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The effects of intermittent exposure to hyperbaric oxygen for the treatment of an acute soft tissue injury 2001

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The effects of intermittent exposure to hyperbaric oxygen for the treatment of an acute soft tissue injury by S H E L I N A BABUL B S c , The University of Brit ish Co lumbia , 1990 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Schoo l of Human Kinet ics W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A October 2001 © She l ina Babu l , 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada o DE-6 (2/88) ABSTRACT This study examined the effects of intermittent exposure to hyperbaric oxygen therapy (HBO) for the treatment of de layed onset musc le so reness ( D O M S ) . It is apparent in the literature that a great deal of controversy exists in using this form of therapy to treat t issue injuries. It was hypothesized that subjects exposed to hyperbaric oxygen would recover from D O M S faster than subjects exposed to normoxic air. Sixteen sedentary, female university students participated in the study and were randomly ass igned to either an experimental or control group. Al l subjects performed 300 maximal voluntary eccentr ic contractions (30 sets of 10 repetitions/minute) of their non-dominant leg (110° - 35° of knee flexion) at a s low speed (30° per second) on the K i n C o m Dynamometer, to elicit muscle damage and injury. H B O treatments consis ted of 100% oxygen for 60 minutes at 2.0 A T A while the control group received 2 1 % oxygen at 1.2 A T A for the same amount of t ime. Both groups received treatment immediately after the induction of D O M S and each day after for a period of 4 days [day 2 post-exercise thru day 5 post-exercise]. Dependent var iables (perceived musc le soreness , isokinetic strength, quadr iceps c i rcumference, creatine k inase (CK) , interleukin-6 (IL-6) and malondia ldehyde (MDA) were a s s e s s e d basel ine (pre- exerc ise, day 1), 4 hours post-exercise (day 2), 24 hours post-exercise (day 3), 48 hours post-exercise (day 4) and 72 hours post-exercise (day 5). MRI [T2 relaxation t ime/STIR]) was a s s e s s e d basel ine (day 1), 24 hours post-exercise (day 3) and 72 hours post-exercise (day 5). Isokinetic strength (p<0.05) and perceived so reness (p<0.05) indicated signif icance for injury to the quadr icep musc le for both groups but no difference was seen between groups (p=0.102, p=0.571 respectively). Quadr icep circumference was measured at the 10 and 20 cm reference point above the superior portion of the patel la. The 10cm girth measurement indicated signif icance (p<0.05) for musc le injury but there was no difference between groups (p=0.815); 20 c m measurement showed no signif icance (p<0.05) for both within and between groups (p=0.677). No signif icance was evident for serum C K (p<0.05), both within and between groups (p=0.647). M D A analys is revealed no signif icance (p<0.05) both within and between groups (p=0.580). Ana lys is of IL-6 demonstrated no signif icance (p<0.05) for both within and between groups (p=0.111). Finally, MRI analys is for T 2 weighted imaging of the rectus femoris, vastus medius and vastus lateralis showed no statistical signif icance (p<0.05) between groups for treatment effects (p=0.800, p=0.361, and p=0.806 respectively). Similarly, analys is of the ST IR images indicated no statistical signif icance (p<0.05) for the s a m e three musc les (p=0.796, p=0.580, and p=0.265 respectively). The findings of this study suggest that hyperbaric oxygen therapy was not effective in the treatment of exerc ise- induced musc le injury as indicated by the markers evaluated. TABLE OF CONTENTS Abstract ii Tab le of Contents iv List of Tab les vi List of F igures viii Acknowledgments x Dedicat ion xi Pre face xii Chapter 1 - General Introduction 1.1 Introduction to Hyperbar ic Oxygen 1 1.2 Combin ing Hyperbar ic Oxygen (HBO) with Delayed Onset 6 Musc le So reness ( D O M S ) : Putting the P ieces of the Puzz le Together 1.3 Hypothesis 7 1.4 Assumpt ions 7 1.5 Limitations 8 1.6 Delimitations 8 Chapter 2 - Review of the Literature 2.1 Effects of Hyperbaric Oxygen In Wound Heal ing & T i ssue Survival 10 2.2 Oxygen Toxicity Rela ted to Hyperbar ic Oxygen Therapy 13 2.3 Prev ious Studies Examin ing Hyperbaric Oxygen and T issue Injuries 18 2.4 The Role of Hyperbaric Oxygen in Sport & Exerc ise Medic ine 25 2.5 Acute Soft T issue Injury: De layed-Onset Musc le S o r e n e s s 28 2.6 The Role of Magnet ic R e s o n a n c e Imaging in the Detection of D O M S 46 2.7 Perce ived Musc le So reness and the V isua l Ana log Sca le (VAS) 48 Chapter 3 - Methodology 3.1 Exper imental Des ign 50 3.2 Subjects 52 3.3 Procedure 52 IV 3.4 Statistical Ana lys is 61 3.5 Statistical Power 61 C h a p t e r 4 - Resu l ts 62 Chapte r 5 - D i s c u s s i o n 82 Chapte r 6 - C o n c l u s i o n s & R e c o m m e n d a t i o n s 100 R e f e r e n c e s 103 A p p e n d i x Appendix A - Consent Form 121 Appendix B - V isua l Ana log Sca le 124 Appendix C - Graphica l Representat ion of R a w Data 125 Append ix D - STIR Images for all subjects (days 1, 3, 5) 152 V LIST OF TABLES Table # Title of Tables Page # Tab le 1. P ressure equivalents for oxygen consumpt ion 4 Tab le 2. Therapeut ic uses of hyperbaric oxygen 4 Tab le 3. Se lec t landmarks in the history and development 5 of hyperbaric medic ine, dating back to the 1660's Tab le 4. Cel lu lar and biochemical benefits of hyperbaric oxygen 13 Tab le 5. Contraindicat ions to H B O therapy 17 Tab le 6. Patient preparation prior to H B O for safety and fire 17 prevention Tab le 7. Benefi ts of H B O in sports injuries 28 Tab le 8. P roposed mechan ism of action in sports injuries 28 Tab le 9. Mechan i sm by which oxygen generates free radicals 44 Tab le 10. Phys ica l characterist ics for both groups (age, height and 62 weight). Tab le 11. Ave rage ratings of perceived soreness for the quadr icep 63 musc le of the non dominant leg before (basel ine) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, fol lowing hyperbaric/normoxic exposure. Tab le 12. Ave rage maximal isokinetic eccentr ic torque of the quadr icep 64 musc le before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure . Tab le 13. Ave rage quadr icep circumference measured at both the 10 66 and 20cm point above the superior portion of the patel la. Measurements were taken before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbar ic/normoxic exposure. Tab le 14. M e a n serum creatine k inase values before (basel ine) and 68 after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, fol lowing hyperbaric/normoxic exposure. Tab le 15. M e a n Malondia ldehyde values before (basel ine) and after 70 (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 16. M e a n interleukin-6 va lues before (baseline) and after 71 (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 17. Ave rage T 2 relaxation t imes of the rectus femoris musc le 73 taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 18. Ave rage T2 relaxation t imes of the vastus intermedius musc le 74 taken before (basel ine) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 19. Ave rage T2 relaxation t imes of the vastus lateralis musc le 76 taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 20. Ave rage signal intensity ratio for ST IR image of the rectus 77 femoris musc le taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Tab le 21 . Ave rage signal intensity ratio for ST IR image of the vastus 79 intermedius musc le taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbar ic/normoxic exposure. Tab le 22. Ave rage signal intensity ratio for ST IR image of the 80 vastus lateralis muscle taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbar ic/normoxic exposure. vii LIST OF FIGURES Figure # Title of Figures Page # Figure 1. Pressure-durat ion relationship for effects of oxygen 14 toxicity when using hyperbaric oxygen therapy. Figure 2. S e q u e n c e of events assoc ia ted with delayed onset 33 musc le soreness , including mechanica l and b iochemical p rocesses . Figure 3. Theoret ical model showing role of cytokines and 41 neutrophils during exerc ise inducing damage to skeletal musc le . F igure 4. B iochemica l mechan ism for oxygen-free radical formation 45 resulting in skeletal muscle damage and inflammation during exerc ise. Figure 5. Ave rage rating of perceived soreness for the quadr icep 63 musc le of the non-dominant leg, according to the v isual ana log sca le (range 1-10), before eccentr ic exerc ise (basel ine) and after hyperbaric/normoxic exposure. Figure 6. Ave rage maximal eccentr ic torque for the quadr icep 65 musc le , before the exerc ise protocol (baseline) and after hyperbaric/normoxic exposure. Figure 7. Ave rage quadr icep circumference (10 c m location), before 67 eccentr ic exerc ise (baseline) and after hyperbar ic/normoxic exposure . Figure 8. Ave rage quadr icep circumference (20 c m location), before 67 eccentr icexerc ise (baseline) and after hyperbar ic/normoxic exposure . F igure 9. Ave rage creatine k inase (CK) levels, before eccentr ic 69 exerc ise (basel ine) and after hyperbaric/normoxic exposure. Figure 10. Ave rage malondia ldehyde (MDA) levels, before eccentr ic 70 exerc ise (basel ine) and after hyperbaric/normoxic exposure. Figure 11. Ave rage interleukin-6 (IL-6) levels, before eccentr ic exerc ise 72 (basel ine) and after hyperbaric/normoxic exposure. Figure 12. M e a n T 2 relaxation t imes (msec) before eccentr ic exerc ise 73 (basel ine) and after hyperbaric/normoxic exposure (days 3, 5) for the rectus femoris muscle. Figure 13. M e a n T 2 relaxation t imes (msec) before eccentr ic exerc ise 75 (basel ine) and after hyperbaric/normoxic exposure (days 3, 5) for the vastus intermedius muscle. Figure 14. M e a n T2 relaxation t imes (msec) before eccentr ic exerc ise 76 (basel ine) and after hyperbaric/normoxic exposure (days 3, 5) for the vastus lateralis muscle. Figure 15. M e a n Short Tip Inversion Recovery (STIR) ratios before 78 eccentr ic exerc ise (baseline) and after hyperbar ic/normoxic exposure (days 3, 5) for the rectus femoris muscle. Figure 16. M e a n Short Tip Inversion Recovery (STIR) ratios 79 before eccentr ic exerc ise (baseline) and after hyperbaric/ normoxic exposure (days 3, 5) for the vastus intermedius musc le . Figure 17. M e a n Short Tip Inversion Recovery (STIR) ratios before 81 eccentr ic exerc ise (baseline) and after hyperbar ic/normoxic exposure (days 3, 5) for the vastus lateralis musc le . ix / A C K N O W L E D G M E N T S I would like to thank my family for their continued support and encouragement during the course of my graduate studies, my c lose friends for all their pat ience and understanding and Pau l for his motivation, words of w isdom and being so supportive, especia l ly when I needed it the most. I would a lso like to thank the following people for their efforts and contributions to the study, because without their ass is tance, this study would never have been completed: Dr. Dennis J a n s e n , Dr. Urs Steinbrecher, C lyde Smith, O r c a Bay Sports & Entertainment, Fahra Rajabal i , T e r e s a Liu, Nabee la Popat ia , as well as the technical staff at St. Pau l 's Hospital for their help. Furthermore, I would a lso like to extend my appreciat ion to the B C Injury Resea rch & Prevention Unit ( B C I R P U ) . Parminder, your support, advice and flexibility in work al lowed me to complete the study and my P h D program. Thank Y o u ! Lastly, I would like to express my most s incere gratitude to my thesis advisor and committee members : Dr. Ted Rhodes , Dr. Jack Taunton & Dr. Mike Lepawsky. It was , without a doubt, their gu idance, pat ience, understanding, enthusiasm and encouragement that helped me to complete my graduate studies. Throughout the course of my thesis, I encountered numerous stumbling blocks and pitfalls and it was with their cont inued support that I was able to persevere and continue the study to its entirety. X DEDICATION One individual who played an enormous part in my life and educational pursuits was my mother whose laughter, love and continuous support made a world of difference in my life. This is for you mom! xi PREFACE During this past decade , society has seen a growing number of individuals participating in sport and recreational activities. However, the number of injuries as a result of this increase in activity has also r isen. Unfortunately, a primary cost related to injury recovery is the time-lost from participating in and resuming normal functional activity. This has compel led health care professionals to seek more efficient and effective therapeutic interventions in treating such injuries. Hyperbaric oxygen therapy may serve to provide a means of therapy to facilitate a speedier resumption to pre-injury activity levels as well as improve both the short and long-term prognosis of the injury. Although a growing interest in sports and exerc ise medicine is becoming evident in the literature, the use of hyperbaric oxygen as an intervention in this field has been controversial . To date, numerous professional athletic teams, ranging from hockey (NHL), football (NFL) , basketbal l (NBA) and socce r (MLS , European League) , utilize and rely on the use of hyperbaric oxygen as adjuvant therapy for numerous sports- related injuries acquired from playing competit ive sports. However, to date, there is a paucity of research on the appl icat ion benefits of hyperbaric therapy and sports injuries. Further research needs to be conducted suggest ing and validating the significant effects of this treatment modality and further grounding its importance in sports and exerc ise medic ine. xii C H A P T E R 1 : G E N E R A L I N T R O D U C T I O N 1.1 Introduction to Hyperbaric Oxygen Although the roots of modern hyperbaric oxygen therapy date back three centuries, it is only in the last few decades that the scientific foundation has been laid recognizing the benefits of inspiring 1 0 0 % oxygen at greater-than-ambient pressures [186]. Hyperbar ic oxygen has been used in a variety of medical condit ions and clinical settings but this form of therapy to treat soft t issue injuries remains a controversial issue. Hyperbaric oxygen (HBO) therapy refers to the therapeutic procedure where patients inspire 100% oxygen while their entire bodies are subjected to pressures greater than ambient barometric pressure at s e a level (the patient encounters pressures greater than one atmosphere absolute [1 ATA] or 760 mmHg). Oxygen is administered at a pressure above normal atmospher ic to increase oxygen delivery to ischemic or hypoxic t issues. Barometr ic pressure changes during hyperbaric oxygen therapy are often expressed in multiples of atmospher ic absolute (ATA) [3]. A change of 1 A T A is equivalent to 14.7 pounds per square inch (PSI), 760 m m H g or 33 feet of seawater (FSW) [3]. Therefore, an individual compressed to 2 or 3 A T A is equivalent to being 33-66 feet below the surface of the ocean [Table 1]. Gain ing medical reputation in the last 35 years, the eff icacy of H B O therapy has widely been accepted and utilized as the primary treatment of decompress ion s ickness (il lness resulting from rapid changes in pressure by divers or aviators), air embol isms (introduction of air into the circulation sys tem, often involuntarily by medical professionals) and carbon monoxide poisoning. It has a lso been used as a successfu l adjunct in crush injuries, compartment syndromes, burns, traumatic ischemias, refractory wounds, necrotizing soft t issue infections, osteomyelit is, radiation t issue damage, compromised skin grafts and f laps and c a s e s of extreme blood loss [3,9,34-35] [Table 2]. 1 The principle of hyperbaric therapy is based on the following two components : 1) the physiologic effects of hyperoxemia ( increase in partial pressure of oxygen d isso lved in arterial blood) and 2) the mechanical effects of increased pressure [3,39]. Through these two components , enough oxygen is d issolved in the p lasma to meet the metabol ic needs of injured t issue. Th is is of extreme importance in soft t issue injuries where the environment is hypoxic. To date, hyperbaric oxygen has been recognized as a significant adjunct in the treatment of a variety of wounds. Amp le ev idence exists that suggests inspiration of oxygen above 2 1 % to injured body t issue can have numerous beneficial effects [5-13]. It has been demonstrated that increasing the partial pressure of oxygen can counter local t issue hypoxia (under-oxygenation of t issue) [5-9], promote peripheral vasoconstr ict ion [5-9], decrease blood flow to the a rea of insult [5,8- 11], promote heal ing of damaged t issue and prevent infection by inhibition of the growth of anaerobic microorganisms [7,11-13]. The application of hyperbaric oxygen (HBO) for the treatment of sports injuries has recently been grounded in the scientific literature as a modality of therapy. However, the concept of using compressed gas for medical purposes has a rich history, with the origins and development of hyperbaric medic ine being c losely tied to the history of diving medicine [9, 31-32] [Table 3]. It was noticed that residents living at high altitudes had wounds that healed more slowly than at s e a level as compared to people living in undersea habitats at hyperbaric pressures (deep s e a divers) whose wounds healed faster at the enhanced pressure environment [8, 33]. Essent ial ly, cl inical hyperbaric medicine can be v iewed as a relatively new application of an old establ ished technology to help resolve selected medical problems [3]. Consequent ly , with this growing popularity, the number of hyperbaric units in the United States has grown from 34 to 260 facilit ies, from 1977-1998, with over 350 single-occupant (monoplace) chambers [36-38]. 