;89:34-41. Kirkpatrick PJ, Lam J, Al-Rawi P, Smielewski P, Czosnyka M. Defining thresholds for critical ischemia by using near-infrared spectroscopy in the adult brain. J Neurosurg. 1997;89,:389–94. Knochel JP. Mechanisms of rhabdomyolysis. Curr Opin Rheumatol. 1993;5:725-31. Koh TJ, Tidball JG. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells. Am J Physiol Cell Physiol. 2000;279:C806-12. Konrad G, Markmiller M, Lenich A, Mayr E, and Ruter A. Tourniquets may increase postoperative swelling and pain after internal fixation of ankle fractures. Clin Orthop Relat Res. 2005;433:189-94. Kostler W, Strohm PC, Sudkamp NP. Acute compartment syndrome of the limb. Injury. 2004;35:1221-7.  131  Kragelj R, Jarm T, Erjavec T, Presern-Strukelj M, Miklavcic D. Parameters of postocclusive reactive hyperemia measured by near infrared spectroscopy in patients with peripheral vascular disease and in healthy volunteers. Ann Biomed Eng 2001;29:311-20. Lampert R, Weih EH, Breucking E. Postoperative bilateral compartment syndrome resulting from prolonged urological surgery in lithotomy position. Serum creatine kinase activity CK as a warning signal in sedated, artificially respirated patients. Anaesthesist. 1995;44:43-7 Lanza IR, Wigmore DM, Befroy DE, Kent-Braun JA (2006) In vivo ATP production during free-flow and ischaemic muscle contractions in humans. J Physiol 577:353–367. doi:10.1113/jphysiol. 2006.114249 Lapinsky S, Easty A. Electromagnetic interference in critical care. J Critic Care. 2006;21:267-70. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxiareoxygenationinjury. Am J Physiol Cell Physiol. 2002;282:C227-41. Lind AR, McNicol GW. Local and central circulatory responses to sustained contractions and the effect of free or restricted arterial inflow on post-exercise hyperaemia. J Physiol Lond. 1967; 192: 575–93. Liu F, Luo Q, G. Xu, Li P. Noninvasive detection of changes in skeletal muscle oxygenation during incremental exercise with near-infrared spectroscopy. Proc SPIE. 2003;5254:573-9. López-Higuera JM. Handbook of optical fibre sensing technology. New York: John Wiley; 2002. Lynch JE, Heyman JS, Hargens AR. Ultrasonic device for the noninvasive diagnosis of compartment syndrome. Physiol Meas. 2004;25:N1-9.  132  MacDonald MJ, Tarnopolsky MA, Green HJ, Hughson RL. Comparison of femoral blood gases and muscle near-infrared spectroscopy at exercise onset in humans. J Appl Physiol. 1999;86:687-93. Macnab AJ, Gagnon RE, Smith M, Moodley M, Roland EH, Hill A. Near Infrared Spectroscopy (NIRS) interference faults during neonatal data collection. Ped Research 1994;36:26. Macnab AJ, Gagnon RE, Gagnon FA, LeBlanc JG. NIRS monitoring of brain and spinal cord – detection of adverse intraoperative events. Spectroscopy. 2003;17:483-90. Macnab AJ. Biomedical Applications of Near Infrared Spectroscopy. In Barth A, Haris PI. Biological and Biomedical Infrared Spectroscopy Volume 2. 1st ed. IOS Press, 2009:355 – 402. Maffulli N, Testa V, Capasso G: Use of a tourniquet in the internal fixation of fractures of the distal part of the fibula. A prospective randomized trial. J Bone Joint Surg. 1993;75A:700-3. Magnusson M. Posture. In: Anderson GBJ. Nordin M, Pope MH, editors. Musculoskeletal disorders in the workplace. St. Louis: Mosby-Year Book; 1997. p. 73 chapter 81. Malinoski DJ, Slater MS, Mullins RJ. Crush injury and rhabdomyolysis. Critic Care Clin. 2004;20:171-92. Mancini DM, Bolinger L, Li H, Kendrick K, Chance B, Wilson J. Validation of nearinfrared spectroscopy in humans. J Appl Physiol. 1994;77: 2740-7. Mars M, Maseko S, Thomson S. Can pulse oximetry detect raised intracompartmental pressure?. South African J Surg. 1994a;32:48-50. Mars M, Hadley GP. Failure of pulse oximetry in the assessment of raised limb intracompartmental pressure. Injury. 1994b;25:379–381.  133  Massey FJ. The Kolmogorov Smirnov test for goodness of fit. J Am Stat Ass. 1951;46:68-78. Mathru M, Huda R, Solanki DR, Hays S, Lang JD. Inhaled nitric oxide attenuates reperfusion inflammatory responses in humans. Anesthesiology. 2007;106:27582. Matsen F, Winquist R, Krugmire R. Diagnosis and management of compartmental syndromes. J Bone Joint Surg Am. 1980;162:286-91. McCormick PW, Stuart M, Goetting MG, Balakrishnan G. Regional cerebrovascular oxygen saturation measured by optical spectroscopy in humans. Stroke. 1991;22:596-602. McCully KK, Iotti S, Kendrick K, Wang Z, Posner JD, Leigh J JR, Chance B. Simultaneous in vivo measurements of HbO2 saturation and PCr kinetics after exercise in normal humans. J Appl Physiol. 1994;77:5–10. McEwen J, Casey V. Measurement of hazardous pressure levels and gradients produced on human limbs by non-pneumatic tourniquets. In: Proceedings of the 32nd Conference of the Canadian Medical and Biological Engineering Society 2009. Calgary, Canada; 2009;20-22:1-4. McLaren AC, Rorabeck CH. The pressure distribution under tourniquets. J Bone Joint Surg Am. 1985;67:433-8. McQueen MM, Christie J, Court-Brown CM. Compartment pressure after intramedullary nailing of the tibia. J Bone Joint Surg Br. 1990;72:395-7. McQueen MM. How to monitor compartment pressures. Tech Orthop. 