2 Currently, hyperbaric oxygen has severa l cl inical indications for which it is the primary treatment modality: decompress ion s ickness , air embol ism and carbon monoxide poisoning [14,15]. Other condit ions such as burns, crush injuries, compartment syndromes and osteomyeli t is have a lso been shown to heal more rapidly with the application of hyperbaric oxygen therapy when used as an adjunct [14,16-20]. There has also been clinical investigation into the beneficial effects of hyperbaric oxygen and brain injuries [21,22]. Further research is underway examining its application for the treatment of multiple sc leros is [23-26] and its use is presently being investigated in chi ldren with cerebral palsy to determine whether it improves the quality of life for these children [27-29]. Recent ly, H B O has also been used in treating patients with HIV/AIDS, as it has been demonstrated to reduce the severity of, and secondary compl icat ions arising from, opportunistic infections [194]. Competi t ive sports have taken a whole new meaning in the last decade. Competi t ion is f ierce as athletes strive to be best in their f ield. However, this competit ive nature in athletes has invoked a higher incidence of injuries in these players. These injuries, ranging from broken bones, torn musc les , tendons and l igaments, may be a result of acute impact forces in contact sports or the everyday rigors of training and condit ioning [30]. Th is is where the field of sports and exerc ise medicine plays a crucial role in the rehabilitation of these athletes of all levels. Phys ic ians are frequently chal lenged by athletes, coaches and trainers to provide new treatments to facilitate a speedier recovery [30]. One such treatment is the use of hyperbaric oxygen to accelerate the recovery process and allow the injured athlete to return to competit ion faster than the normal course of rehabilitation. j V 3 Table 1: Pressure Equivalents for Oxygen Compress ion A T A mmHg FSW (atmosphere absolute) (feet of sea water) 1 760 0 2 1520 33 3 2280 66 6 4560 165 Table 2: Therapeut ic U s e s of Hyperbaric Oxygen* Strong Scientific Evidence M a l t r e a t m e n t Decompress ion s ickness Arterial gas embol ism v Severe carbon monoxide poisoning and smoke inhalation Adjunct ive treatment Prevent ion and treatment of osteoradionecrosis Improved skin graft and flap heal ing Clostr idial myonecros is Suggestive Scientific Evidence Adjunctive treatment Refractive osteomyeli t is Radiat ion induced injury Acute traumatic ischemic injury Pro longed failure of wound heal ing Except ional anemia from blood loss * adapted from Leach RM et al40 Tab le 3: Select landmarks in the history and development of hyperbaric medic ine, dating back to the 1600's Date Key Landmark 1662 J . Henshaw used compressed air for the treatment of a variety of d i seases 1 1775 Discovery of oxygen by J . Priest ley 1796 T. Beddoes and J . Watt wrote first book on medica l application of oxygen 1837 C . G . Pravaz of F rance constructed largest hyperbaric chamber of that time to treat a variety of ai lments ^ 1860 First hyperbaric chamber on North Amer ican continent in O s h a w a , C a n a d a 1895 J . S . Haldane showed that a mouse p laced in a jar containing oxygen at 2.0 A T A failed to develop C O poisoning 1921 O . J . Cunn ingham built a hyperbaric chamber in Lawrence, K a n s a s used to treat a variety of ai lments 1928 O . J . Cunn ingham builds the largest chamber in the world in C leve land 1937 A . R . Benke and L.A. S h a w first used H B O for treatment of decompress ion s ickness 1956 1. Boerema, father of modern hyperbaric medic ine, performed ,„ cardiac surgery in a hyperbaric chamber 1963 First International C o n g r e s s on Hyperbaric Medic ine in Amsterdam 1967 Undersea Medica l Society founded in the U S A . Now known as the Undersea and Hyperbar ic Medica l Society. 1970s Extensive expans ion of hyperbaric facilities in J a p a n and U S S R 1983 Formation of the Amer ican Co l lege of Hyperbaric Medic ine 1987 K.K. Ja in demonstrated H B O integration with physical therapy 1988 Formation of the International Society of Hyperbar ic Medic ine 5 1.2 Combining Hyperbaric Oxygen with Delayed Onset Muscle Soreness: Putting the Pieces of the Puzzle Together The delayed-onset muscle so reness ( D O M S ) model will elicit musc le injury and t issue inflammation in humans and serve as an excellent model to a s s e s s the eff icacy of using this form of treatment in the rehabilitation of exercise-related injuries. D O M S , character ized by high intensity eccentr ic contractions, clinically presents as increased stiffness, a dec reased range of motion, tenderness, decl ine in force, swel l ing, electrically silent muscle shortening and a release of musc le enzymes in the blood [119, 182]. Rodenburg et al [224] has stated that " D O M S ultimately ar ises from a ' sequence of events ' occurr ing after eccentr ic exerc ise, including myofibrillar disruption, increased permeabil i ty of the sa rco lemma to musc le proteins, free radical re lease and inflammatory p rocesses , with the latter possibly leading to D O M S . Th is ' sequence of events ' is initiated by mechanica l stress on the musc le f ibres, metabolic over load or a combinat ion of both". Due to the lack of scientific ev idence support ing the efficacy of using hyperbaric oxygen therapy in sport and exercise-related injuries, the effects of using this treatment modality was looked at in an acute soft t issue inflammatory condit ion, namely D O M S . This premise is based on the mechan ism of action assoc ia ted with H B O , and the mechanica l and b iochemical p rocesses that occur during a bout of D O M S . Musc le damage, musc le so reness and loss of musc le function will be determined by assess ing isokinetic strength decrements, assoc ia ted perceived musc le soreness , elevation in blood enzyme levels of creatine k inase, interleukin 6 and malondia ldehyde, as well as magnet ic resonance imaging of edematous t issue. E a c h piece of the "puzzle" can be put together to determine whether, in fact, hyperbaric oxygen does have an important role in the future of sports and 6 exerc ise medic ine. If it does, further research can then be conducted on a wide array of other injuries acquired in competit ive and recreational activities as well as other cl inical disorders 1.3 Hypothesis The purpose of this study was to determine whether intermittent exposure to hyperbaric oxygen in the treatment group, compared to the control group, would increase the rate of recovery from D O M S , thus: 1. Reduc ing perceived musc le soreness over the f ive-day testing period. 2. Signif icantly improving eccentr ic musc le strength during the recovery of D O M S . 3. Decreas ing elevated creatine k inase (CK) levels assoc ia ted with skeletal musc le damage over the five days, thereby bringing C K levels c lose to pre- exerc ise levels by day 5. 4. Decreas ing elevated interleukin-6 (IL-6) levels assoc ia ted with exerc ise- induced musc le injury over the five days, thereby bringing IL-6 levels c lose to basel ine by day 5. 5. Decreas ing elevated malondia ldehyde (MDA) levels assoc ia ted with lipid peroxidation as a result of musc le injury over the five days, thereby bringing M D A levels c lose to pre-exercise levels by day 5. 6. Reduc ing e d e m a in the quadr icep muscle of the non-dominant leg over the f ive-day testing period, as ev idenced by magnet ic resonance imaging (MRI). 1.4 Assumptions The following assumpt ions were made when designing the study: 1. Al l subjects will respond honestly, to the best of their knowledge, regarding the amount of activity that they perform on a weekly basis as well as their involvement in competit ive sporting activities. 2. All subjects will report accurate pain scores , to the best of their ability, when filling out the visual analog sca le . 7 3. Performing 300 eccentr ic contractions will create musc le injury and t issue inflammation ( D O M S ) in the quadr icep musc le of the non-dominant leg. 1.5 Limitations The study, at the present time, was limited by the following: 1. The severity of so reness , as perceived by individual subjects, may be prone to inter-subject variability. 2. Subject recruitment: It was extremely difficult to recruit subjects for the study due to the time commitment required over the 5 days of treatment and the travel d is tances required by the subjects (Allan McGav in Sports Medic ine Cl in ic, Buchanan Exerc ise Sc ience Laboratory, U B C , St. Pau l ' s Hospital and MRI Vancouver) . 1.6 Delimitations 1. Samp le select ion; sedentary or relatively sedentary individuals, between the ages of 18-40 years (N=16; n=8 per group), were recruited to maximize the extent of D O M S s ince their quadr icep musc le will not be adapted to eccentr ic loading. 2. The Kinetic Communicator (KinCom) Dynamometer was used to test the subject 's musc le strength through a specif ic range of motion while performing isolated knee extensions. 3. The D O M S protocol (eccentric exerc ise protocol) al lowed the investigator to control the insult of the injury. 4. The c i rcumference measurement of the quadricep musc le evaluated the girth of the thigh, including skin, fat, musc le , bone and possib le e d e m a occurr ing within the musculature. 5. IL-6 measurements taken at basel ine and repeated throughout the five- day testing period demonstrated the cytokine response during inflammation and D O M S . 8 Malondia ldehyde measurements taken at basel ine and repeated throughout recovery were indicative of lipid peroxidation during musc le injury. CHAPTER 2: REVIEW OF THE LITERATURE 2.1 Effects of Hyperbaric Oxygen in Wound Healing and Tissue Survival During a soft t issue injury, a disruption of cel ls and blood vesse ls occur within the t issue, resulting in hypoxia. Th is is fol lowed by a subsequent aggregation of platelets and col lagen to the area of injury. In addit ion, an increase in extracellular fluid and vascu lar dilation occurs, fol lowed by an influx of neutrophils, macrophages, f ibroblasts, smooth muscle cel ls and endothelial cel ls to c leanse and reconstruct the insult. Lactate, hypoxia and the production of cytokines further c a u s e s growth stimulation, which leads to angiogenes is and the production of co l lagen. The wound is essential ly cons idered healed after the occurrence of this c a s c a d e of events [41-42, 50]. T i ssue damage can lead to edema , compl icat ions with blood flow and eventual t issue death, including ischemia [8, 41-42]. The increase in extracellular fluid and vascular dilation impairs oxygen delivery from capil lar ies to cel ls because of an increase in the diffusion distance [9]. Result ing dec reases in oxygen tensions (e.g. below 30 mmHg) make the cel ls more suscept ib le to infection [7, 43] and the eff icacy of leukocytes in reducing invading organisms becomes defective [12, 44]. Furthermore, host repair p rocesses are also impaired during this time [7]. Oxygen , therefore, plays a critical role in the wound heal ing process. It serves as a catalyst and energy source for maintenance, metabol ism and repair [45, 46]. In wound heal ing, oxygen serves to provide the addit ional energy source necessary for the reparative process. Early in the repair of wounds, f ibroblasts begin to migrate, divide and produce col lagen, which is an essent ial matrix for wound heal ing. Oxygen must a lso be present in sufficient quantit ies for fibroblast proliferation and col lagen production to occur. Adequate amounts of proline and lysine (two amino ac ids incorporated by oxygen) must be hydroxylated with oxygen for col lagen to be synthes ized by fibroblasts [47]. Oxygen must a lso be present in adequate amounts during the repair p rocess to provide energy for protein synthesis. It has been demonstrated that raising oxygen tension in 10 tissues increases the ratio of RNA/DNA [48]. Pal et al [49] have stated that an increase of 150% above the normal physiologic range for oxygen (PO 2=40 mmHg) increases the rate of collagen production by seven times. An adequate delivery of oxygen via an extensive capillary network is essential since the diffusion of oxygen through tissues is limited [46]. Disruption of the capillaries from trauma produces hypoxia and the release of hormonal mediators. Macrophages release an angiogenesis factor, which is a potent stimulus for endothelial cell activity [50]. Polymorphonuclear cells (PMN) which locate, identify, phagocytose, kill and digest microorganisms, require oxygen to kill organisms by producing superoxide, hydrogen peroxide, singlet oxygen [ and other products via the respiratory burst [51]. Detoxifying free radicals by superoxide dismutase, catalase and glutathione protect the PMNs. Therefore, the degree of PMN cell function in killing of bacteria is directly dependent on oxygen tensions [44, 52]. Hyperbaric Oxygen has two components in wound healing and tissue survival: acting mechanically, due to its pressure component and physiologically, due to its oxygen component [39]. The oxygen content is determined by a combination of oxygen that is bound to hemoglobin plus the amount of oxygen that is dissolved in the plasma. [Total 0 2 = h e m o g l o b i n b o u n d o x y g e n + o x y g e n d i s s o l v e d in p lasma] The application of hyperbaric oxygen increases the amount of available oxygen to the hypoxic area of injury, increasing oxygen tensions that make host repair processes functional [8, 9]. Increased availability causes a shift in the oxygen cascade (a gradient from the partial pressure of oxygen in the ambient air to that available immediately to the tissues on a cellular level). This effect of hyperoxygenation is based on a combination of two laws, namely Henry and Dalton's Laws. Henry's Law states that as the P 0 2 increases during compression, 11 the amount of oxygen dissolved directly into the p lasma increases [3, 53]. By increasing the partial pressure of oxygen in the air, a significant amount of oxygen becomes d issolved in the blood p lasma. Furthermore, Dalton's Law states that air is a mixture of gases and that the total pressure exerted is the sum of the partial pressures of each of the g a s e s in the mixture [53]. T h e s e two laws combined produce the effect of hyperoxemia, which al lows enough oxygen to d issolve in the p lasma to meet metabol ic needs . At s e a level in room air, there is 0.32 ml of oxygen d issolved in each 100 ml of whole blood (0.32 vol.%). W h e n breathing 1 0 0 % oxygen, each addit ional a tmosphere of pressure produces an addit ional 2.3 vo l .% oxygen d issolved in p lasma. At 2.0 A T A , the blood oxygen content increases 2 . 3 % while p lasma and t issue oxygen tensions increase tenfold (1000%) [8,9,11,22,54]. Consequent ly , sufficient oxygen becomes physical ly d issolved in the p lasma to keep t issues alive despite the inability of hemoglobin-bound oxygen to reach the insulted area [17, 55]. At 3.0 A T A , the partial pressure of oxygen in the blood can increase to as much as 2200 m m H g (p lasma contains 6.8 vo l .% oxygen). Th is elevated oxygen pressure increases the oxygen diffusion gradient and improves oxygen delivery to relatively ischemic t issues [53-56]. At this pressure, enough oxygen can be d issolved in the p lasma to sustain life temporarily without any red blood cel ls [57]. Secondary effects of hyperbaric oxygen therapy include vasoconstr ict ion, gas vo lume reduction, inhibition of anaerobic organisms and neovascular izat ion. Vasoconstr ic t ion causes a decrease in e d e m a at the wound site leading to dec reased t issue perfusion without sacrif icing oxygenation [53]. Sufficient amounts of oxygen are d issolved in the blood p lasma to adequately compensate for the decrease in blood flow to the area of injury [9, 58]. This reduction in blood flow produces a subsequent 2 0 % reduction in post-traumatic vasogen ic edema [59]. Hyperbaric oxygen therapy a lso c a u s e s a reduction in gas vo lume by reducing the s ize of the gas bubbles. This physiological effect is based on Boy le 's law stating, "the volume of a gas is inversely proportional to its pressure at a constant temperature". Therefore, as the pressure increases, the vo lume of 12 a gas dec reases , reducing the s ize of gas bubbles that impede circulation [53]. B e c a u s e of this effect, H B O is used as the primary treatment in air embo l isms and decompress ion s ickness [60]. Al though aerobic organisms continue to thrive when given increased amounts of oxygen, their growth may be inhibited when the partial pressure of oxygen exceeds 1.3 A T A [57, 61]. However, inhibition of aerobic organisms is best ach ieved when they are superficial [62] (e.g. a superficial ulcer or burn). Hyperbar ic oxygen therapy used as an adjunct has also been demonstrated to be beneficial in treating anaerobic infections such as gas gangrene and inhibition of a lpha toxin production [62] [Table 4]. Tab le 4: Cel lu lar and Biochemica l Benefi ts of Hyperbaric Oxygen* • Promotes ang iogenes is and wound heal ing • Kil ls certain anaerobes • Prevents growth of spec ies such as P s e u d o m o n a s • Prevents production of clostridial a lpha toxin • Restores neutrophil mediated bacterial killing in previously hypoxic t issues • R e d u c e s leukocyte adhesion in reperfusion injury, preventing re lease of proteases and free radicals which cause vasoconstr ict ion and cel lular damage *adapted from Leach RM 2.2 Oxygen Toxicity Related to Hyperbaric Oxygen Therapy Oxygen in large amounts, like most therapeutic modalit ies, can be toxic and life threatening. However, it has widely been reported that toxicity is related to both the duration of exposure and pressure [63] (Figure 1). Oxygen toxicity affects both central and pulmonary nervous sys tems. Oxygen toxicity involving the central nervous system is termed the Paul Bet effect and results in se izures [205]. Oxygen toxicity affecting the pulmonary system is termed the Lorrain-Smith effect, and c a u s e s e d e m a in the lungs and subsequent alveolar co l lapse [206]. 