1996;11:99-101. Mc Queen MM, Christie J, Court-Brown CM. Acute compartment syndrome in tibia diaphyseal fractures. J Bone Joint Surg Br. 1996a;78:95-8.  134  McQueen MM, Christie J, Court-Brown CM. Compartment pressure monitoring in tibial fractures. J Bone Joint Surg. 1996b;78:99-104. McQueen MM, Gaston P, Court-Brown CM. Acute compartment syndrome. Who is at risk? J Bone Joint Surg Br. 2000;82:200-3. McQueen MM. Acute Compartment Syndrome. In: Buchholz RW, Heckman JD, CourtBrown CM, eds. Rockwood and Green's Fractures in Adults. Philadelphia: Lippincott Williams & Wilkins; 2006:425-43. McPherson G, Wolfe JHN. Acute ischaemia of the leg. In: Wolfe J, ed. ABC of vascular diseases. London: BMJ Publishing, 1992:15-8. Middleton RW, Varian JP. Tourniquet paralysis. Aust N Z J Surg. 1974;44:124-8. Mitsuta H, Ohdan H, Fudaba Y, Irei T, Tashiro H, Itamoto T, Asahara T. Near-infrared spectroscopic analysis of hemodynamics and mitochondrial redox in right lobe grafts in living-donor liver transplantation. Am J Transplant. 2006;6:797-805. Moalla W, Dupont G, Berthoin S, Ahmaidi S. Respiratory muscle Deoxygenation and ventilatory threshold assessments using near infrared spectroscopy in children. Int J Sports Med. 2005;26:576-82. Mohler LR, Pedowitz RA, Myers RR, Ohara WM, Lopez MA, Gershuni DH. Intermittent reperfusion fails to prevent posttourniquet neurapraxia. J Hand Surg Am. 1999;24:687-93. Mubarak SJ, Hargens AR, Owen CA. The wick catheter technique for measurement of intramuscular pressure. A new research and clinical tool. J Bone Joint Surg Am. 1976;58:1016-20. Mubarak SJ, Owen CA, Hargens AR. Acute compartment syndromes: diagnosis and treatment with the aid of the wick catheter. J Bone Joint Surg Am. 1978;60:10915.  135  Muehlemann T, Haensse D, Wolf M. Wireless miniaturized in-vivo near infrared imaging. Opt Express. 2008;16:10323-30. Murphy CG, Winter DC, Bouchier-Hayes DJ. Tourniquet injuries: pathogenesis and modalities for attenuation. Acta Orthop Belg. 2005;71:635-45. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36. Murthy G, Kahan NJ, Hargens AR, Rempel DM. Forearm muscle oxygenation decreases with low levels of voluntary. contraction. J Orthop Res. 1997;15:507-11. Murthy G, Hargens AR, Lehman S, Rempel DM. Ischemia causes muscle fatigue. J Orthop Res. 2001;19:436-40. Nakayama M, Iwasaki S, Ichinose H. Intraoperative acute lower extremity ischemia detected by near-infrared spectroscopy. J Cardiothorac Vasc Anesth. 2001;15:624-5. Neary JP. Application of near infrared spectroscopy to exercise sports science. Canadian J Appl Physiol. 2004; 29:488-503. Niwayama M, Hamaoka T, Lin L, Shao J, Kudo N, Katoh C, Yamamato K. Quantitative muscle oxygenation measurement using NIRS with correction for the influence of a fat layer: comparison of oxygen consumption rates with measurements by other techniques. Proc. SPIE, Vol. 3911, 256 (2000); doi:10.1117/12.384911 Noordin S, McEwen JA, Kragh Jr. JF, Eisen A, Masri BA. Surgical Tourniquets in Orthopaedics. J Bone Joint Surg Am. 2009;91:2958-67. Nylander G, Otamiri T, Lewis DH, and Larsson J. Lipid peroxidation products in postischemic skeletal muscle and after treatment with hyperbaric oxygen. Scand J Plast Reconstr Surg Hand Surg. 1989;23:97-103.  136  Obrig H, Villringer A. Beyond the visible – imaging the human brain with light. J Cereb Blood Flow Metab. 2003;23:1-18. Ochoa J, Danta G, Fowler TJ, Gilliatt RW. Nature of the nerve lesion caused by a pneumatic tourniquet. Nature. 1971;233:265-6. Odinsson A, Finsen V. Tourniquet use and its complications in Norway. J Bone Joint Surg Br. 2006;88:1090-2. Odland R, Schmidt AH, Hunter B. Use of tissue ultrafiltration for treatment of compartment syndrome: a pilot study using porcine hindlimbs. J Orthop Trauma. 2005;19:267-75. Olson SA, Glasgow RR. Acute compartment syndrome in lower extremity musculoskeletal trauma. J Am Academy of Orthop Surg. 2005;13:436-44. Ömeroğlu H, Günel U, Biçimoğlu A, Tabak AY, Uçaner A, Güney Ö. The relationship between the use of tourniquet and the intensity of post-operative pain in surgically treated malleolar fractures. Foot Ankle. 1997;18:798–802. Ozyurt B, Iraz M, Koca K, Ozyurt H, and Sahin S. Protective effects of caffeic acid phenethyl ester on skeletal muscle ischemia-reperfusion injury in rats. Mol Cell Biochem. 2006;292:197-203. Pang CY, Yang RZ, Zhong A, Xu N, Boyd B, Forrest CR. Acute ischaemic preconditioning protects against skeletal muscle infarction in the pig. Cardiovasc Res 1995;29(6):782–788. Papanastasiou S, Estdale SE, Homer-Vanniasinkam S, Mathie RT. Protective effect of preconditioning and adenosine pretreatment in experimental skeletal muscle reperfusion injury. Br J Surg 1999;86:916–922. Pasupathy S, and Homer-Vanniasinkam S. Ischaemic preconditioning protects against ischaemia/reperfusion injury: emerging concepts. Eur J Vasc Endovasc Surg. 2005a;29:106-15.  137  Pasupathy S, Homer-Vanniasinkam S. Surgical implications of ischemic preconditioning. Arch Surg. 2005b;140:405-10. Pedowitz RA, Gershuni DH, Friden J, Garfin SR, Rydevik BL, Hargens AR. Effects of reperfusion intervals on skeletal muscle injury beneath and distal to a pneumatic tourniquet. J Hand Surg Am. 1992;17:245-55. Pemberton M, Anderson G, Vetvicka V, Justus DE, Ross GD. Microvascular effects of complement blockade with soluble recombinant CR1 on ischemia/reperfusion injury of skeletal muscle. J Immunol. 1993;150:5104-13. Pereira MIR, Gomes PSC, Bhambhani YN. A brief review of the use of near infrared spectroscopy with particular interest in resistance exercise. Sports Med. 2007;37:615-24. Petrova A, Mehta R. Near-infrared spectroscopy in the detection of regional tissue oxygenation during hypoxic events in preterm infants undergoing critical care. Ped Crit Care Med. 2006;7:449-54. Powers SK, Kavazis AN, McClung JM. Oxidative stress and disuse muscle atrophy. J Appl Physiol. 2007;102:2389-97. Pucar D, Dzeja PP, Bast P, Juranic N, Macura S, Terzic A. Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR. J Biol Chem 2001;276:44812–44819. Quaresima V, Lepanto R, Ferrari M. The use of near infrared spectroscopy in sports medicine. J Sport Med Phys Fitness. 2003;43:1-13. Qvarfordt P, Christenson JT, Eklof B. Intramuscular pressure, muscle blood flow, and skeletal muscle metabolism in chronic anterior tibial compartment syndrome. Clin Orthop Rel Res. 1983;179:284-90. Reilly T, Drust B, Clarke N. Muscle fatigue during football match-play. Sports Med. 2008;38:357-67.  138  Rolfe P. In vivo near-infrared spectroscopy. An Rev Biomed Eng. 2000;2:715-54. Rominger M, Lukosch C, Bachmann G. Compartment syndrome: value of MR imaging. Radiology. 1995;197:296. Rominger MB, Lukosch CJ, Bachmann GF. MR imaging of compartment syndrome of the lower leg: a case control study. Eur Radiol. 2004;14:1432-9. Rorabeck CH, Castle GSP, Hardie R. Compartmental pressure measurements: an experimental investigation using the slit catheter. J Trauma 1981;21:446. Roy D, Quiles J, Sharma R. Ischemia-modified albumin concentrations in patients with peripheral vascular disease and exercise-induced skeletal muscle ischemia. Clin Chem. 2004;50:1656-60. Rubin BB, Romaschin A, Walker PM, Gute DC, Korthuis RJ. Mechanisms of postischemic injury in skeletal muscle: intervention strategies. J Appl Physiol. 1996;80:369-87. Sako T, Hamaoka T, Higuchi H, Kurosawa Y, Katsumura T. Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise. J Appl Physiol. 2001;90: 338-44. Sanz A, Hiona A, Kujoth GC, Seo AY, Hofer T, Kouwenhoven E, Kalani R, Prolla TA, Barja G, Leeuwenburgh C. Evaluation of sex differences on mitochondrial bioenergetics and apoptosis in mice. Exp Gerontol. 2007;42:173-82. Sapega AA, Heppenstall RB, Chance B, Park YS, Sokolow D. Optimizing tourniquet application and release times in extremity surgery. A biochemical and ultrastructural study. J Bone Joint Surg Am. 1985;67:303-14. Saunders KC, Louis DL, Weingarden SI, Waylonis GW. Effect of tourniquet time on postoperative quadriceps function. Clin Orthop Relat Res. 1979;143:194–9.  139  Scheufler O, Exner K, Andresen R. Investigation of TRAM flap oxygenation and perfusion by near-infrared reflection spectroscopy and color-coded duplex sonography. Plast Reconstr Surg. 2004;113:141-52. Schoen M, Rotter R, Gierer P, Gradl G, Strauss U, Jonas L, Mittlmeier T, Vollmar B. Ischemic preconditioning prevents skeletal muscle tissue injury, but not nerve lesion upon tourniquet-induced ischemia. J Trauma. 2007;63:788-97. Segal B, Retfalvi S, Pavlasek T. Silent” malfunction of a critical-care device caused by electromagnetic interference. Biomed Instrum Technol. 1995;29:350 -4. Shadgan B, Menon M, O’Brien PJ, Reid WD. Diagnostic Techniques in Acute Compartment Syndrome of the leg. J Orthop Trauma. 2008a;22:581-7. Shadgan B, Stothers L, Macnab A. A Transvaginal Probe for Near Infrared Spectroscopic Monitoring of the Bladder Detrusor Muscle and Urethral Sphincter. Spectroscopy. 2008b;22:429-436. Shadgan B, Reid WD, Gharakhanlou R, Stothers L, Macnab A. Wireless near-infrared spectroscopy of skeletal muscle oxygenation and hemodynamics during exercise and ischemia. Spectroscopy 2009;23:233-41. 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. 2010a; Vol.755512:doi:10.1117/12.842712. Shadgan B, Afshar K, Stothers L, Macnab A. Near-infrared spectroscopy of the bladder: a new technique for studying lower urinary tract function in health and disease. Proc SPIE. 2010b; Vol. 7548, 75480U:doi:10.1117/12.841066. Shadgan B, Menon M, Sanders D, Berry G, Martin C Jr, Duffy P, Stephen D, O'Brien PJ. Current thinking about acute compartment syndrome of the lower extremity. Canadian J of Surg. 2010c;53:329-34.  