13 Figure 1: Pressure-durat ion relationship for effects of oxygen toxicity when using hyperbaric oxygen therapy [63] 0.5 ATM 3 6 12 15 18 21 2 4 Exposure Duration (hours) Central nervous system assoc ia ted grand mal se izures have been documented at a pressure of 3.0 A T A , [62, 63] while pulmonary e d e m a has been detected at 2.0 A T A [54, 64-66]. However, both toxic effects occurred at prolonged exposures of three or more hours [9, 65, 66, 68]. Davis [67] est imated the incidence of oxygen se izures to be one in 11,000 treatments. Thorn [207] reported that the l ikelihood of central nervous system agitation is approximately 0.0009%. Factors contributing to a seizure are fever, exerc ise, apprehension and CO2 buildup [57]. Typical warning signs may include tunnel v is ion, shortness of breath, tinnitus, nausea and extreme apprehension. If a seizure occurs during treatment, the patient should be removed from the chamber but not until the seizure has stopped. To decompress the patient during the tonic phase of the seizure can put the patient at risk of air or oxygen embol ism [57]. Se izures , if promptly treated, have no permanent seque lae. Pneumothorax can occur under hyperbaric condit ions due to predisposing lung pathology such as gas trapping in a local ized portion of the lung [57]. The patient may exper ience respiratory distress, sudden stabbing chest pain, a shift of the t rachea toward the unaffected side, lack of chest movement on the affected side, decreased breath sounds on the affected side and increased tympany [57]. W h e n a mult iplace chamber is used, treatment of pneumothorax can be 14 managed by inserting a needle or chest tube into the affected s ide. For treatment in a monoplace chamber , the patient must be decompressed before treatment. Th is may cause the pneumothorax to double or triple in volume but respiratory distress may subs ide when recompression occurs . Pulmonary oxygen toxicity may be a considerat ion in patients maintained on an inspired oxygen fraction of greater than 4 0 % between hyperbaric oxygen sess ions [208]. In this instance, the cl inician on hand must be aware of the hyperbaric oxygen treatment to monitor for toxicity and intervene if the need ar ises. The uses of aspir in, insulin, steroids, epinephrine and norepinephrine have all been noted to increase the onset of oxygen toxicity [9, 34]. Concurrent therapy with doxorubicin (Adr iamycin R ) , cis-plat inum and disulf iram (Antabuse R ) are a lso incompatible with H B O [3]. W h e n combined with H B O therapy, doxorubicin has produced a high mortality rate in animals. Furthermore cis-plat inum used concurrently with H B O therapy dec reases the strength of healing wounds and disulfiram blocks production of superoxide d ismutase, the body's major protection against oxygen toxicity [3]. Conce rns have also been expressed as to whether physical exerc ise pred isposes patients to oxygen toxicity [9] but a study conducted by S tevens et al [66] conc luded that there were no adverse effects of oxygen toxicity with exerc ise. Converse ly , antioxidants such as vitamin C , vitamin E and mexamine, lithium, and magnes ium have been used to prevent or delay the onset of toxicity [202-204]. The best way, however, to prevent prolonged oxygen toxicity is to take periodic breaks, inspiring normal air (21% oxygen) [9]. A five year prospect ive study expos ing 12 468 patients to 100% oxygen at 2.0, 2.2 and 2.4 A T A showed no oxygen-related compl icat ions or se izures at 2.0 A T A [68]. Mild aural barotrauma was the only complicat ion observed in this study. Barotrauma is defined as any injury to structures such as the ear due to dif ferences between atmospher ic and intratympanic pressures (commonly referred to as "squeeze") [57]. Due to pressure changes assoc ia ted i 15 with hyperbaric treatment, the patient must be taught to equal ize the pressure in the ear by swal lowing, yawning or using the va lsa lva manoeuver . The primary symptom of "squeeze" is pain in the ear while the chamber is being compressed [57]. Barot rauma can a lso occur in the s inus cavity, primarily the frontal s inus. Decongestants are usually prescr ibed when this occurs . In a ser ies of studies conducted, Dav ies reported that one in 270 c a s e s had barotrauma significant enough to interrupt treatment [67]. Others have stated compl icat ions such as nausea , tooth and s inus pain and blurred vision [9]. Therefore, it can be conc luded that no toxic effects are seen within the current therapeutic range of 2.0 A T A at a 60-90 minute exposure time, accompan ied by frequent air breaks during treatment. Proper screening is essent ia l before hyperbaric treatment can be administered. Contraindicat ions for H B O therapy include upper respiratory tract infections, d iabetes, pregnancy, conf inement syndrome, pneumothorax, sinusit is and fever [Table 5]. Fire in the 100% 0 2 environment of a mono/mult iplace chamber is, although rare, a concern that should be addressed . Al l efforts should be maintained to minimize the risk, including the exclusion of items that could be assoc ia ted with heat or f lame. These include velcro, glycerin (including hair products and cosmet ics) , perfumes, lotions and non-cotton fabrics (should be 100% cotton to reduce static) . [Table 6]. Oxygen toxicity is a ser ious consequence of treatment with hyperbaric oxygen. However, this can be controlled with proper screening of the patient prior to administration of treatment and appropriate control over length of exposure and pressure levels. Ideally, it can be conc luded that treatments for 60-90 minute durations are within a reasonable level ' to avoid compl icat ions that may ar ise. Furthermore, treatment should be administered at" 2.0-2.5 A T A , accompan ied by frequent air breaks. 16 Table 5: Contraindicat ions to H B O Therapy* • Pneumothorax • Uncontrol led high fevers • Severe chronic obstructive pulmonary d isease • Opt ic neuritis • Acute viral infection • Congeni ta l spherocytosis • Upper respiratory tract infections • Pregnancy • Psychiatr ic problems • Prior thoracic or ear surgery • Acute seizure disorders * adapted from Foster J Table 6: Patient Preparat ion Prior to H B O for Safety and Fire Prevent ion • The patient should shower or bathe daily and hair should be / shampooed if oily before H B O • Body oils, lotions, talc, petroleum products, make-up, co logne, perfume, deodorants, nail pol ish, hair oils and spray are not to be used before treatment. • Only all cotton clothing and l inens are al lowed in the chamber to prevent development of static electricity. • W igs or hairpieces, hearing aids, jewelry and contact lenses are not to be worn in the chamber. *adapted from Curtis et al 17 2.3 Previous Studies Examining HBO and Tissue Injuries Burns A vast amount of research exists on the effects of H B O on burns, especia l ly thermal burns. First documented in the 1970's, patients exposed to H B O found that burns dried sooner, had fewer infections and healed more quickly [18, 70]. Heal ing time was shown to dec rease by 3 0 % in more recent control led studies [10, 71-73]. Other authors examining second degree burns report increased rates of epithelial ization, dec reased fluid requirements, preservation of microcirculat ion, decreased convers ions from partial to full th ickness injury, reduction in e d e m a and inflammatory response and dec reases in grafting and surgical procedures, hospital t ime and relevant assoc ia ted costs [3, 5, 72-74]. In a model sca ld burn, Nylander et al [76] demonstrated a decrease in global e d e m a following an ear burn. Stewart et al [75] examined A T P , phosphocreat ine and col lagen synthesis in burn wounds of rats. They conc luded that as an adjunct, H B O could preserve or enhance energy rich phosphate compounds and col lagen synthesis in model burn wounds. Al though large amounts of research exist demonstrat ing the heal ing potential and beneficial effects on epithelial ization, some d iscrepancy does exist in the literature with respect to the eff icacy of H B O therapy as an adjunct treatment. Other control led, randomized human studies show no effect in the heal ing process [43, 76]. This d iscrepancy may be attributable to the time lag from injury to the initiation of treatment, possibly 24-48 hours post-injury. Infections The role of oxygen in fighting infections has also been establ ished. Oxygen acts as a potent antibiotic, improving the ability of specia l scavenger white blood cel ls (phagocytes) to rid the body of bacteria and other foreign proteins. Hyperbar ic oxygen has a distinct antimicrobial effect that is equal to or better than that of numerous antibiotics [29]. This form of treatment may also enhance the act ions of certain antibiotics and thereby increase their ef fect iveness in overcoming 18 infections [29]. B e c a u s e of its immune system-enhanc ing effects, hyperbaric oxygen therapy helps fight all microorganisms, both anaerobic and aerobic organ isms. The literature has reported over 4,000 c a s e s of gas gangrene treated with hyperbaric oxygen therapy. O n e study reported a survival rate of 8 8 . 3 % among 248 patients who received treatment [209]. Another study of 139 patients reported a survival rate of 70 percent, with 8 0 % of the survivors able to avoid amputat ions [210]. Ell is and Manda l ana lyzed 58 patients who fai led to respond to convent ional treatment of antibiotics and surgery. After treatment with hyperbaric oxygen, there was a survival rate of 8 4 % [211]. Wound Healing A wound is any disruption in the body's t issues. It is often assoc ia ted with the loss of skin (and underlying t issue), musc le or bone. Many wounds respond to convent ional medic ine while others do not. Primarily, a wound lacks the necessary oxygen required for heal ing to take place. This may be due to a blood clot, which interferes with the circulation. A s a result, the oxygen supply to the area of insult is diminished and toxic materials accumulate s ince waste products cannot be removed. This starts a cycle of damage to affected t issues. W o u n d s are a lso prone to infection s ince the lack of oxygen in the t issues can reduce the injured person 's defense by decreas ing the activity of infection-fighting white blood cel ls [12]. Underoxygenat ion can also deactivate the cel ls that produce granulation t issue and interfere with col lagen production [194]. Therefore, it can be conc luded that a lack of oxygen in the wounded t issue can interfere with the entire wound heal ing process. In the treatment of open wounds, H B O plays a vital role in col lagen synthesis, hydroxylation of the col lagen molecule and intracellular A T P production, thus promoting t issue repair and growth [7, 8]. To summar ize, H B O promotes granulation t issue formation, revascular izat ion, epithelial ization, enhanced fibroblastic activity and leukocyte killing, fibroblast migration, ang iogenes is as well as capil lary budding [3, 36, 78-80]. Ample clinical investigations have 19 demonstrated that in hypoxic wounds (PO2 = 5-20 mmHg), t issue heal ing occurs with sufficient t issue oxygenat ion, which is enhanced by H B O [81]. In a study of patients with chronic diabetic foot lesions, Doctor et al [184] observed a better control of infection and less need for amputation in the group treated with convent ional management and sess ions of H B O . Bone Healing and Hyperbaric Oxygen Therapy Bone is c o m p o s e d of three layers: a spongy inner layer, a rigid middle layer and a tough outer layer. W h e n bone is injured, spec ia l cel ls cal led osteoclasts work to repair the damage . T h e s e cel ls carve paths through the bone t issue around the brake and cause dead bone to be reabsorbed by the body. Osteoblasts , another group of cel ls, then create new bone [29]. T h e s e osteoclasts depend and utilize oxygen for proper function. Therefore, hyperbaric oxygen may facilitate bone heal ing by stimulating both osteoclasts and osteoblasts [7, 8]. It may also stimulate the production of new blood vesse ls , so that the growing bone receives a steady supply of nutrients, including oxygen. This b lood-vessel network helps support the function of the osteoclasts and brings infection-fighting white blood cel ls to the area of injury [29]. Osteomyelitis Osteomyel i t is is a bacterial infection that usually occurs on the outer layers of bone as well as the inner bone marrow. Staphy lococc i bacteria are primarily implicated in this type of infection. The germs that cause this infection can enter the bone during an injury or surgery. Furthermore, it may also reach the bone directly from a nearby infection or indirectly through the b loodstream. Osteomyel i t is may be either acute or chronic, with severe pain, swel l ing and redness at the site of infection in the acute phase of infection. High fever is a lso prominent in patients with osteomyelit is. In the chronic stage, symptoms include bone pain, tenderness and local musc le s p a s m . 20 Convent ional treatment for osteomyeli t is includes antibiotics and severa l weeks of bed rest. Surgery may be required to take out dead bone and soft t issue, fill ho les and impjant artificial dev ices des igned to keep the d i seased bones and joints from moving [29]. It is imperative to treat the bone infection promptly and vigorously in order to prevent it from spreading to other parts of the body. Hyperbaric oxygen has three main functions in treating osteomyeli t is. First it helps strengthen the bone cel ls (osteoclasts) that reabsorb dead bone, removing bony debris more effectively. S e c o n d , it enhances the function of the immune system's white blood cel ls, s ince they depend on oxygen. This is especia l ly effective when used with antibiotics [29]. Lastly, hyperbaric oxygen helps the body to create new blood vesse ls . Through these mechan isms, hyperbaric oxygen enab les the body to get rid of the d iseased bone and replace it with healthy bone [29]. An imal research on the effect iveness of hyperbaric oxygen for the treatment of osteomyelit is dates back to the late 60 's . T h e s e investigators demonstrated that animals were cured of bone infections with the use of hyperbaric oxygen alone (i.e. no antibiotics or surgery). Furthermore, they t reated another group of rats prior to inducing infection but found that it did not prevent the infection from taking hold. The authors conc luded that hyperbaric oxygen was an effective treatment modality in treating osteomyelit is because it enhances the host 's own immune system and not because it kills the bacteria directly [212]. The benefits of hyperbaric oxygen have also been establ ished in human treatments. Neubauer et al [29] reported that the overall s u c c e s s rate in var ious investigations using hyperbaric oxygen therapy on osteomyelit is ranges from 60 to 85%, with a lower rate of recurrence. Davis [213] demonstrated in a five year follow-up study, that in 136 patients with refractory chronic osteomyeli t is (cases which failed to heal with antibiotic treatment or surgery) of the spine, extremities, pelvis, skull and chest wall, over half of these patients had their infection clear up. 21 Another study demonstrated a cure rate of 8 5 % in a two-year follow-up study of 40 patients with chronic osteomyelit is. Hyperbar ic oxygen in this study was used as an adjunct to surgery and antibiotics [214]. Aseptic Bone Necrosis Asept ic bone necros is (ABN) occurs-when bone becomes inflamed without being infected. It usually occurs as a complicat ion of decompress ion s i ckness but can occur in diabetes, hepatitis, rheumatoid arthritis and sickle cell anemia . It may also result as a s ide effect of var ious therapeutic procedures such as radiation therapy and steroid treatment [29]. A B N can also ar ise spontaneously in the general population, especial ly in chi ldren ages eight to fourteen [215-216]. A B N is essential ly a blood supply problem [217]. Th is blood vesse l disruption results in ischemia or a reduced blood supply resulting in bone not getting enough nutrients and oxygen. In addit ion, cellular wastes accumulate [217]. Asept ic bone necrosis should be treated properly as multiple joints are often involved and may lead to permanent disability. Widespread A B N requires a great deal of joint-replacement surgery, caus ing a large amount of expense , pain and disability [29]. This is where hyperbaric oxygen therapy fits in. It s e e m s logical that increasing ^ the amount of oxygen in the affected t issues may halt deterioration and promote heal ing. Neubauer and co l leagues [29] have conc luded that hyperbaric oxygen does indeed help patients with A B N , however treatment should be long-term. Short-term treatment does relieve pain and disability but no lasting cure takes place [218-219]. Fractures Clin ical investigation on the use of H B O on fractures has been promising, especial ly where non-union and compl icated fractures with increased chance of 22 infection are involved [82]. Findings such as increases in osteoblast ic D N A and R N A , bone mineral izat ion, hematoma and cal lus formation, cal lus nitrogen content, capacity for protein synthesis, alteration of the homeostat ic environment, reduction in osteoblast formation, col lagen formation, capil lary budding, fibroblastic activity and proliferation and os teogenes is have all led to the conclus ion that H B O speeds recovery in fractures and reduces heal ing time [75, 83 , 85]. Researchers have conc luded that hyperbaric oxygen therapy leads to greater carti lage production and bone formation [220]. It has a lso been demonstrated to help in bone grafts [221]. In an animal study of 487 fractures in and around the joints, researchers conc luded that hyperbaric oxygen therapy used as an adjunct to conventional orthopedic methods, shortened the process of bone regeneration and wound heal ing by ten to twelve days [222]. Aga in , contradiction does exist in the literature whereby other authors observe no decreases in heal ing time [86, 87]. Th is may be due to the time of onset of treatment from the actual injury time (12 hours or more after the injury). Crush