140  Shaw JA, Murray DG. The relationship between tourniquet pressure and underlying softtissue pressure in the thigh. J Bone Joint Surg Am. 1982;64:1148-52. Schoen M, Rotter R, Gierer P, Gradl G, Strauss U, Jonas L, Mittlmeier T, Vollmar B. Ischemic preconditioning prevents skeletal muscle tissue injury, but not nerve lesion upon tourniquet-induced ischemia. J Trauma. 2007;63:788-97. Shenton DW, Spitzer SA, Mulrennan BM. Tourniquet-induced rhabdomyolysis. A case report. J Bone Joint Surg Am. 1990;72:1405-6. Sheridan GW, Matsen FA 3rd, Krugmire RB Jr. Further investigations on the pathophysiology of the compartmental syndrome. Clin Orthop Rel Res. 1977;123:266-70. Shiga T, Yamamoto K, Tanabe K, Nakase Y, Chance B. Study of an algorithm based on model experiments and diffusion theory for a portable tissue oximeters. J Biomed Optics. 1997;2:154-61. Shuler MS, Reisman WM, Kinsey TL, Whitesides TE Jr, Hammerberg EM, Davila MG, Moore TJ. Correlation between muscle oxygenation and compartment pressures in acute compartment syndrome of the leg. J Bone Joint Surg Am 2010;92:86370. Silberberg JL. Performance degradation of electronic medical devices due to electromagnetic interference. Compliance Eng. 1993;10:1-8. Sjøgaard G, Kiens B, Jorgensen K, Saltin B. Intramuscular pressure, EMG and blood flow during low-level prolonged static contraction in man. Acta physiol scand. 1986;12:475-84. Smuder AJ, Kavazis AN, Hudson MB, Nelson WB, Powers SK. Oxidation enhances myofibrillar protein degradation via calpain and caspase-3. Free Radic Biol Med 2010;49:1152-1160.  141  Sorichter S, Mair J, Koller A. Early assessment of exercise induced skeletal muscle injury using plasma fatty acid binding protein. B J Sports Med. 1998;32:121-4. Sowa MG, Leonardi L, Payette JR, Fish JS, Mantsch HH. Near infrared spectroscopic assessment of hemodynamic changes in the early post-burn period. Burns. 2001;27:241-9. Sowa MG, Leonardi L, Payette JR, Cross KM, Gomez M, Fish JS. Classification of burn injuries using near-infrared spectroscopy. J Biomed Opt. 2006;11:054002. Steinberg BD. Evaluation of limb compartments with increased interstitial pressure. An improved noninvasive method for determining quantitative hardness. J Biomechanics. 2005;38:1629-35. Steinbrink J, Fischer T, Kuppe H. Relevance of depth resolution for cerebral blood flow monitoring by near-infrared spectroscopic bolus tracking during cardiopulmonary bypass. J Thorac Cardiovasc Surg. 2006;132:1172-8. Stothers L, Shadgan B, Macnab A. Urologic Applications of Near Infrared Spectroscopy (NIRS). Canadian J of Urol. 2008;15:4399-409. Styf J. Compartment Syndromes Diagnosis, Treatment and Complications. CRC Press LLC; 2004. Sugiura T, Ito N, Goto K, Naito H, Yoshioka T, Powers SK. Estrogen administration attenuates immobilization-induced skeletal muscle atrophy in male rats. J Physiol Sci. 2006;56:393-9. Suzuki S, Takasaki S, Ozaki T, Kobayashi Y. A tissue oxygenation monitor using NIR spatially resolved spectroscopy, Proc. SPIE 1999;3597:582–92. Tachtsidis I, Tisdall M, Leung TS, Cooper CE, Delpy DT, Smith M, Elwell CE. Investigation of in vivo measurement of cerebral cytochrome-c-oxidase redox changes using near-infrared spectroscopy in patients with orthostatic hypotension. Physiol Meas. 2007;28:199-211.  142  Tamas R, Nemeth N, Brath E, Sasvari M, Nyakas C, Debreczeni B, Miko I, Furka I. Hemorheological, morphological, and oxidative changes during ischemiareperfusion of latissimus dorsi muscle flaps in a canine model. Microsurgery. 2010;30:282-8. Terakado S, Takeuchi T, Takeshi M, Sato H, Nishioka N, Fujieda Y, Kobayashi R, Ibukiyama C. Early occurrence of respiratory muscle deoxygenation assessed by near-infrared spectroscopy during leg exercise in patients with chronic heart failure. Jpn Circ J. 1999;63,97-103. Tesch PA, Karlsson J. Muscle metabolite accumulation following maximal exercise. A comparison between short-term and prolonged kayak performance. Eur J Appl Physiol Occup Physiol. 1984;52:243-6. The Euroqol group. EuroQol: a new facility for measurement of health related quality of life. Health Policy 1990;16:199-208. Tiidus PM. Estrogen and gender effects on muscle damage, inflammation, and oxidative stress. Can J Appl Physiol. 2000;25:274-87. Tobias JD, Hoernschemeyer DG. Near-infrared spectroscopy identifies compartment syndrome in an infant. J Pediatr Orthop. 2007;27:311-3. Tredwell SJ, Wilmink M, Inkpen K, McEwen JA. Pediatric tourniquets: analysis of cuff and limb interface, current practice, and guidelines for use. J Pediatr Orthop. 2001;21:671-6. Ueno T, Ballard RE, Macias RB. Non-invasive measurement of pulsatile intracranial pressure using ultrasound. Acta Neurochir. 1998;71:66-9. Ulmer T. The clinical diagnosis of compartment syndrome of the lower leg: are clinical findings predictive of the disorder? J Trauma. 2002;16:572-7.  143  Usaj A, Jereb B, Robi P, Von Duvillard SP. The influence of strength-endurance training on the oxygenation of isometrically contracted forearm muscles. Eur J Appl Physiol. 