Injuries / Traumas / Compartment Syndrome Crush injuries involve diffuse blood loss resulting in t issue hypoxia, which severely affects cel lular function [19]. In addit ion, increased blood flow resulting in edema accompan ies the insult. Th is e d e m a c a u s e s an increased diffusion distance for oxygen to travel to the capi l lar ies [9]. In c losed crush injuries, all circulation to the injured area is diminished [88]. H B O serves to cause a 2 0 % reduction in blood flow in addition to providing a sufficient medium for macrophage to function [44, 45]. Cl in ical trials have demonstrated beneficial effects such as 1) countering wound infection accompany ing open traumas of the extremities 2) accelerat ing the recovery of neutrophil phagocyt ic activity 3) prevention of limb amputation and 4) heal ing the open fracture without suppuration (dead skin with a distinct line of demarcation) [13, 66]. However, the above studies failed to compare the H B O treated patients with those treated more conservatively, thus not illustrating a c lear difference in heal ing patterns. In v. 23 a study demonstrat ing the effects of H B O on the management of severe t rauma of the l imbs in older patients with grade III soft-t issue injuries, Bouchard et al [89] found H B O to improve wound heal ing and reduce the repetitive surgery necessary in c a s e s of aggravat ion of crushing t issue damage. Nylander et al [90] exposed rat hind l imbs to 3 hours of temporary ischemia fol lowed by 45 minutes of H B O sess ions at 2.5 A T A . The results showed a significant reduction in post- ischemic e d e m a and thus conc luded that H B O was a useful adjunct in the treatment of acute ischemic condit ions when surgery couldn't be attempted or failed to reverse ischemia [76, 90, 91]. J o n e s et al [92] conducted a preliminary study on the effects of H B O on ten patients with compress ive lesions of the spinal cord leading to paralysis. The results suggested that by support ing injured spinal cord t issue with oxygen under pressure, improvement might occur. A large ser ies of patient, in this type of study, will be necessary before definite conc lus ions can be drawn on whether H B O therapy improves recovery in the paralyzed patient with a bruised spinal cord. Studies conducted on animal models for compartment syndrome ( increased pressure in skeletal musc le compartments caus ing reduced capil lary perfusion, leading to ischemia, nonfunctional and necros is of t issue) have all showed promising results of reduced muscular necros is and e d e m a in H B O treated versus control groups [81, 84]. Zambon i et al [93,94] have also conducted severa l exper iments examining the effects of hyperbaric oxygen on ischemic musc le on animal models , again with positive results showing a reduction in e d e m a and improved microvascular perfusion. Further research, however, needs to supplement the existing body of literature in terms of human, control led, double-bl inded studies. Hyperbaric oxygen therapy can provide var ious benefits in the treatment of wounds. To summar ize : it encourages the growth of new t issue by providing extra oxygen, it counteracts the chance of infection by indirectly providing cel ls (i.e. white blood cells) the extra oxygen that they need and directly by killing anaerobic organisms, stopping their.multiplication and neutralizing the toxins that some produce [12], it encourages bone repair by supplying osteoclasts and 24 osteoblasts the rich supply of oxygen that they require and lastly it provides a c lear line of demarcat ion between t issue which is beyond repair and that which can be saved [223]. Therefore, the effect iveness of hyperbaric oxygen therapy has been beneficial in minimizing t issue death, reduction in swell ing and promoting heal ing. 2.4 The Role of Hyperbaric Oxygen in Sports and Exercise Medicine There is a considerable amount of significant research suggest ing the eff icacy of H B O therapy as both a primary and adjunct in the treatment of a variety of i l lnesses and injuries. However, only a paucity of knowledge exists in terms of its use, benefits and mechan ism of action in sport and exercise-related injuries [Table 7,8]. Although the few studies conducted thus far looking at acute injuries in sports and exerc ise have proven to be promising in terms of using H B O as a treatment modality, these studies have been limited by their sample s ize and study design [1, 2]. Leach et al states, "the gap between the knowledge gleaned from the laboratory and severely traumatized patients and the athlete in the locker room remains vast [40]". Th is statement clearly summar izes how' much research needs to be conducted to establ ish or refute the role of hyperbaric oxygen therapy in sports and exerc ise medicine. Hyperbaric Oxygen - "Applications of HBO arise as a result of medical adventurism; A therapy in search of a disease" Gabb G & Robin ED70 Studies to date Oriani et al [88] first suggested the use of H B O to accelerate the rate of recovery from injuries suffered in sports. However, it wasn't until recently that a study on H B O was first publ ished. This study looked at the number of days lost to injury in professional soccer players in Scot land [1]. The results suggested a 5 5 % reduction in days-lost-to-injury based on a physiotherapists estimation of the t ime-course for the injury versus the actual number of days lost with routine therapy and H B O treatment sess ions . Although promising, this study was subjective in nature, needed a control group, input from an objective third party 25 and required a greater homogenei ty of injuries. Randomized , control led, double- bl inded studies with quantif iable injuries are required for significant validity of results. A n ankle inversion study conducted at Temp le University sugges ted that patients exposed to H B O treatments returned to activity 3 0 % faster than control groups [2]. Unfortunately, the results were inconclusive in this study s ince a large amount of variability existed in the study des ign. The authors attributed much of this observed variability to the difficulty in quantifying the severity of ankle sprains [33]. Furthermore, this study has been countered by a recent randomized double- blind design in which H B O treatment did not improve time to recovery after an acute ankle sprain injury [95]. Stap les et al [96, 97] conducted both an animal and human study on a musc le injury model . The animal model , which measured myeloperox idase levels in treated versus untreated rats, was suggest ive of an inhibitory effect of hyperbaric oxygen on the inflammatory p rocess or the ability of H B O to actually modulate the injury to the t issue. The human study employed a randomized, double- bl inded design with controlled start and end points. The promising results revealed that the treatment group with H B O had a greater recovery of eccentr ic strength from delayed-onset musc le so reness ( D O M S ) . However, this treatment modality had no effect on pain levels [96, 97]. O n e of the best quantitative studies to date c o m e s from a rat model of surgically lacerated medial collateral l igaments. Th is study compared l igament strength and stiffness in injured and uninjured l igaments over an 8-week period and conc luded that the H B O appears to have promoted the return to normal stiffness of the l igaments at 4 weeks as well as enhanced recovery of l igament strength [98]. 26 Another cl inical study completed by S o o l s m a et al [32] at the University of British Co lumb ia examined the short-term recovery of grade II medial collateral l igament injuries of the knee. Posit ive results suggest that recovery was more rapid in the H B O exposed individuals as compared to the control group [32]. A recent study completed by Best et al [99], demonstrated in a rabbit model , that a 5-day treatment regimen with H B O appears to improve functional and morphologic recovery at 7 days after a controlled reproducible musc le stretch injury. Counter ing the above studies is the work conducted by Harr ison B C et a l , at the University of Co lorado, which examined the effects of treating exerc ise- induced musc le injury v ia hyperbaric oxygen. T h e s e researchers conc luded that H B O was not effective in the treatment of musc le injury as ev idenced by MRI , C K response, isometric strength testing and perceived soreness [246]. Mekjavic et a l , have also recently reported that H B O is not effective therapy for the treatment of D O M S based on maximal isometric muscle strength of the elbow flexor musc les , right upper arm circumference and ratings of perceived musc le so reness [225]. 1 Although few in number, all of the above studies provide some insight into the application of H B O as an adjunctive treatment in sports and exerc ise medic ine, and warrants the need for addit ional research to better define the therapeutic indications of hyperbaric oxygen. The demand for hyperbaric oxygen therapy is increasing throughout the medical field. Medica l professionals, cl inicians and researchers have been and are currently venturing on new areas that haven't previously been investigated. A growing interest in sports and exerc ise medicine is appear ing throughout the literature. However, with this new spark of enthusiasm comes a high degree of 27 skept ic ism that has been developed regarding its use. To date only a handful of studies exist in this a rea and of these studies, only a select few support the benefits of utilizing this intervention. Table 7: Benefits of HBO in Sports Injuries* • Reduction of pain and swelling in the acute stage • Speeds up recovery and return to activity training • Improves fracture healing • Aids in the recovery from exhaustion and collapse *JainKK9 • Table 8: Proposed Mechanism of Action in Sports Injuries* • Vasoconstriction • Reduction in neutrophil-adhesion • Free radical quenching ability • Enhancement of leukocyte killing and hydroxyproline formation *JainKK9 2.5 Acute Soft Tissue Injury: Delayed Onset Muscle Soreness Characteristics of DOMS At some point in time, nearly all of us have exper ienced the sensat ion of de layed- onset muscle soreness ( D O M S ) ; many of which have had numerous encounters with this common self-limiting ailment. Even trained individuals will exper ience some soreness following a novel bout of unaccustomed exerc ise [100]. Th is condition is usually character ized by a sensat ion of pain and discomfort that occurs in skeletal musc les following a bout of unaccustomed exerc ise and exertion, and is often accompan ied by tenderness and stiffness, with a reduction in mobility or flexibility of the musc les involved. This reduced mobility and flexibility is exacerbated during palpation, pass ive stretching and contraction of the involved musc les [101-103]. The pain may be slight and may d isappear upon repeated activity or may be severe enough to interfere with and limit 28 movement. Th is pain and tenderness is usual ly local ized to the distal third portion of the musc le , in the region of the muscular- tendinous junction where musc le pain receptors are most concentrated, with eventual spreading to the center of the musc le belly by 48 hours [104]. Newham et al [104] have reported that the pain assoc ia ted with D O M S appears medial ly, laterally and then distally, becoming more diffuse throughout the musc le 24-48 hours post-exerc ise. General ly , however, the pain is evident throughout most of the affected musc le belly. The so reness that is prominent with D O M S initially appears 8-24 hours after exerc ise, increases in intensity within the first 24 hours, peaks from 24-72 hours and finally subs ides 5-7 days post-exerc ise [102, 105, 106]. Type of Activity Inducing Muscle Injury High intensity, short duration exerc ise results in the largest increase in musc le damage and D O M S [107]. Eccentr ic exerc ise (e.g. downhil l running - involving forced lengthening of a muscle as it deve lops tension [108]) has been shown to produce greater damage to the musc le fibres as opposed to concentr ic contractions (e.g. uphill running), s ince the former requires lower energy costs as fewer motor units are activated for a given load [104, 109, 110]. At a given submaximal force or power output, E M G activity in musc le has been demonstrated to be lower during eccentr ic (negative) work than concentr ic [111, 112]. This leads to higher tensions per cross-sect ional area of active skeletal muscle fibres [104, 110]. The increased tension could cause mechanica l disruption of the structural e lements in the musc le f ibres themselves [102, 104, 110, 113] or in the connect ive t issue that is in ser ies with the contractile e lements [114]. Evans [115, 116] suggested that the reason why eccentr ic exerc ise causes far greater amounts of muscle damage than concentr ic exerc ise might be due to different fiber recruitment patterns. In other words, eccentr ic contraction condit ions provide a situation where relatively few fibres are recruited and are producing relatively large forces [110]. C leak [105] reported that lengthening muscle fibres imposes additional tension and stretch on connect ive t issue and appears to increase local ized soreness in the region of the musc le involved. 29 C a n n o n et al [108] cite that repeated force lengthening of a musc le as it deve lops tension c a u s e s immediate ultrastructural damage to the sarcomeres , fol lowed by delayed-onset musc le so reness and a re lease of myocel lular enzymes . Consequent ly , it has been reported that eccentr ic work requires less oxygen, lower amounts of A T P , produces less lactate and shows lower motor unit activity than concentr ic work [114, 117-120]. Furthermore, eccentr ic contractions require less time to reach peak tension [113]. More recent ev idence suggests that muscle damage is not a function of absolute force or tension generated but the magnitude of strain during active lengthening [121]. T h e s e authors determined that active strain during eccentr ic work was related to the speed at which a musc le was lengthened while it was contracting. It has been suggested that the greater damage elicited by faster eccentr ic contractions was due to the fact that cross-br idge cycl ing could not keep pace with the change in length of the musc le [122]. Musc le fiber type has also been reported to be a c a u s e for musc le damage. It has been suggested that specif ic damage can be noticed in the type 2 B fast glycolytic f ibers, thus hypothesizing that musc le injury may be a result of the musc le fiber, oxidative capaci ty [227]. It is bel ieved that type 2 fibers might be more suscept ib le to stretch induced injury because of a less deve loped endomys ium that type 1 fibers [228]. From a review of the literature, it s e e m s apparent that musc le damage may not only be a result of high force generation but the magnitude of strain during active lengthening may also play a role in the injury process . Eccentric Strength Loss Accompanying DOMS Throughout the literature, it is evident that D O M S is a consequence of muscle "over-use" [100-107]. Any activity in which the musc le produces high forces or forces over a longer period of t ime is capable of producing the sensat ion of 3 0 D O M S [107]. In addit ion, although the degree of so reness is related to both the intensity of the muscular contractions and the duration of exerc ise, intensity s e e m s to be the more important determinant of the two [101, 107, 123]. Hough [124] was the first to descr ibe in detail the phenomenon of D O M S , hypothesiz ing the etiology and mechan isms involved. He demonstrated that D O M S was a result of structural damage to the musc le and/or connect ive t issue and to a reduction in muscular performance [124]. This reduction in performance may result from a reduction in voluntary effort due to the sensat ion of so reness and/or a lowered inherent capaci ty of the musc le to produce force [101]. Therefore, D O M S is a lso accompan ied by a significant decrease in force-generat ing capaci ty of the muscle resulting in eccentr ic strength decrements [125]. The time course for the development of so reness and loss of strength suggests little or no relationship between the two parameters [112]. Newham and co l leagues [110] found that maximal voluntary force of the knee extensors had returned to normal by 24 hours after exerc ise while so reness at this time was most intense. Furthermore, Hough showed that the occurrence of de layed pain was directly related to the peak forces developed and to the rate of force development in rhythmic contractions, but not to the rate of fatigue [124, 126]. A reduction in max imum force production has been observed after eccentr ic exerc ise, as early as 1-hour post-exercise [113, 127]. Al though Newham and co l leagues have suggested that strength returns to pre-exercise levels within 24 hours [110], others have reported a return to basel ine levels as long as 1 week [113]. G leeson and co l leagues [128] have reported that decrements in max imum isometric force (50% of normal) are greatest immediately following eccentr ic exerc ise, with recovery taking place by days 4-7. In addit ion, they report that a decrease in max imum dynamic power output (80% of normal) persists up to 4 days. * Maclntyre and colleague's [129] have reported a bimodal pattern of eccentr ic torque that occurs 0 and 20-24 hours post-exercise. Th is bimodal pattern was the first report demonstrat ing this pattern in humans. Faulkner et al [130] reported 31 two decl ines in the musc le force in an animal model . They suggest that the initial decl ine in force may be a function of mechanica l injury and fatigue (including myofibrillar disruption at the level of the Z-line), leading to an acute inflammatory response [129]. Faulkner and co l leagues [130] further suggest that the second decl ine in force occurs in response to phagocyt ic activity at the site of the initial damage . This deficit in force does not appear to be