2007;100:685-92. Uslu MM, Apan A. Can skin surface pressure under a cast reveal intracompartmental pressure? Arch Orthop Trauma Surg. 2000;120:319-22. Uzun H, Kayali R, Cakatay U. The chance of gender dependency of oxidation of brain proteins in aged rats. Arch Gerontol Geriatr. 2010;50:16-9. van Beekvelt MC, Colier WN, Wevers RA, van Engelen BG. Quantitative measurement of oxygen consumption and forearm blood flow in patients with mitochondrial myopathies. Adv Exp Med Biol. 1999a;471:313-9. van Beekvelt MC, van Engelen BG, Wevers RA, Colier WN. Quantitative near-infrared spectroscopy discriminates between mitochondrial myopathies and normal muscle. Ann Neurol. 1999b;46:667-70. van Beekvelt MCP, Borghuis MS, van Engelen BGM, Wevers RA, Colier WNJM. Adipose tissue thickness affects in vivo quantitative near-infrared spectroscopy in human skeletal muscle. Clin Sci. 2001a;101:21-8. van Beekvelt MCP, Colier WNJM, Wevers RA. Performance of near-infrared spectroscopy in measuring local O2 consumption and blood flow in skeletal muscle. J Appl Physiol. 2001b;90:511–19. van Beekvelt MCP, van Baziel CP, van Engelen GM, Wevers RA, Colier WNJM. In vivo quantitative near-infrared spectroscopy in skeletal muscle during incremental isometric handgrip exercise. Clin Physiol Funct Imaging 2002a;22:210-7. van Beekvelt MCP, Wevers RA, van Engelen BG, Colier WNJM. Muscle tissue oxygenation as a functional tool in the follow up of dermatomyositis, J Neurol Neurosurg Psychiatry. 2002b;73:93–94.  144  van Beekvelt MCP, Orbon K, van Engelen BGM, Wevers RA, Colier WNJM. NIR spectroscopic measurement of local muscle metabolism during rhythmic, sustained, and intermittent handgrip exercise. Proc SPIE. 2003;5138:35. van den Brand JG, Verleisdonk EJ, van der Werken C. Near infrared spectroscopy in the diagnosis of chronic exertional compartment syndrome. Am J Sports Med. 2004;32:452-6. van der Sluijs MC, Colier NJM, Houston RJF, Oesburg B. A new and highly sensitive continuous wave near infrared spectrophotometer with multiple detectors. Proc SPIE. 1997;3194:63-72. van der Togt R van Lieshout EJ, Hensbroek R. Electromagnetic interference from radio frequency identification inducing potentially hazardous incidents in critical care medical equipment. JAMA 2008;299:2884-90. van dervelpen G, Goris L, Broos PLO. Functional sequelae in tibial shaft fractures with compartment syndrome following primary treatment with urgent fasciotomy. Acta Chir Belg. 1992;92:234-40. van Nieuwenhoven FA, Kleine AH, Keizer HA. Comparison of myoglobin and fatty acid-binding protein as plasma marker for muscle damage in man. Eur J Physiolo. 1992;421:40. van Nieuwenhoven FA, Kleine AH, Wodzig KWH. Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein. Circulation. 1995;92:2848-54. van Oostrom J, Mahla M, Gravenstein D. The Stealth Station Image Guidance System may interfere with pulse oximetry. Canadian J Anesth; 2005;52:379-82. Vo TV, Hammer PE, Hoimes ML, Nadgir S, Fantini S. Mathematical model for the hemodynamic response to venous occlusion measured with near-infrared spectroscopy in the human forearm. IEEE Trans Biomed Eng. 2007;54:573-84.  145  Vrouenraets BC, Kroon BB, Klaase JM. Value of laboratory tests in monitoring acute regional toxicity after isolated limb perfusion. Ann Surg Oncol. 1997;4:88-94. Wakai A, Winter DC, Street JT, Redmond PH. Pneumatic tourniquets in extremity surgery. J Am Acad Orthop Surg. 2001;9:345-51. Walker TG, Acute limb ischemia. Tech Vasc Interv Radiol. 2009;12:117-29. Ward KR, Ivatury RR, Barbee RW, Terner J, Pittman R, Torres Filho IP, Spiess B. Near infrared spectroscopy for evaluation of the trauma patient: a technology review. Resuscitation. 2006;68:27-44. Wassenaar EB, van den Brand JG. Reliability of near-infrared spectroscopy in people with dark skin pigmentation. J Clin Monit Comput. 2005;19:195–9. Watkin SL, Spencer SA, Dimmock PW, Wickramasinghe YA, Rolfe P. A comparison of pulse oximetry and near infrared spectroscopy (NIRS) in the detection of hypoxaemia occurring with pauses in nasal airflow in neonates. J Clin Monit Comput. 1999;15: 441–7. Weinmann M. Compartment syndrome. Emerg Med Serv. 2003;32:36. Wickramasinghe YA, Livera LN, Spencer SA, Rolfe P, Thorniley MS. Plethysmographic validation of near infrared spectroscopic monitoring of cerebral blood volume. Arch Dis Child. 1992;67:407-11. Whitesides TEJ, Haney TC, Morimoto K, Hirada H. Tissue pressure measurements as a determinant for the need of fasciotomy. Clin Orthop. 1975;113:43. White TO, Howell GED, Will EM. Elevated intramuscular compartment pressures do not influence outcome after tibial fracture. J Trauma Inj Infec Critic Care. Wiemann JM, Ueno T, Leek BT. Noninvasive measurements of intramuscular pressure using pulsed phase-locked loop ultrasound for detecting compartment syndromes: a preliminary report. J Orthop Trauma. 2006;20:458-63.  