related to the level of so reness s ince it occurs prior to the soreness and can remain for a greater period [131]. Maclntyre et al [132] have also reported that no relationship exists between the development of so reness and loss of muscle strength s ince the latter appears immediately after exerc ise. Therefore, this bimodal pattern of eccentr ic torque further grounds support for the theory that more than one mechan ism is involved in exerc ise- induced musc le so reness [129,130] . Theories Associated with DOMS The pain and discomfort assoc ia ted with D O M S has been studied extensively s ince 1902 and as a consequence , several theories have been suggested to explain this condit ion. Theor ies such as the lactic acid theory (lactic acid accumulat ion in the muscle) , muscle spasm theory (originally descr ibed by DeVr ies [133], in 1961 that exerc ise causes ischemia which results in the production of a pain substance) , connective tissue damage theory (rupture and damage of the musc le , predominantly the connect ive t issue), muscle damage theory (skeletal muscle damage the primary mechan ism contributing to muscle soreness) and inflammation (tissue damage triggering an inflammatory response) as causes of D O M S have been postulated [105, 134]. B a s e d on a review of the avai lable publ ished literature, it appears that D O M S is a result of two processes , a mechanica l and biochemical p rocess [102, 132]. A sequence of events representing these two p rocesses during fatiguing exerc ise is outlined in Figure 2 [101,126, 135]. \ 3 2 Figure 2: S e q u e n c e of events assoc ia ted with delayed onset musc le soreness , including mechanica l and b iochemical p rocesses . S e q u e n c e Of Events High Mechanical- Force macrophage disruption of —) structural proteins in muscle fibre and connective tissue attraction of monocytes structural — damage to sarcolemma degradations- structural components of contractile apparatus influx Ca2+ from- interstium to muscle fibre activation of proteolytic enzymes mitochondrial accumulation of ions i inhibition of cellular respiration accumulation of histamine and " kinins increased pressure from edema activation of nociceptors D O M S 'adapted from Appell et al126, Armstrong et al i101, 102 33 Stauber and co l leagues [136] have suggested that the pain and inflammation assoc ia ted with D O M S may be due to the swell ing and disruption of the extracellular matrix. In contrast, J o n e s et al [137] have reported the stiffness to be a result of connect ive t issue damage . Pyne [138, 139] & Ebbel ing and C la rkson [112] have suggested that mechan ica l stress (mechanical shear force production during exercise) and metabol ic st ress (disturbances in normal cel lular metabol ism provoked by exhaust ive exercise) account for exercise-init iated damage to skeletal musc le fibres. Within the connect ive t issue of the musc les are myel inated group III (A-delta) and unmyel inated group' IV (C) afferent receptors. The large myel inated group III f ibers are bel ieved to transmit "sharp" local ized pain, whereas the group IV fibers carry "dull", diffuse pain. Therefore, it s e e m s likely that the group IV receptors carry the sensat ion of D O M S s ince the pain is usually dull and diffuse, with the free nerve endings responding to mechan ica l and chemica l (metaboceptors) as well as noxious stimuli (nociceptors) [112]. Bradykinin, serotonin and histamine may activate the free nerve endings of nociceptors to produce soreness and pain exper ienced after exerc ise [138]. Warho l et al [140] reported considerable d isturbances in the contractile apparatus of the gastrocnemius muscle during competit ive marathon running. Z-band streaming, myofibrillar lysis and contracture bands were noted upon histological examinat ion. Furthermore, pathological changes occurred in the mitochondria, showing focal swell ing and crystall ine inclusions and the sa rco lemma and sarcotubular system showed dilatation and disruption [126, 141]. C r e n s h a w and co l leagues [103, 142] investigated whether D O M S of the vastus lateralis musc le was assoc ia ted with elevated intramuscular pressure. Intramuscular pressure (IMP) is def ined as the fluid pressure created by a muscle during contraction [142] and is correlated linearly with the force of contraction during isometric and isokinetic exerc ise [142]. B a s e d on their f indings, they first conc luded that D O M S of the vastus lateralis muscle is assoc ia ted with extensive intracellular swell ing and with elevated IMP [103], however in a follow-up study, they reported that IMP was not an etiologic indicator of D O M S [142]. Newham [143] suggested that 34 intramuscular pressures are raised in some , but not all, painful compartments and even when raised, follow a different time course to the pain that appears . Biochemical ly , a variety of cl inical measures have been assoc ia ted with D O M S . Musc le f ibres contain proteolytic enzymes that are re leased following injury and initiate degradat ion of lipid and protein structures in the injured cel l [144]. The presence of intramuscular enzymes in the blood has been considered to be indicative of damage to muscle f ibres, particularly to the sa rco lemma [101]. Numerous reports suggest the time courses of increased levels of p lasma enzymes are similar to the time course of D O M S following exerc ise and the intensity of so reness and level of p lasma enzymes are a lso correlated [107, 119, 145]. However, Donnel ly et al [146] has sugges ted that musc le enzyme re lease and muscle soreness are unrelated. This suggest ion was based on the f indings that decl ine in musc le strength and in 5 0 % endurance time did not differ between the first and second period (10 week gap) of the study, indicating 1) that the repeat bout effect for muscle enzyme re lease was not demonstrated and 2) musc le soreness reached the same level after both exerc ise bouts. In contrast, Armstrong et al [101] demonstrated in an animal model that elevat ions in p lasma enzymes might occur simultaneously with exerc ise- induced necrosis of skeletal musc le fibers. P l a s m a enzymes such as myoglobin (18,000-Da heme-containing oxygen carrier protein of skeletal musc le cel ls [147]), hydroxyprol ine, creatine k inase (80,000-Da enzyme found in large concentrat ions in musc le t issue [147]), and hyperkalemia have been indicative of musc le injury [101]. Increases in serum activities of enzymes glutamic-oxaloacet ic t ransaminase (GOT) , lactic dehydrogenase (LDH) and aspartate aminot ransaminase (AST) has also been reported to reflect musc le fibre damage involving increased membrane permeabil i ty [138, 139, 148-151]. Mair and co l leagues [152] have reported that an increase in myosin heavy chain (MHC) fragment p lasma concentrat ions is demonstrated after eccentr ic exerc ise. 3 5 Creat ine k inase (CK) is found almost exclusively in musc le t issue and is therefore cons idered the most common indicator of skeletal musc le damage [153]. The increase in serum or p lasma C K activity after exerc ise is de layed and the extent of delay depends upon the type of exerc ise [112]. To explain this delay in C K re lease, C larkson and Tremblay [154] have theorized that exerc ise- induced damage may cause an accumulat ion of C a 2 + resulting in; a) production of noxious stimuli such as bradykinin and histamine caus ing musc le soreness , b) musc le contractures leading to dec reased range of motion, c) impairment of sarcop lasmic reticulum and mitochondrial functioning and d) activation of sarcop lasmic proteases resulting in loss of sarco lemmal integrity and delayed release of C K . Al though C K has been widely used as a clinical marker for damage to the musc le , the relationship between the magnitude of C K release and histological ev idence of the extent of musc le damage has not been establ ished [155]. Smith [156] proposed that the most likely chemica l stimulant for the induction of D O M S may be prostaglandin E 2 ( P G E 2 ) that c a u s e s increased sensitivity of pain receptors and that invading macrophage have the capabil ity of synthesiz ing P G E 2 [156]. Similarit ies in time course for increases in P G E 2 and D O M S (p<0.05), 24 hours after a bout of eccentr ic exerc ise was reported by Smith and col leagues [157]. Furthermore, Sa lminen and co l leagues [158] have demonstrated in an animal study that Indomethacin, a non-steroidal anti-inflammatory drug that inhibits prostaglandin synthesis, had reduced damage to the muscle in exerc ised mice. O n the other hand, Kuipers and co l leagues [159] found that prostaglandins are not involved in an exerc ise- induced inflammatory response s ince flurbiprofen (cyclo-oxygenase inhibiting drug) did not have any effect on muscle so reness . Smith and co l leagues [160] a lso studied whether there would be a reduction in total cholesterol levels in response to microtrauma induced by eccentr ic activity. Th is was founded on the notion that cholesterol is a component of the cell membrane and that cholesterol levels are temporari ly reduced in response to t rauma and post-surgery. Interestingly, the findings from this study showed a significant decrease in total cholesterol for both groups examined [160]. It has also been suggested that thiol proteases, such as 36 calpain, degrade structural proteins such as alpha-act inin, resulting in Z-l ine streaming and disorganizat ion of the normal al ignment of the myofi laments in condit ions of altered metabol ic and/or, functional demands [132]. In an animal model involving the rat hindlimb muscle, Belcastro and co l leagues [161] have reported increased calpain activity following level treadmill running, proposing that this increased activation of skeletal musc le calpain may result from increased intracellular ca lc ium. Appel l et al [126] showed that nifedipine, a ca lc ium channel blocker, diminished exerc ise- induced musc le damage in a mouse model , support ing the notion that C a 2 + ions are implicated in the mediat ion of t issue damage . Inflammation and Delayed-Onset Muscle Soreness It has been suggested that muscle so reness is related to an inflammatory response. Inflammation is the body's normal response to an insult, such as an injury, infection or antigen. The purpose of this response is to c lear and eliminate damaged t issue and microbial invaders, thus leading to t issue reparation. The main pathological feature of inflammation consis ts of leukocyte infiltration and exudation of p lasma into the lesion in the early stage fol lowed by proliferation of connect ive t issue including fibroblasts, which leads to the formation of granulation t issue. Two sub-classi f icat ions of inflammation exist. First and foremost is the acute inflammatory process with local and systemic changes character ized by a rapid change in blood flow and accompan ied by an immigration of neutrophils (histological hallmark of acute inflammation) and monocytes. Neutrophils are key nonspecif ic host defense cel ls responsible for phagocytosis of microbial, bacterial and viral pathogens [139, 162]. They play both efferent (phagocytosis and degranulation) and afferent (release of immunomodulatory molecules) roles in the immune response [139]. The cardinal s igns of redness, swel l ing, heat and pain; "rubor et tumor cum calore et dalore" (Cornelius Celsus) are noted in the acute- local inflammatory phase [163, 164]. In addit ion, the induction of fever 37 (due to the production of IL-1, T N F , IFN-alpha) and the production of p lasma proteins constitute the systemic reaction. Chronic inflammation is character ized by the presence of lymphocytes and monocytes [132]. Monocytes and macrophages are primarily responsib le for the removal of neutrophils and necrotic t issue [116, 132, 165]. After an insult occurs to the t issue, the body responds both at a vascu lar and cellular level. The former involves vasoconstr ict ion (5-10 minutes), fol lowed by vasodi lat ion and increased vascu lar permeabil i ty [156, 166]. The latter response involves the interaction of var ious inflammatory mediators, mainly neutrophils and monocytes. A short time after the t rauma occurs , circulating neutrophils dramatically increase in number, aggregating at the site of injury, reaching peak concentrat ions approximately 1-4 hours post-injury. Th is initial increase in neutrophils in the capil lary bed of the injured t issue is due to the slowing down of blood flow within the capil lary and the leak of p lasma proteins from the capil lary [132]. Smith et al [167] demonstrated a marked increase, above basel ine, in neutrophil levels between 1 and 2 hours post-exercise. It has been demonstrated that during th is .per iod, neutrophils become intimately assoc ia ted with the endothelial cel ls, upregulating adhes ion proteins (CD11 /CD18) that al low the neutrophils to role along the endothelial surface, become adherent and eventually migrating out into the t issue [132]. It is important to realize that only the presence of neutrophils in the interstitum or musc le is indicative of severe inflammation [132]. After this peak, the concentrat ion decl ines rapidly and is fol lowed by the migration of monocytes. Th is immigration rises in concentrat ion and is maintained for 48 hours, after which the monocytes mature into macrophage. Macrophages play a pivotal role in the recovery process following exerc ise- induced musc le injury [132]. These adult macrophage remove necrotic t issue and foreign bodies [156, 166]. Monocytes /macrophage are responsible for the resorption of neutrophils in necrotic t issue and the sequestrat ion of foreign material or ant igens [132]. Furthermore, they are capable of producing a wide variety of cytokines in large amounts, thus contributing to the cytokine network. In addit ion, they play a pivotal 38 role in the specif ic response of T and B- lymphocytes to antigen [132]. There is a great deal of ev idence suggest ing the role of monocytes /macrophage during eccentr ic exerc ise, both in animal and human models. Bendstrup [168] was the first to hypothesize that t issue damage resulting from intense exerc ise may trigger an inflammatory response and the time required for the response to occur expla ins the soreness delay [169]. It has been demonstrated that exerc ise- induced musc le injury triggers mobil ization of some aspec ts of the inflammatory response [132], however the specif ic events initiating this s e e m s somewhat unclear. It may be possib le that the inflammatory response may be responsible for initiating, amplifying and/or resolving skeletal musc le injury [132]. It has been demonstrated that cytokines play a role in the immune response following strenuous exerc ise. However, Northoff et al [234] has reported that changes in cytokine levels observed in serum or p lasma are a lways subtle, thus explaining signi f icance in s o m e studies while being borderl ine significant or undetectable in others. Cytok ines are essent ia l components of our defense and repair sys tems but a lso potentially harmful mediators of infectious and immunoinf lammatory reactions [245]. Cytok ines are re leased at the site of inflammation when there is a local response to an infection or t issue injury. They facilitate an influx of lymphocytes, neutrophils, monocytes and other cel ls into the t issue, and these cel ls participate in the c learance of the antigen and the heal ing of the t issue [187, 188]. Accompany ing this local inflammatory response is a systemic response known as the acute phase response. The "inflammatory" cytokines produced as a result of this response include tumor necrosis factor-a (TNF-a) , interleukin 1 (IL-1) and interleukin 6 (IL-6). IL-1 and T N F increase after exerc ise and induce the release of a third cytokine, IL-6. Both IL-1 and T N F have proinflammatory effects while IL-6 has been cited to be restorative in nature, with anti-inflammatory and immunosuppress ive effects [139, 231]. 39 First introduced in 1980 by W e i s s e n b a c h [235], IL-6 is a pleiotropic, "multifunctional" cytokine involved in the regulation of immune responses , the acute phase response (APR) and hematopoies is [238]. Many different cel ls produce it after stimulation during infection, t rauma or immunological chal lenge [231]. Its receptor system consis ts of two molecules: a l igand-binding 80 -kDa molecule and a non-l igand binding signal t ransducer, gp 130, both of which were found to belong to the cytokine receptor family [232]. A theoretical model of the immunological and inflammatory responses to exerc ise and musc le damage showing the central role of cytokines and neutrophils in the repair of damaged t issue was proposed by Pyne [138, 139] [Figure 3]. Severa l authors have reported that eccentr ic exerc ise caus ing musc le damage is assoc ia ted with an increase in serum IL-6 concentrat ions and this increase is significantly correlated with the concentrat ion of creatine k inase in the days following exerc ise [150, 187, 188, 189]. The time course of cytokine production, the c lose associat ion with musc le damage and the finding of increased IL-6 after intense exerc ise support the idea that during eccentr ic exerc ise, myofibers are mechanical ly damaged , thus stimulating the local production of inflammatory cytokines [187]. Rohde et al [188] have reported that eccentr ic exerc ise induced an increase in p lasma concentrat ions of IL-6 by 570%, 2 hours post-exercise and return to pre-exerc ise levels by day 2. Bauer et al [233] have cited that elevated levels of IL-6 can be found as early as a few hours after the onset of a pathogenic event and may persist for only a few hours or up to a few days. Furthermore, Bruunsgaard et al [189] demonstrated that IL-6 levels increased five-fold and C K levels increased almost 40 fold, 4 days post-eccentr ic exerc ise. Furthermore, IL-6 s e e m s to be the one cytokine that provides the most reliable results, being elevated shortly after strenuous exerc ise [234]. 