146  Williams PR, Russel ID, Mintowt-Czyz WM. Compartment pressure monitoring - current UK orthopaedic practice. Injury. 1998;29:229-32. Wolf M, Duc G, Keel M, Niederer P, von Siebenthal K, Bucher HU. Continuous noninvasive measurement of cerebral arterial and venous oxygen saturation at the bedside in mechanically ventilated neonates. Crit Care Med. 1997;25:1579–82. Wolf M, Ferrari M, Quaresima V. Progress of near-infrared spectroscopy and topography for brain and muscle clinical applications. J Biomed Optics. 2007;12:062104. Wyatt JS, Cope M, Delpy DT, Wray S, Reynolds EOR. Quantification of cerebral oxygenation and haemodynamics in sick newborn infants by near infrared spectrophotometry. Lancet. 1986;2:1063-6. Yadav SS, Sindram D, Perry DK, Clavien PA. Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway. Hepatology 1999;30:1223–1231. Yamada T, Mishima T, Sakamoto M, Sugiyama M, Matsunaga S, Wada M. Oxidation of myosin heavy chain and reduction in force production in hyperthyroid rat soleus. J Appl Physiol. 2006;100:1520-6. Yamada E, Kusaka T, Arima N, Isobe K, Yamamoto T, Itoh S. Relationship between muscle oxygenation and electromyography activity during sustained isometric contraction. Clin Physiol Funct Imaging. 2008;28:216-21. Younger AS, McEwen JA, Inkpen K. Wide contoured thigh cuffs and automated limb occlusion measurement allow lower tourniquet pressures. Clin Orthop Relat Res. 2004;428:286-93. Yoshitani K, Kawaguchi M, Okuno T, Kanoda T, Ohnishi Y, Kuro M, Nishizawa M. Measurements of optical pathlength using phase-resolved spectroscopy in patients undergoing cardiopulmonary bypass. Anesth Analg. 2007;104:341-6.  147  Yu G, Durduran T, Lech G, Zhou C, Chance B, Mohler ER 3rd, Yodh AG. Timedependent blood flow and oxygenation in human skeletal muscles measured with noninvasive near-infrared diffuse optical spectroscopies. J Biomed Opt. 2005;10:24-7. Zapico-Muniz E, Santalo-Bel M, Merce-Muntanola J. Ischemia-modified albumin during skeletal muscle ischemia. Clin Chem. 2004;50:1063-5. Zergeroglu MA, McKenzie MJ, Shanely RA, van Gammeren D, DeRuisseau KC, and Powers SK. Mechanical ventilation-induced oxidative stress in the diaphragm. J Appl Physiol. 2003;95:1116-24.  148  APPENDIX I  Informed Consent Forms  149  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.  150  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 NearInfrared 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 151  §  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  152  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  153  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  154  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.  155  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:  156  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.  157  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  158  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.  159  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-  160  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  161  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.  162  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]. 163  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.  164  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  165  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.  166  167  168  169  170  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.  171  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.  172  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). 173  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,  174  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.  175  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. 176  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  177  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.  178  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  179  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 180  (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  181  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 % predicted FEV1, liters % predicted FEV1/FVC % predicted  6.0 ± 0.4 (4.2-7.9) 109 ± 6 (88-147) 4.7 ± 0.3 (3.4-6.36) 101 ± 5 (74-133) 0.79 ± 0.01 (0.70-0.82) 93 ± 2 (74-98)  FVC, forced vital capacity; FEV1, forced expired volume in 1 second. Values are mean ± SE. Ranges are presented in parentheses.  182  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).  183  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). 184  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).  185  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).  186  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). 187  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.  188  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).  189  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  190  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.  