40 Figure 3: Theoretical model showing role of cytokines and neutrophils during exercise inducing damage to skeletal muscle , 1 3 9 . Theoretical Model Showing Role Cytokines & Neutrophils Mechanical Stress pain macrophage Exercise Oxidative Damage Cytokines — • (IL-1, 2, 6, TNF) 1 acute phase response I — chemoattractants i neutrophils I repair tissue damage — 1 growth/repair/restorative Metabolic Stress _^ hormonal activation .'"adapted from Pyne DB [138, 139) 41 Free Radicals and Exercise-Induced Muscle Damage During intense exerc ise, whole body oxygen uptake can increase 20-fold above resting levels and in active musc le f ibers, oxygen consumpt ion may rise 200-fold [237, 238]. It has been est imated that 4 - 5 % of the oxygen consumed during respiration is not completely reduced to water, instead forming free radicals [240]. A growing amount of ev idence indicates that free radicals play an important role as mediators of skeletal musc le damage and inflammation [171]. Free radicals are chemica l spec ies with one or more unpaired electrons in their outer orbit making them highly reactive [112] s ince they strive to balance their unpaired electrons by combin ing with electrons with opposite sp ins in other subs tances [239]. Oxygen free radicals are increased during exerc ise as a result of increases in mitochondrial oxygen consumpt ion and electron transport flux, inducing lipid peroxidation [239] [Table 9]. The cascade of lipid peroxidation is characterist ic of inflammation in D O M S . Lipid peroxidation initiated by free radicals dec reases the barrier function of cell membranes and may b e a s s o c i a t e d with musc le fibre necros is and enzyme re lease following damaging exerc ise [112]. During exerc ise, two potentially harmful free radical generating sources are semiquinone (in the mitochondria) and xanthine ox idase (in the capil lary endothelial cel ls). \ W h e n the microcirculation is damaged , free radical formation may activate proteolytic enzymes [126, 229, 230]. React ive oxygen spec ies are a lso activated during ischemia/hypoxia and subsequent reperfusion/oxygenation in skeletal musc le [229]. Degradat ion of adenos ine tr iphosphate (ATP) forms xanthine, which leads to a c a s c a d e effect on xanthine dehydrogenase, xanthine ox idase and uric ac id , caus ing malfunctioning of the ion pumps and increasing intracellular levels of calc ium [111, 229]. The resulting effect is the generat ion of oxygen free radicals, which induce disruption of phosphol ipid layers and lipid peroxidation [229]. 4 2 Sjodin et al [171] have proposed that high intensity exerc ise increases the flow of oxygen through the skeletal musc les , caus ing metabol ic st ress and b iochemical changes leading the skeletal musc le damage and inflammation [Figure 4]. Controversy a lso exists on the role of hyperbaric oxygen therapy in free radical- mediated t issue injury. Hyperbaric oxygen has been shown to enhance the antioxidative defense mechan isms in some animal models , but it has a lso been reported to increase the production of oxygen free radicals [193]. Severa l authors have reported that oxygen under certain condit ions can generate highly reactive free radicals that mediate t issue injury and impair the process of wound heal ing while others have reported that hyperoxia increases the b iochemical defense mechan isms against free radicals [193]. To a s s e s s the level of lipid peroxidation that occurs during oxidative st ress on t issue, several reliable, analytical methods are avai lable. The assessmen t of malondia ldehyde (MDA) , a product of lipid peroxidation, has become the most common technique to measure the degree of oxidative damage in biological sys tems [190]. Malondia ldehyde is one of severa l low-molecular weight end products formed v ia the decomposi t ion of certain primary and secondary lipid peroxidation products [191, 192]. M D A , which may exist in a free form or as a complex with var ious t issue constituents, is formed during the oxidative degradation of some macromolecules, as a product of free radical generation by ionizing radiation in vivo, and as a by-product of prostaglandin biosynthesis [190]. M D A is formed during the last s tages of the breakdown of endoperox ides formed during intramolecular rearrangements in the structure of. polyunsaturated fatty ac ids [190]. A m o n g the various methods to evaluate malondia ldehyde, which include direct spectrophotometry or high-pressure liquid chromatography, the reaction with thiobarbituric acid (TBA) to form a colored adduct appears as a more rapid, inexpensive and sensit ive technique [190, 191]. The sample under investigation is heated with T B A at low p H , and a pink chromogen (a T B A 2 - 43 malondia ldehyde adduct) is measured by its absorbance at or c lose to 532 nm or by f luorescence at 553 nm [236]. The f luorescence technique will be appl ied for our purposes in examining lipid peroxidation resulting from exerc ise- induced musc le damage. > Tab le 9: Mechan ism by which exerc ise generates free radicals * • Increases in epinephrine and other catecholamines that can produce oxygen radicals when they are metabolical ly inactivated • Product ion of lactic acid that can convert a weakly damaging free radical (superoxide) into a strongly damaging one (hydroxyl) • Inflammatory responses to secondary musc le damage incurred with overexertion *Clarkson PM, et al 2000 44 Figure 4: B iochemica l mechan ism for oxygen-free radical formation resulting in skeletal musc le damage and inflammation during exerc ise Biochemical Mechanism For Oxygen-Free Radical Formation During Exercise High intensity exercise — Increased flow of ~ " oxygen thru skeletal muscle cells Skeletal muscle damage and inflammation (lipid peroxidation of cell membrane) Cellular necrosis ATP utilization — greater than ATP generation Loss of cell viability 4* Metabolic Stress ™ Biochemical changes — Attack of free radicals on <- cell membrane Increase in oxygen free radical Decrease in cellular defense system *adapted from Sjodin etal[171] 4 5 <- Training and Adaptation Effect A training and adaptation response has been reported in the literature. S c h w a n e et al [145] suggested that training could reduce the magnitude of pathological alterations that occur after eccentr ic exerc ise. Newham et al [131] further suggested a few possib le explanation for this training effect: 1) there is a change in the pattern of motor unit recruitment (i.e. either suscept ib le f ibres are spared on second and subsequent occas ion or more fibres are recruited and the force- fibre ratio is reduced), 2) there may be musc le fibre adaptation so that they become more resistant to the fatiguing and damaging effects of eccentr ic exerc ise and 3) the first bout of exerc ise had caused damage and destruction to a population of suscept ible fibres, possibly those near the end of their life cyc le. The time between exerc ise bouts would then be sufficient for regeneration thus making avai lable a fibre population of high mechanica l resistance [126]. Byrnes et al [172] and N o s a k a et al [173] found that the effect of training on reducing indicators of eccentr ic exerc ise- induced musc le damage might be observed after only a single exerc ise bout. Simi lar results were also evident showing that training a lso reduces the increase in serum C K and myoglobin, upon repeat bouts of activity [112, 172, 173]. Byrnes et al [172] suggest that the mechan isms responsible for soreness and enzyme re lease are the same but the magnitude of the change between repeat exerc ise sess ions can be altered by the performance on the first bout of work. In addit ion, C larkson and co l leagues [127] showed a dec rease in response to a second bout of exerc ise after a 9 week separat ion between exerc ise while Byrnes et al [172] and N o s a k a et al [173] reported that this prophylactic effect on the generation of musc le soreness and serum protein response may last up to 10 weeks . 2.6 The Role of Magnetic Resonance Imaging in the Detection of DOMS Discovered in the 1940's, the phenomenon of nuclear magnet ic resonance (NMR) has become a valuable instrument for the non-invasive study of muscle bioenerget ics during exerc ise [176]. N M R can be used to produce high quality images using magnetic resonance imaging (MRI) and has been useful in 4 6 examining healthy and d iseased skeletal musc le [174]. MRI , used to study protons, uses large volume radiofrequency coi ls and gradient magnet ic f ields that allow 2D and 3D images to be constructed [174]. The intensity of the generated images depends on the rate of relaxation of the protons in the sample area. Observ ing different types of nuc leus relaxation, namely T1and T2 relaxation, can generate images of varying contrast [174]. T2-weighted proton images have been particularly useful in detecting dif ferences in the chemica l environment of cel lular water, although T1 relaxation has a lso been investigated. Exerc ise changes the chemica l properties of water molecu les in musc le (e.g. increase in total water content [extracellular fluid]). The resulting effect is longer T1 and T2 relaxation t imes and brighter s ignals within musc le immediately following exercise [177-179]. Furthermore, the magnitude of these immediate post-exercise changes have been shown to be linearly related to exerc ise intensity [177-179]. F leckenste in et al [179] found that after sports- related musc le injuries, delayed increases in musc le T2 relaxation t imes and signal intensities were demonstrated and that this outlasted all other indicators of musc le injury. Th is has also been reported in numerous other studies of eccentr ic exerc ise [180-183]. Nurenburg et al [182] has suggested that the sensitivity of MRI-guided biopsy for the detection of exerc ise induced musc le damage is by far more accurate in determining the extent and location of muscle injury than b iopsies guided by D O M S . T h e s e investigators cite that there is a poor correlation between D O M S and C K and the extent of ultrastructural musc le injury but that there is a good correlation between signal intensity grades by MRI and the degree of ultrastructural damage. Severa l studies have used MRI to investigate changes in cross sect ional area of muscle following a protocol of lengthening contractions [181, 183]. These studies demonstrate that the cross sect ional a rea of muscle increases in a de layed manner with a time course similar to the changes in signal intensity and T2 relaxation in muscle. 47 Funct ional MRI refers to imaging not only the anatomy of a t issue but a lso the extent to which the t issue is involved in performing some task [226]. Musc le functional magnet ic resonance imaging is used to compare the relative involvement of different musc les recruited during exerc ise. Th is method relies on the activity-induced increase in the nuclear magnet ic t ransverse relaxation time (T2) of the musc le water, which is caused by osmotical ly driven shifts of fluid into the myofibrillar s p a c e [226]. In addition to imaging of whole musc le recruitment, musc le MRI may reveal changes in motor unit organizat ion during d i sease [226]. The availability and sophist icat ion of magnet ic resonance imaging scanners have increased enormously during the last decade and has now become the method of choice for cl inical imaging of most soft t issue pathologies, including sports- related injuries of musc le and joints [226]. B e c a u s e of its noninvasive technique and its independence from ionizing radiation, researchers now use this imaging technique for basic and appl ied morphometric studies of human subjects. For the purpose of our study, MRI can easi ly measure the effects of exerc ise training on musc le and fat vo lume and the inflammation assoc ia ted with delayed-onset muscle soreness . 2.7 Perceived Muscle Soreness and the Visual Analog Scale Pain is very subjective in nature, thereby making it very difficult to objectively measure and quantify. The visual analog sca le is one measure for quantifying pain [155, 195-197, 199, 201]. It consists of a 10 cm long line with marked endings indicating no pain at one end and extreme pain at the other end [ A P P E N D I X B]. Th is line may be horizontal or vertical in nature with both showing a high correlation (r=0.99) [200]. In order to quantify and objectively measure pain, subjects are asked to place a mark along this line with respect to their level of pain at the time of measurement . This distance is then measured from one extreme of no pain to the area marked. Although a great deal of crit icism exists in the literature with respect to the high variability inherent in this pain rating sys tem, the visual analog sca le has been found to bring greater 4 8 sensitivity and statistical power to data collection and analys is by al lowing a broader range of responses than traditional categorical responses . It a lso removes the bias brought on by examiner quest ioning and al lows for graphical temporal compar isons, thus minimizing bias and boost ing statistical power [155, 198]. 49 C H A P T E R 3: M E T H O D O L O G Y 3.1 Experimental design The experiment was divided into four s tages: • STAGE 1 (baseline/pre-exercise) Evaluation period - Height - Weight - Perceived muscle soreness (VAS) - Eccentric strength (torque) assessment - Quadricep circumference - Blood analysis - Creatine Kinase - Interleukin 6 - Malondialdehyde - Magnetic resonance imaging • STAGE 2 (eccentric exercise protocol/DOMS) Familiarization and warm-up period Exercise period (inducing muscle injury (DOMS)) • STAGE 3 (treatment) Treatment period (HBO therapy) • STAGE 4 (post-exercise evaluation - days 2-5) Evaluation period (post-exercise) - Height - Weight - Perceived muscle soreness (VAS) - Eccentric strength (torque) assessment - Quadricep circumference - Blood analysis - Creatine Kinase - Interleukin 6 - Malondialdehyde - Magnetic resonance imaging (days 1, 3 and 5) 50 1ft Q o _. °- 3 •? E oo — </) Q N " >- a 3 < x: £ a 4 UJ < CQ ro a z w U J _ j L u < CL CC UJ < Q > LU -I CL < X X X X X X X X X X X X X X X X X X X X X X X CD o 0) < a5 Q p CD o - a . * £ 8 9. c n ' i = C TD CU CD O — 3 O 11 0- CO O 00 l7rT' c -2 CO a) c CL g UJ CD co 2 Q . O CD 3.2 Subjects Sixteen female volunteers between the ages 18-40 participated in this study. Subjects were recreationally active with no weight training, running, or team sports as part of their physical regimen. Any previous exper ience with eccentr ic exerc ise would have caused adaptation of the musc le such that the muscle would have been more resistant to the effects of subsequent bouts of intense exerc ise. Subjects did not engage in physical activity for more than 3 hours a week and had to have met both inclusion and exclusion criteria to participate. Exc lus ion from participation in the study included athletes who actively weight train, run, jog, play in team sports, and/or ski . These activities involve repetitive eccentr ic loading of the quadr iceps and therefore the eccentr ic exerc ise would not have produced the desired effect of D O M S . In addit ion, individuals who had exper ienced delayed-onset musc le soreness to their quadr iceps in the previous three months prior to participating or who had a past history of severe joint injury, arthritis or other chronic i l lnesses were exc luded. Subjects taking ana lges ics or prescription drugs were also exc luded. Contraindicat ions to hyperbaric oxygen were a lso a s s e s s e d ( H B O contraindications: diabetes, lung cysts, epi lepsy, upper respiratory tract infections, pregnancy, fever). After recruitment to the study, the subjects who met the above criteria were then required to carefully read and sign a consent form and fill out a questionnaire that was approved by the University of British Co lumb ia Cl in ical Ethics Commit tee for research involving human subjects ( A P P E N D I X A) . 3.3 Procedure The research protocol was a randomized double-bl ind des ign. Subjects were brought into the Al lan McGav in Sport Medic ine Cl in ic where they were screened and examined thoroughly to ensure that all the inclusion and exclusion criteria had been met and that they were not at any risk by undergoing H B O treatment. The subjects were then randomly ass igned to one of two groups: a control (N=8) 52 and experimental group (N=8) and bl inded to their specif ic treatment and group assignment . Eccentr ic torque (strength), perceived musc le so reness and quadr icep c i rcumference measurements were taken at basel ine (Day 1) and after each of the four treatment sess ions (i.e. Days 2-5). B lood samp les were col lected by antecubital venipuncture for the purpose of serum creatine k inase, interleukin- 6 and malondia ldehyde assessmen t at basel ine (Day 1), 4-hours post-exercise (Day 2) and each day following treatment sess ions (Day 3, 4, 5). Magnet ic resonance images were col lected at basel ine (Day 1), 24-hours post-exercise (Day 3) and 72-hours (Day 5) post-exerc ise. STAGE 1 Initially subjects were given a brief explanat ion of the experimental process. The subjects were then evaluated pre-exerc ise for perceived musc le soreness , eccentr ic musc le strength (torque) and quadr icep c i rcumference. B lood samp les were col lected to a s s e s s levels for C K , IL-6 and M D A and an MRI was taken of their quadr icep musc les (both dominant and non-dominant). The height and weight of each subject was also recorded daily. Perceived muscle soreness Subjects were requested to give a subjective rating of musc le so reness in their quadr icep of the non-dominant leg. The testing occurred pre-exerc ise and immediately after each hyperbaric exposure on days 2-5. They were instructed to complete four deep knee bends and then rate the soreness they exper ienced during these squats. The ratings were completed on the visual analog sca le (VAS) . The V A S is a 10cm line with "no pain or discomfort" (e.g. 0) at one end of the line and "worst pain or discomfort" (e.g. 10) at the other end . The subjects were given a form and asked to place a mark on the line as to where they felt the level of perceived muscle soreness ( A P P E N D I X B). The V A S was used to record the subjects' perception of so reness of the quadr icep on the exerc ised leg due to exerc ised- induced injury and not so reness exper ienced upon recollection of past injuries. 