191  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  192  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 193  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 194  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  195  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.  196  REFERENCES Aldrich, T.K., Spiro, P., 1995. Maximal inspiratory pressure: does reproducibility indicate full effort? Thorax 50, 40–43. Aora, N.S., Rochester, D.F., 1984. Effect of chronic airflow limitation (CAL) on sternocleidomastoid muscle thickness. Chest 85, 58S-59S. ATS, 2002. ATS/ERS Statement on respiratory muscle testing. Am. J. Respir. Crit. Care Med. 166, 518–624. ATS, 2005. Standardization of spirometry, Eur Respir. J. 26, 319–338. Boushel, R., Piantadosi, C.A., 2000. Near-infrared spectroscopy for monitoring muscle oxygenation. Acta Physiol. Scand. 168, 615-622. Boushel R., Langberg H., Olesen J., Gonzales-Alonzo J., Bulow J., Kjaer M., 2001. Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease. Scand. J. Med. Sci. Sports 11, 213-222. Butler, J.E., De Troyer, A., Gandevia, S.C., Gorman, R.B., Hudson, A.L., 2007. Neuromechanical matching of central respiratory drive: a new principle of motor unit recruitment? Physiol. News 67, 22-24. Campbell, E.J., 1970. Accessory muscles. In The Respiratory Muscles: Mechanics and Neural Control, 2 edn, ed. Campbell EJ, Agostoni E & NewsomDavis J, LloydLuke Ltd., London, pp. 181–193. Cannon, D.T., Grout, S.L., May, C.A., 2007. Recruitment of the Serratus Anterior as an Accessory Muscle of Ventilation during Graded Exercise. J. Physiol. Sci. 57, 127131. Clanton, T.L., Dixon, G., Drake, J., Gadek, J.E., 1985. Inspiratory muscle conditioning using a threshold loading device. Chest 87, 62–66.  197  Costa, D., Vitti, M., de Olivera Tossello, D., Costa, R.P., 1994. Participation of the sternocleidomastoid muscle on deep inspiration in man, an electromyographic study. Electromyogr. Clin. Neurophysiol. 34, 315-320. Cram, J.R., Kasman, G.S., Holtz, J., 1998. Introduction to surface electromyography. Gaithersburg: Aspen Publishers Inc. Delpy, D.T., Cope, M., 1997. Quantification in tissue near-infrared spectroscopy. Phil. Trans. R. Soc. Lond. B. 352, 649-659. de Mayo, T., Miralles, R., Barrero, D., Bulboa, A., Carvajal, D., Valenzuela, S., Ormeno, G., 2005. Breathing type and body position effects on sternocleidomastoid and suprahyoid EMG activity. J. Oral. Rehabil., 32, 487-494. De Troyer, A., Peche, R., Yernault, J.C., Estenne, M., 1994. Neck muscle activity in patients with severe chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 150, 41-47. De Troyer, A., Kirkwood, P.A., Wilson, T.A., 2005. Respiratory action of the intercostals muscles. Physiol. Rev. 85, 717-756. Eastwood, P.R., Hillman, D.R., Finucane, K.E., 1994. Ventilatory responses to inspiratory threshold loading and role of muscle fatigue in task failure. J. Appl. Physiol. 76, 185-189. Epstein, S.K., 1994. An overview of respiratory muscle function. Clin. Chest Med. 15, 619–639. Ferrari, M., Mottola L., Quaresima, V., 2004. Principles, techniques and limitations of near infrared spectroscopy. Can. J. Appl. Physiol. 29, 463-487. Gandevia, S.C., Leeper, J.B., McKenzie D.K., De Troyer, A., 1996. Discharge frequencies of parasternal intercostal and scalene motor units during breathing in normal and COPD subjects. Am. J. Respir. Crit. Care Med. 153, 622-628.  198  Grassi, B., Quaresima, V., Marconi, C., Ferrari, M., Cerretelli, P., 1999. Blood lactate accumulation and muscle deoxygenation during incremental exercise. J. Appl. Physiol. 87, 348-355. Hamaoka T., McCully, K.K., Quaresima, V., Yamamoto, K., Chance, B., 2007. Nearinfrared spectroscopy/imaging for monitoring muscle oxygenation and oxidative metabolism in healthy and diseased humans, J. Biomed. Opt. 12, 062105, 1–12. Harms, C.A., Wetter, T.J., St Croix, C.M., Pegelow, D.F., Dempsey, J.A., 2000. Effects of respiratory muscle work on exercise performance. J. Appl. Physiol. 89, 131– 138. Homma, S., Fukunaga, T., Kagaya, A., 1996. Influence of adipose tissue thickness on near infrared spectroscopic signals in the measurement of human muscle. J. Biomed. Optics 1, 418–424. Hudson, A.L., Gandevia, S.C., Butler, J.E., 2007. The effect of lung volume on the coordinated recruitment of scalene and sternomastoid muscles in humans. The Journal of Physiology. 584, 261-270. Kirkpatrick, P.J., Lam J., Al-Rawi, P., Smielewski, P., Czosnyka, M., 1998. Defining thresholds for critical ischemia by using near-infrared spectroscopy in the adult brain J. Neurosurg. 89, 389–394. Knudson, R.J., Lebowitz, M.D., Holberg, C.J., Burrows, B., 1983. Changes in the normal maximal expiratory flow-volume curve with growth and aging. Am. Rev. Respir. Dis. 127, 725–734. Laghi, F., Jubran, A., Topeli, A., Fahey, P.J., Garrity, E.R., Arcidi, J.M., DePinto, D.J., Edwards, L.C., Tobin, M.J., Effect of lung volume reduction surgery on neuromechanical coupling of the diaphragm. Am. J. Respir. Crit. Care Med. 157,475-483. Loring, S.H., DeTroyer, A., 1985. Actions of the respiratory muscles, in: Roussos, C., Macklem, P.T., (Eds.), The thorax. Marcel Dekker Inc., New York, pp. 327-349. 