53 Eccentric strength Isokinetic strength was measured using the K i n C o m Dynamometer . The K i n C o m Dynamometer (Chattecx Corp . , Chat tanooga, Tennessee) is a hydraulically powered, computer control led exercise-test ing device. This apparatus was used to measure and record the eccentr ic torque (Newton - metres, Nm) of the quadr icep musc le as well as create the exerc ise- induced musc le so reness ( D O M S ) in the subjects. Subjects were first instructed to ride a stationary bike for 5 minutes fol lowed by a stretching exerc ise (lunges) before being seated on the exerc ise equipment. The subjects were then instructed to sit on the isokinetic dynamometer, with their hips at 80°, their back supported and their pelvis stabi l ized on the bench with strapping (fastening all three seatbelts (waist belt, left shoulder and right shoulder belt) to limit movement during the testing protocol). The researcher set the lever arm length of the K i n C o m dynamometer for each subject to 7 5 % of the length from the head of the fibula to the lateral mal leolus with the lateral joint line of the knee in al ignment with the center of the rotational axis point of the machine. Secur ing the test leg on the upper third of the quadricep and the lower leg by a tight Velcro strapped shin pad ensured stabil ization and limited the movement of the subjects ' leg during the exerc ise protocol. They were further instructed to hold the s ides of the seat for addit ional stability. The angular velocity was set at 30° through a range of 60° at a long musc le length (110 - 35° of knee flexion). The subjects were then given a practice trial consist ing of three submaximal and one maximal contraction (both concentr ic and eccentr ic contractions), fol lowed by four maximal test contractions. A 2-minute rest period occurred between the practice and test contract ions, with the practice serving as the warm-up for each test sess ion . For the purpose of this study, only eccentr ic torque of the knee extensors was col lected. The basel ine mean torque value was col lected from the average of three maximal efforts of the four repetitions. 54 Quadricep circumference The measurements of quadr iceps c i rcumference were taken before the eccentr ic exerc ise protocol and after each treatment sess ions using a standard anthropometric Gul ick measur ing tape (JUZO®). The s a m e Gul ick measur ing tape was used throughout the entire testing protocol and the same s ide of tape was used consistently (i.e. the centimetre side of the tape would be on the superior s ide of the quadricep). Establ ished landmarks were identified at the 10 and 20cm point above the superior border of the patel la, as the subject lay supine on an examining table. A permanent, waterproof pen mark was p laced at these two points so that the investigator could measure the s a m e points throughout the testing period and ensures accuracy over the five days. This mark was reinforced on a daily bas is . A mean of two measurements was obtained during every testing period. Th is measurement was used to evaluate changes in the c i rcumference of the quadricep muscle, indicating the presence of edema . Blood analysis Thirty minutes after each treatment sess ion , subjects were driven to St. Pau l ' s Hospital (SPH) where twelve millilitres (mi's) of blood was withdrawn each day, during the five day testing period. Tra ined hospital laboratory technic ians col lected blood samp les by standard antecubital venipuncture. S ix milliliters of blood was col lected in an S S T (serum separator) tube for C K and M D A analys is and the remaining six milliliters was col lected in an E D T A (ethylene diamine tetra-acetic acid) tube for IL-6 analysis. The blood samples were then spun to isolate the p lasma, separated in 1.5 ml cryotubes and frozen at - 7 0 ° C . Ana lyses of all blood measurements were conducted at the phlebotomory laboratory at St. Pau l ' s Hospital and Vancouver Hospital & Health Sc iences Centre. The levels for each parameter (i.e. CK /MDA/ IL -6 ) were carefully quantified and measured by highly qualif ied technic ians in the laboratory as well as using state-of-the-art measurement dev ices (e.g. instruments measur ing reflection densit ies, f luorescence, etc.). 55 Quality control was maintained for all b lood ana lyses in the study. Th is al lowed the investigator to be certain that the a s s a y was reproducible and va lues obtained were consistent on a day-to-day bas is . Cal ibrat ion of instruments used in the laboratory is conducted every six months or when changes occur to the sl ide generator (reagent s l ides of the instrument). Adjustments to the instrument are made internally by the computer operating sys tem. Internal Quality Control (IQC) and checks are carried out three t imes a day and are. subsequent ly monitored on a monthly bas is . Evaluat ions are sent out for compar ison with other laboratories worldwide to further ensure reliability and validity of results. External Quality Control ( E Q C ) is monitored by CEQAL®' which is mandated by the B C Medica l Assoc ia t ion, Department of Diagnost ic Accreditat ion Program. Subscr ibers from C a n a d a and the United States provide samp les every 2 months and evaluat ions are p rocessed with the results, including statistical ana lyses , being reported. Finally, the laboratory is fully accredi ted, with a f ive-year accreditation period. To ensure reliability and validity of M D A samples , a standard curve was constructed prior to a s s a y ana lyses . Us ing a standardized curve ensured good reproducibility and precision of results on a daily bas is . Al l samples were ana lyzed in a batch to further ensure reliability of the results. In addit ion, every subject served as their own control to examine changes of malondialdehyde levels post-injury, therefore showing lipid peroxidation changes over the f ive-day treatment period. Creatine Kinase Analys is of C K activity in serum p lasma was conducted using the Vitros Chemistry Cal ibrator Kit 3™ and the Vitros C K Sl ide. The Vitros C K Sl ide is a dry, multi layered, analytical element coated on a polyester support. A n 11 uL drop of sample is deposi ted on the slide and evenly distributed by spreading the layer to the underlying layers. This layer contains N-acetylcysteine (NAC) to activate C K without pretreating the sample. W h e n the sample is deposi ted on the sl ide, creatine k inase cata lyzes the convers ion of creatine phosphate and A D P to creatine and A T P . In the presence of glycerol k inase (GK), glycerol is 56 phosphorylated to L-a-g lycerophosphate by A T P . Oxidat ion of L-oc- glycerophosphate to dihydroxyacetone phosphate and hydrogen peroxide occurs in the presence of L-a-g lycerophosphate ox idase (oc-GPO). Finally leuco dye is oxid ized by hydrogen peroxide in the presence of perox idase to form a dye. Reflect ion densit ies are monitored during incubation and the rate of change in reflection density is then converted to C K enzyme activity. Interleukin-6 Serum IL-6 was ana lyzed using a standard E L I S A kit obtained by ChemiKine™. This kit is a sandwich enzyme immunoassay (EIA), which measures the "free" forms of the cytokine IL-6. With this assay sys tem, pre-coated mouse monoclonal antibodies generated against human IL-6 are used to capture human IL-6 in a sample. Simul taneously, IL-6 specif ic rabbit polyclonal antibodies detect IL-6 in the sample. With the addition of goat anti-rabbit conjugated-alkal ine • phosphatase (which binds to the rabbit anti-human polyclonal cytokine antibody), fol lowed by the addition of the suppl ied color generat ing solut ion, the amount of IL-6 is detected. Malondialdehyde Finally, the analysis of serum p lasma for M D A levels was completed using the standard T B A assay and fluorimetric analys is. Tetramethoxy propane was diluted with ethanol. A n aliquot (250 ul) with distil led water was treated with 1.5 ml of 2 0 % trichloroacetic ac id (TCA) and then mixed with 1.5 ml of 0 .67% thiobarbituric acid solution. The mixture was heated for 30 minutes and then spun at 2500 rpm for 10 minutes. Relat ive f luorescence intensity of the reaction product was measured at 515 nm excitation and 553 nm emiss ion . Magnetic resonance imaging All s c a n s were performed on a 1.5 tesla S iemens Symphony MRI sys tem. The patients were posit ioned supine, and centered on the MRI scanning table, with the legs adducted. Corona l and axial scout images were obtained for initial 57 local izat ion. Axia l images were obtained from the level of the lesser trochanter to the superior pole of the patel la. The field of view included both thighs. The posit ioning and sl ice locations were identical for all subjects and scans . The body coil was used for signal reception. T2 relaxation time and Short Tip Inversion Recovery (STIR) images were a s s e s s e d . Sur face coi ls of the radiofrequehcy sys tem (i.e. body array coil) for magnet ic resonance imaging were tested and rotated on a daily basis to maintain quality control for this measurement . The pulse sequences were axial T 2 , axial ST IR and coronal ST IR. The axial T2 images used a T R 5500 msec , T E 110 msec , echo train 11, matrix 256 by 256, 10 mm sl ice th ickness, 5 mm sl ice gap, one signal averaged. The axial STIR images used a T R 4300 msec , T E 30 msec , T l 160 msec , echo train 18, matrix 256 by 256, 10 mm slice th ickness, 5 mm slice gap, one signal averaged. The coronal ST IR images used the s a m e parameters as the axial STIR images, except that the sl ice th ickness was 5 mm, with no interslice gap. The images were ana lyzed on a P C - b a s e d computer workstation using eFi lm (eFilm 1.5.0 software version). Th is workstation software al lows s imul taneous viewing, windowing, and measurements to be performed on multiple s l ices from multiple imaging ser ies. For measurement of muscle signal intensity, the images from the 24-hour post exerc ise scan were visually inspected to identify any areas of increased muscle signal (indicative of edema). The area of maximal edema was identified. A n oval region of interest was manually t raced around the area of maximal musc le edema. The region of interest was greater that 1cm in area, thus it represented at least 1 cc vo lume of musc le t issue. C a r e was taken to not include non-musc le t issues such as l igament, blood vesse ls , or fat in the region of interest. The corresponding area was identified in the opposite (dominant) leg, and in the identical anatomic location of the left leg on the first and third MRI scans . The signal intensity of the muscle was recorded. The ratio of signal intensity of the exerc ised (non-dominant)/signal intensity of the identical anatomic location in the dominant leg was calculated. The same process was repeated for 58 all three musc les : rectus femoris musc le , vastus intermedius and vastus lateralis musc le . A subjective score based on visual assessmen t was also recorded: 0 = no visible edema , 1 = minimal musc le edema , 2 = moderate musc le edema , 3 = marked musc le e d e m a . STAGE 2 Delayed-onset musc le so reness was induced on the K i n C o m Dynamometer in the quadricep musc le of the non-dominant leg. Subjects were seated on the exerc ise equipment as outlined in stage 1. The subjects were given a warm-up sess ion to acquaint them with the eccentr ic exerc ise. Th is warm-up sess ion consis ted of one set of 10 repetitions at a submaximal effort. After this warm-up period, subjects were instructed to perform repeated eccentr ic contract ions of their non-dominant leg (110° - 35° of knee flexion) at a s low speed (30° per second) on the K i n C o m Dynamometer . The subject were instructed not to resist the concentr ic movement on the way up but to resist the mach ine 's eccentr ic force on the way down. The exerc ise required the complet ion of 300 maximal voluntary eccentr ic contractions. The subjects completed 30 sets of 10 repetitions with each set beginning every minute for 30 minutes, al lowing for a 15 second rest between each set. They received verbal feedback from the researcher (verbally encouraged to ensure that they are giving maximal effort) as well as biofeedback from the resistive force or force versus velocity curve displayed on the K i n C o m monitoring unit. STAGE 3 Immediately after the exerc ise protocol and for the following 3 days, subjects were exposed to a hyperbaric environment (a total of 4 hyperbaric/normoxic exposures) . Subjects were required to read a two-page information sheet on the hyperbaric unit and procedures for compress ion and decompress ion , prior to the first treatment sess ion . Subjects were a lso required to try the avai lable aviator- style gas mask to ensure a comfortable fit and to ensure that a tight sea l was maintained between the mouth and nose. This mask was steri l ized after each 5 9 treatment sess ion to make sure that proper hygiene was maintained throughout the study period. Emergency equipment and clearing techniques were a lso expla ined (i.e. swal lowing, va lsa lva manoeuvre, etc). Subjects were verbal ly instructed as to how they could decompress the chamber on their own and let themselves out if necessary . A microphone in the chamber was continually open and pointed out to the subjects to allow them to communicate any problems that they may exper ience during compress ion . Control group For each treatment sess ion , the subjects were seated inside the H Y O X monoplace chamber (Aberdeen, Scot land). The chamber was then compressed to a pressure of 1.2 A T A (an increase of 140 mm Hg). During compress ion , the subjects breathed the ambient air in the chamber. O n c e at a pressure of 1.2 A T A , the subjects were instructed to wear the gas mask, inspiring normoxic air (21% oxygen). The chamber was then reduced to barometric pressure for the remainder of the 60-minute treatment sess ion . After 60 minutes, the subjects were instructed to remove the mask and again began breathing the ambient air of the chamber while decompress ion was initiated. This increase in pressure to 1.2 A T A was sufficient to cause the subjects to exper ience the .common tympanic membrane sensat ions assoc ia ted with increasing ambient pressure. Therefore, it was unlikely for subjects to determine their group designat ion. The air was del ivered through a ser ies of regulators to the mask from high-pressure cyl inders external to the chamber. Experimental group The compress ion and decompress ion procedure for the hyperbaric oxygen group was identical with the procedures for the control group, with the except ion that this group was compressed to 2.0 A T A . O n c e at 2.0 A T A , the subjects in the experimental group received 100% oxygen, which was del ivered to the aviator- style mask from high-pressure cyl inders external to the chamber. 60 STAGE 4 The s a m e evaluation was performed as stage 1 after each treatment sess ion . Al l dependent var iables were measured immediately pos t -HBO/normox ic treatment sess ions (days 2-5). 3.4 Statistical Analysis The study design involved 2 groups (experimental and control group) with a number of dependent var iables (i.e. perceived soreness , eccentr ic strength, quadricep c i rcumference, blood enzymes and MRI) that was measured on a number of repeated occas ions . A two way (group x time) A N O V A was performed with repeated measures on the dependent var iables, with the except ion of perceived musc le pain ( V A S scores) . V A S scores were ana lyzed using a nonparametr ic test (Fr iedman test). For all statistical ana lyses, the significant level was set at p<0.05. Statistical ana lyses were performed using an IBM- compatible computer and S P S S 9.0 statistical software. 3.5 Statistical Power Power for the study (0.76) was based on calculat ions of Cohen ' s Dc (deita/s(i-r)1/z). Calculat ions were based on an a lpha level set at 0.05, a 2 0 % expected change, correlations of 0.6 and a standard deviation of 3 3 % for the individual var iables. These va lues were determined from previous literature and assumpt ions were based on clinical s ignif icance. 61 CHAPTER 4: RESULTS Anthropometric Data The mean physical characterist ics of the 16 subjects who completed the study are listed in Table 10. No significant dif ferences were detected between subjects in their height, weight and age (p<0.05). T a b l e 10: Phys ica l characterist ics for both groups (age, height and weight). Va lues reported as Mean ± S D . AGE (yr*) HEIGHT (cm) WEIGHT (kg) Control (n=8) 25.25 ± 4 . 1 0 162.31 ± 6 . 0 3 57.0 ± 12.8 Experimental (n=8) 25.49 ± 4.24 160.90 ± 3 . 2 7 58.5 ± 10.1 Note: no significant differences between groups on any of the variables Muscle Pain Resul ts for the rating of perceived so reness of the non-dominant leg are illustrated in Figure 5. Al l groups showed a significant increase in soreness after the exerc ise protocol (p<0.05, p=0.0001) but there was no statistical difference between groups for treatment effects (p=0.571). A significant interaction effect between treatment and time was evident (p<0.05, p=0.010). The control group showed an increase in pain from basel ine, at 4 hours post- exerc ise, peaking at 24 hours and 48 hours post-exerc ise and decreasing 72 hours post-insult ( A P P E N D I X C - Figure A) . The experimental group had pain levels increase from basel ine, 4 hours post-exerc ise, peaking at 24 hours and decreas ing 48 hours and further decreas ing 72 hours post-exercise ( A P P E N D I X C - Figure B) [Table 11]. Both groups exper ienced less pain over time even though the peak time period for pain differed between groups. 