199  Mananas, M., Jane, R., Fiz, J., Morera, J., Caminal, P., 2000. Study of myographic signals from sternomastoid muscle in patients with chronic obstructive pulmonary disease. IEEE Trans. Biomed. Eng. 47:674-681. Marras, W.S. and Davis, K.G., 2001, A non-MVC EMG normalization technique for the trunk musculature: Part 1. Method development. Journal of Electromyography and Kinesiology, 11, 1– 9. Martyn, J.B., Moreno, R.H., Pare, P.D., Pardy, R.L., 1987. Measurement of inspiratory muscle performance with incremental threshold loading. Am. Rev. Respir. Dis. 135, 919-23. Masubuchi, Y., Abe, T., Yokoba, M., Yamada, T., Katagiri, M., Tomita, T., 2001. Relation between neck accessory inspiratory muscle electromyographic activity and lung volume. Nihon Kokyuki Gakkai Zasshi 39, 244-249. Mathur, S., Sheel, A.W., Road, J.D., Reid, W.D., 2010. Delayed Onset Muscle Soreness after Inspiratory Threshold Loading in Healthy Adults, Cardiopulm. Phys.Ther. J. 21, 5-12. Moalla, W., Dupont, G., Berthoin, S., Ahmaidi, S., 2005. Respiratory muscle Deoxygenation and ventilatory threshold assessments using near infrared spectroscopy in children. Int. J. Sports Med. 26, 576-582. Nielsen, H.B., Boesen, M., Secher, N.H. 2001. Near-infrared spectroscopy determined brain and muscle oxygenation during exercise with normal and resistive breathing. Acta. Physiol. Scand. 171, 63-70. Pardee, N.E., Winterbauer, R.H., Allen, J.D., 1984. Bedside evaluation of respiratory distress. Chest 85, 203-206. Parthasarathy, S., Jubran, A., Laghi, F., Tobin, M.J., 2007. Sternomastoid, rib cage and expiratory muscle activity during weaning failure. J. Appl. Physiol. 103, 140-147.  200  Raper, A.J., Thompson, W.T. Jr., Shapiro, W., Patterson, J.L. Jr. 1966. Scalene and sternomastoid muscle function. J. Appl. Physiol. 21, 497–502. St Croix, C., M., Morgan, B. J., Wetter, T. J., Dempsey, J. A., 2000. Fatiguing inspiratory muscle work causes reflex sympathetic activation in humans. J. Physiol. 529, 493504. Sheel, A.W., Derchak, P.A., Morgan, B.J., Pegelow D.F., Jacques, A.J., Dempsey, J.A., 2001. Fatiguing inspiratory muscle work causes reflex reduction in resting leg blood flow in humans. J. Physiol. 537, 277–289. Sheel, A.W., Derchak, P.A., Pegelow, D.F., Dempsey, J.A., 2002. Threshold effects of respiratory muscle work on limb vascular resistance. Am. J. Physiol. Heart Circ. Physiol. 282, 1732-1738. Tachtsidis, I., Tisdall, M., Leung, T.S., Cooper, C.E., Delpy, D.T., Smith, M., Elwell, C.E., 2007. Investigation of in vivo measurement of cerebral cytochrome-coxidase redox changes using near-infrared spectroscopy in patients with orthostatic hypotension. Physiol. Meas. 28,199-211. Terakado, S., Takeuchi, T., Takeshi, M., Sato, H., Nishioka, N., Fujieda, Y., Kobayashi, R., Ibukiyama, C., 1999. Early occurrence of respiratory muscle deoxygenation assessed by near-infrared spectroscopy during leg exercise in patients with chronic heart failure. Jpn. Circ. J. 63, 97-103. Tobin, M. J., Laghi, F., Brochard, L., 2009. Role of the respiratory muscles in acute respiratory failure of COPD: lessons from weaning failure. J. Appl. Physiol. 107, 962-970. Turner, D., Jackson, S., 2002. Resistive loaded breathing has a functional impact on maximal voluntary contractions in humans, Neurosci. Lett. 326, 77-80. van Beekvelt, M.C.P., Borghuis, M.S., van Engelen, B.G.M., Wevers, R.A., Colier, W.N.J.M., 2001. Adipose tissue thickness affects in vivo quantitative nearinfrared spectroscopy in human skeletal muscle, Clin. Sci. 101, 21-28. 201  van Beekvelt, M.C.P., Orbon, B. G. M., van Engelen, B.G.M., Wevers, R.A., Colier, W.N.J.M., 2003. NIR spectroscopic measurement of local muscle metabolism during rhythmic, sustained and intermittent handgrip exercise, Proc. Soc. Photo Opt. Instrum. Eng. 5138, 35–45. van der Sluijs, M.C., Colier, W.N.J.M., Houston, R.J.F., Oesburg, B., 1997. A new and highly sensitive continuous wave near infrared spectrophotometer with multiple detectors, Proc. SPIE 3194, 63–72. Wetter T.J., Harms C.A., Nelson W.B., Pegelow D.F., Dempsey J.A., 1999. Influence of respiratory muscle work on ˙VO2 and leg blood flow during submaximal exercise. J. Appl. Physiol. 87, 643-651. Wright P.E., Basic surgical technique and aftercare. In: W.C. Campbell, S.T. Canale and J.H.  Beaty,  Editors,  Campbell's  Operative  Orthopaedics  (11th  ed.),  Mosby/Elsevier, Philadelphia, PA (2008), p. 3805. Yokoba, M., Abe, T., Katagiri, M., Dobashi, Y., Yamada, T., Tomita, T., 1999. Electromyographic activity of neck muscles during the production of inspiration pressure. Nihon Kokyuki Gakkai Zasshi, 37, 102-107.  202  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0071639/manifest

Comment

Related Items