62 T a b l e 11: Average ratings of perceived so reness for the quadr icep muscle of the non-dominant leg before (baseline) and after (days 2, 3, 4, 5) the eccentric exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as visual ana log score (1-10) ± S E M . Baseline (Day 1) 4-Hours Post- Exercise 24 Hours Post- Exercise 48 Hours Post- Exercise 72 Hours Post- Exercise (Day 5) U « A w l V> 1 U v./ (Day 2) (Day 3) (Day 4) Control (n=8) 0.00 ± 0.00 2.38 ± 0.57 3.25 ± 0.45 3.38 ± 0.71 1.88 ±0.52 Experimental (n=8) 0.00 ± 0.00 2.75 ± 0.65 4.38 ± 0.57 2.12 ±0.44 0.25 ±0.16 Figure 5: Average rating of perceived soreness for the quadricep muscle of the non-dominant leg, according to the visual analog scale (range 1-10), before (baseline) and after hyperbaric/normoxic exposure. 5.0 4.0 3.0 2.0 LO H • Control Group Hi—Experimental Group Baseline 4 Hours Post-Ex 24 Hours Post-Ex Day(s) 48 Hours Post-Ex 72 Hours Post-Ex 63 Eccentric Strength Ana lys is of the mean eccentr ic torque indicated that there were significant di f ferences within the groups (p<0.05, p=0.0001); however, there was no statistical difference between groups and there was no significant interaction effect (p<0.05, p=0.102; p=0.100) (Figure 6). The control group showed eccentr ic torque to be significantly lower 24 hours post-exerc ise, with gradual recovery by day 5 (72 hours post-exercise) ( A P P E N D I X C - Figure C) . The experimental group demonstrated a decrement in eccentr ic torque at 4 hours post-exerc ise, fol lowed by gradual recovery 24, 48 and 72 hours post-exercise ( A P P E N D I X C - Figure D) [Table 12]. Both groups demonstrated immediate eccentr ic strength decrements, fol lowed by a gradual pattern of recovery. Table 12: Average maximal eccentr ic torque for the quadr icep musc le before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Strength values reported as maximal torque (Nm) ± S E M . Baseline (Day 11 4-Hours Post- Exercise (Dry 2) 24 Hours :;Rpst-Exercise (Day 3) 48 Hours Post-Exercise (Day 4) 72. Hours Post- Exercise f Day 5) Control (n=8) 136.0 + 13.75 91.0 + 9.02 87.25 + 9.92 95.38 ± 10.13 98.38 ± 1 1 . 1 0 Experimental (n=8) 142.38+7.44 109.50 ± 7 . 9 5 113.0 ± 7 . 8 2 125.38 ± 9 . 5 6 125.63 ± 8 . 4 9 64 Figure 6: Average maximal eccentric torque for the quadricep muscle, before the exercise protocol (baseline) and after hyperbaric/normoxic exposure. Quadricep Circumference Analys is of the quadricep c i rcumference at the 10cm reference point above the superior portion of the patella indicated that quadricep circumference was significantly different within the groups (p<0.05, p=0.005); however, there was no significant difference between groups and there was no interaction effect (p<0.05, p=0.815; p=0.939) (Figure 7). At the 20cm point above the knee, quadr icep circumference was not significantly different within the groups (p<0.05, p=0.253) and again there was no significant difference between groups. A lso , there was no interaction effect (p<0.05, p=0.677, p=0.676) (Figure 8). Both groups (control and experimental), for the 10cm measurement point, demonstrated a slight increase in e d e m a at 4 hours post-exercise, peaking at day 65 3 (24 hours post-exercise), decreas ing on day 4 (48 hours) and finally showing a further dec rease on day 5 (72 hours) post-exercise ( A P P E N D I X C - Figure E, F). The 20 cm measurement for both groups was slightly more variable. The control group had a slight decrease 4 hours post-exercise, increasing 24 and 48 hours post-insult and further decreas ing 72 hours post-exercise ( A P P E N D I X C - Figure G) . The experimental group showed a slight increase in c i rcumference 4 hours post-exerc ise, decreas ing below basel ine 24 hours post-insult, increasing 48 hours and finally decreas ing 72 hours post-exercise ( A P P E N D I X C - Figure H) [Table 13]. i T a b l e 13: Average quadricep c i rcumference measured at both the 10 and 20cm point above the superior portion of the patel la. Measurements were taken before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Quadr icep circumference reported as cent imetres (cm)) ± S E M . 10 cm Baseline (Day 1) 4-Hours Post- Exorciso (Day 2) 24 Hours Post- Exercise (Day 3) 48 Hours Post- Exercise (Day 4) 72 Hours Post- Exe-cise (Day 5) Control (n=8) 44.99 ± 1.93 45.10 ± 1.78 45.31 ±1 .71 44.82 ± 1.91 44:13 ± 1.86 Experimental (n=8) 45.42 ± 2.04 45.73 ± 1.99 45.96 ± 2 . 1 8 45.71 ± 2.05 44.79 ± 1 . 9 9 Baseline (Day 1) . 4-Hours 24 Hours 48 Hours on ^ P o s t - P'wt- : P o s t " 20 cm ,_ ,_ ,_ Exercise Exorcise Exorcifn (Day 2i (Day 3) (Day 4) 72 Hours Post- Exercise (Day 5) Control (n=8) 53.49 ± 1 . 9 6 52.97 ± 1.99 53.19 ± 1.99 53.21 ± 2.01 52.89 ± 2.01 Experimental (n=8) 54.49 ± 2 . 1 7 54.64 ± 2 . 2 8 54.38 ± 2.38 54.68 ± 2.28 53.91 ± 2 . 1 5 66 Figure 7: Average quadricep circumference (10 cm location), before eccentric exercise (baseline) and after hyperbaric/normoxic exposure. 42.0 -I 41.0 40.0 39.0 -•—Control Group - • — Experimental Group Baseline 4 Hours Post-Ex 24 Hours Post-Ex 48 Hours Post-Ex Day(s) 72 Hours Post-Ex F igure 8: Ave rage quad r i cep c i r c u m f e r e n c e (20 c m locat ion) , before eccen t r i c e x e r c i s e (base l ine) a n d after hype rba r i c / no rmox i c e x p o s u r e . a i » - — - - - — _ _ _ _ _ _ 1 | —i I • " 1 •Control Group -Experimental Group Baseline 4 Hours Post-Ex 24 Hours Post-Ex Day(s) 48 Hours Post-Ex 72 Hours Post-Ex 67 B l o o d E n z y m e s Creatine Kinase (CK) Ana lys is of the creatine k inase (CK) measurements indicated that C K was not significantly different within the two study groups (p<0.05, p=0.538). No significant difference was demonstrated between groups and there was no significant interaction effect (p<0.05, p=0.647; p=0.570) (Figure 9). The control group demonstrated an increase in C K levels 4 hours post-exerc ise, fol lowed by a decrease at 24 hours and 48 hours post-exerc ise, and an increase at 72 hours post-insult ( A P P E N D I X C - Figure I). The experimental group showed a marked decrease from basel ine at 4, 24, 48 and 72 hours post exerc ise ( A P P E N D I X C - Figure J) [Table 14]. O n e subject had an unusual ly high C K response be fo r^ the eccentr ic exerc ise protocol (>2500 uL). C K analys is was performed with and without this subject to examine whether the high variability introduced into the data by this subject was masking any significant statistical f indings. Remov ing this subject from the statistical analysis did not alter the overall f indings. Furthermore, removing this outlier from the data revealed a more stable curve, showing an increase from basel ine at 4 and 24 hours post- exerc ise, fol lowed by a gradual decrease at 48 and 72 hours post-injury [Table 14]. T a b l e 14: Mean serum creatine k inase va lues before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. C K va lues reported as microlitres (u./L) ± S E M . Baseline (Day1) 4-Hours Post- Exercise (Day 2) 24 Hours Post- Exercise (Day 3) 48 Hours Post- Exercise (Day 4) 72 Hours Post- Exercise (Day 5) Control (n=8) 150.50 ± 69.20 208.13 ±76.76 178.58 ±39.85 139.13 ±32.22 168.13 ±56.93 Experimental (n=8) 396.50 ±316.42 271.63 ± 147.45 192.63 ±52.75 143.88 ±25.89 124.50 ±22.38 Experimental (minus outlier) 80.14 ±7.27 125.43 ±22.11 145.30 ±26.87 120.57 ± 13.02 110.43 ±20.10 68 Figure 9: Average creatine kinase (CK) levels, before (baseline) and after hyperbaric/normoxic exposure. 800.0 700.0 600.0 500.0 400.0 300.0 200.0 i 100.0 •Control Group - Experimental Group Baseline 4 Hours Post-Ex 24 Hours Post-Ex Day(s) 48 Hours Post-Ex 72 Hours Post-Ex Malondialdehyde (MDA) Malondia ldehyde (MDA) results indicate no significant difference within the two study groups (p<0.05, p=0.08). No significant difference was demonstrated between groups for treatment effects and there were no significant interaction effects (p<0.05, p=0.58; p=0.56) (Figure 10). The control group demonstrated very little variability between basel ine values and post-exercise levels, increasing slightly days 3 and 4 and decreas ing c lose to basel ine by day 5 ( A P P E N D I X C - Figure K). The experimental group, however, increased day 2, dec reased slightly day 3, increased again on day 4 and slightly dec reased by day 5. The va lues for the experimental group remained slightly higher than what was observed at basel ine ( A P P E N D I X C - Figure L) [Table 15]. 69 T a b l e 15: Mean malondia ldehyde values before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. M D A va lues reported as nmoles M D A / m L ± S E M . Baseline (Day1) 4-Hours Post- Exercise (Day 2) 24 Hours Post- Exercise (Day 3) 48 Hours Post- Exercise (Day 4) 72 Hours Post- Exercise (Day 5) Control (n=8) 3.98 ± 0.48 4.0 ± 0.34 4.28 ± 0.35 4.34 ± 0.58 4.03 ± 0.36 Experimental (n=8) 4.26 ± 0.38 4.55 ± 0.47 4.46 ± 0.52 4.58 ± 0.43 4.46 ± 0.44 Figure 10: Average malondialdehyde (MDA) levels, before (baseline) and after hyperbaric/normoxic exposure . Normal levels 3.9-4.2 nmol MDA/mL • Control - » — Experimental Baseline 4 Hous Post-Ex 24 Hours Post-Ex 48 Hours Post-Ex 72 Hours Post-Ex Day(s) 70 Interleukin 6 (IL-6) Interleukin 6 (IL-6) results indicate no significant difference within the two groups (p<0.05, p=0.400). No significant difference was demonstrated between groups for treatment effects and there was no significant interaction effect (p<0.05, p=0.111; p=0.451) (Figure 11). Both groups demonstrated marked dif ferences in the pattern of recovery from exerc ise- induced musc le injury and there was a great deal of variability. The control group fluctuated markedly; decreas ing from basel ine, 4 hours post- exerc ise, increasing above basel ine 24 hours post-exercise, further decreas ing at 48 hours to levels similarly to those seen at 4 hours post-exercise and increasing, although below basel ine, by 72 hours post-insult ( A P P E N D I X C - Figure M). The experimental group was more stable with less variability and fluctuations in IL-6 levels. From basel ine, the IL-6 levels slightly decreased 4 hours post-exerc ise, remained at the same level 24 hours post-exercise, dec reased further by 48 hours and increased above basel ine levels by 72 hours post-exercise ( A P P E N D I X C - Figure N) [Table 16]. Table 16: Mean interleukin-6 va lues before (baseline) and after (days 2, 3, 4, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. IL-6 values reported as pg/mL ± S E M . Baseline .Day 1) 4-Hours Post- Exercise (Day 2) 24 Hours Post- Exercise (Day 3) 48 Hours 72 Hours Post- Post- Exercise Exercise (Day 5) (Day 4) Control (n=4) 618.13 ±517.87 67.11 ±57.83 873.19 ± 519.41 24.54 ± 11.71 459.26 ± 405.81 Experimental (n=4) 59.73 ±21.98 44.80 ± 14.33 46.98 ± 20.94 26.08 ± 5.75 86.23 ±21.03 71 Figure 11: Average interleukin-6 (IL-6) levels, before eccentric exercise (baseline) and after hyperbaric/normoxic exposure. 1 6 0 0 . 0 i 1 4 0 0 . 0 1 2 0 0 . 0 1 0 0 0 . 0 8 0 0 . 0 6 0 0 . 0 i 4 0 0 . 0 2 0 0 . 0 .0 i -•—Control Group HH— Experimental Group Normal levels <50 pg/ml Baseline 4 Hours Post-Ex 2 4 Hours Post-Ex 4 8 Hours Post-Ex 7 2 Hours Post-Ex Day(s) Magnetic Resonance Imagine (MRI) T2-weighted images Rectus Femoris Analys is of the rectus femoris musc le using T2 weighted imaging revealed that this muscle was not significantly different within the two groups (p<0.05, p=0.108). No significant difference was demonstrated between groups for treatment effects and there was no significant interaction effect (p<0.05, p=0.800; p=0.799) (Figure 12). The control group demonstrated very little variability between basel ine ratios and post-exercise ratios, revealing a slight increase by day 5 ( A P P E N D I X C - Figure O). The experimental group also showed very little variability between days 1 72 through 5, however a slight increase was noted on day 3, fol lowed by another increase on day 5 ( A P P E N D I X C - Figure P) [Table 17]. Table 17: Average T2 relaxation t imes of the rectus femoris muscle taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as T2 Relaxat ion T ime (msec) ± S E M . Day 1 (Baseline) Day 3 (24 hours post-exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.91 ±7.02 x ^0~'^ 0.92 ± 4.34 x 10"2 1.03 ±0.10 Experimental Group (n=8) 0.89 ± 6.13 x 10"2 0.97 ±0.10 1.09 ±0.19 Figure 12: Mean T2 relaxation times (msec) before (baseline) and after hyperbaric/normoxic exposure (days 3, 5) for the rectus femoris muscle o a> to E <D E c o ra x ro a DC 1.200 1.000 .800 .600 .400 - •— Experimental Group HI—Control Group .200 .000 Baseline 24 Hours Post-Ex Day(s) 72 Hours Post-Ex 73 Vastus Intermedius Analys is of the vastus intermedius musc le using T2 weighted imaging revealed that this musc le was significantly different within the two study groups (p<0.05, p=0.007) but no significant difference was evident between groups for treatment effects and group interaction effects (p<0.05, p=0.361; p=0.259) (Figure13). The control group demonstrated an increase in s ignal intensity 24 hours post- exerc ise, fol lowed by a further increase at 72 hours post-insult ( A P P E N D I X C - Figure Q) . The experimental group also showed an increase on day 3, but on day 5, a decrease was noted ( A P P E N D I X C - Figure R) [Table 18]. Tab le 18: Average T2 relaxation t imes of the vastus intermedius musc le taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as T2 Relaxat ion T ime (msec) ± S E M . Day 1 (Baseline) Day 3 (24 hours post-exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.95 ± 3.66 x 10" 1 .26±9 .16x10 1.36 ±0.15 Experimental Group (n=8) 0.96 ± 4.85 x10" 2 1.25 ±0.13 1.104 ±5.91 x 10"2 74 Figure 13: Mean T2 relaxation times (msec) before (baseline) and after hyperbaric/normoxic exposure (days 3, 5) for the vastus intermedius muscle. 1.600 1.400 1 1.200 fi 1 1.000 fi E c o a a CM .800 .600 - •— Experimental Group -•—Control Group .400 .200 .000 4- Baseline 24 Hours Post-Ex Day(s) 72 Hours Post-Ex Vastus Lateralis Analys is of the vastus lateralis muscle using T2 weighted imaging revealed that there was a significant difference within the groups for this muscle (p<0.05, p=0.038) but no significant difference was evident between groups for treatment effects and group interaction effects (p<0.05, p=0.806; p=0.258) (Figure 14). The control group demonstrated an increase in s ignal intensity 24 hours post- exerc ise, fol lowed by a slight decrease at 72 hours post-insult ( A P P E N D I X C - Figure S) . The experimental group also showed an increase 24 hours post-injury, fol lowed by a further slight increase 72 hours post-insult ( A P P E N D I X C - Figure T) [Table 19]. 75 T a b l e 19: Average T2 relaxation t imes of the vastus lateralis muscle taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as T2 Relaxat ion Time (msec) ± S E M Day 1 (Baseline) Day 3 (24 hours post-exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.88 ± 8.65 x 10~2 1.10±7.12x 10"2 1.05 ± 7.54 x 10"2 Experimental Group (n=8) 0.98 ± 7.75 x 10' 2 1.02 ± 7.55 x 10"2 1.09 ± 8.59 x 10"2 Figure 14: Mean T2 relaxation times (msec) before (baseline) and after hyperbaric/normoxic exposure (days 3, 5) for the vastus lateralis muscle. 76 STIR IMAGES Rectus Femoris Analys is of the rectus femoris musc le using STIR imaging demonstrated that there was a significant difference within the groups (p<0.05, p=0.018) but no significant difference was evident between groups for treatment effects and interaction effects (p<0.05, p=0.796; p=0.733) (Figure 15). Both control and experimental groups demonstrated a similar pattern over the 3 measurements taken over 5 days. The control group demonstrated an increase 24 and 72 hours post-exercise from basel ine measurements ( A P P E N D I X C - Figure U). The experimental group also showed an increase 24 hours post-injury, fol lowed by a further slight increase 72 hours post-insult ( A P P E N D I X C - Figure V) [Table 20]. Table 20: Average signal intensity ratio for STIR image of the rectus femoris musc le taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as signal intensity ratio - STIR (msec) ± S E M . ' Day 1 (Baseline) Day 3 (24 hours post- i exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.88 ± 5.95 x 10"2 1.00 ± 6.26 x 10"2 1.07±6.94x 10"J Experimental Group (n=8) 0.91 ±2.01 x 10"2 0.98 ±3.81 x 10"2 1.02 ± 9.06 x 10"2 / 77 Figure 15: Mean Signal Intensity Ratio for STIR image before (baseline) and after hyperbaric/normoxic exposure (days 3, 5) for the rectus femoris muscle. Vastus Intermedius Analys is of the vastus intermedius musc le using STIR imaging demonstrated that there was a significant difference within the groups (p<0.05, p=0.014) but no significant difference was evident for between groups for treatment effects and group interaction effects (p<0.05, p=0.580; p=0.451) (Figure 16). The control group demonstrated an increase in signal intensity 24 hours post- exerc ise, fol lowed by a slight increase at 72 hours post-insult ( A P P E N D I X C - Figure W) . The experimental group also showed an increase 24 hours post- exerc ise, fol lowed by a slight dec rease 72 hours after injury ( A P P E N D I X C - Figure X) [Table 21]. 78 Table 21: Average signal intensity ratio for STIR image of the vastus intermedius muscle taken before (baseline) and after (days 3, 5) the eccentric exercise protocol, following hyperbaric/normoxic exposure. Values reported as signal intensity ratio - STIR (msec) ± S E M . Day 1 (Baseline) Day 3 (24 hours post-exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.97 ± 2.96 x 10"2 1.07 ± 4.99 x 10"2 1.07 ± 5.87 x 10"2 Experimental Group (n=8) 0.97 ± 1.98 x10" 2 1.06 ± 5.73 x10" 2 1.00 ± 2.78 x 10"2 Figure 16: Mean Signal Intensity Ratio for STIR image before (baseline) and after hyperbaric/normoxic exposure (days 3, 5) for the vastus intermedius muscle. 1.150 .800 1 , Baseline 24 Hours Post-Ex 72 Hours Post-Ex Day(s) 79 Vastus Lateralis Analys is of the vastus lateralis musc le using STIR imaging demonstrated statistical s igni f icance within the groups (p<0.05, p=0.004) but no significant difference was evident between groups for treatment effects and group interaction effects (p<0.05, p=0.265; p=0.560) (Figure 17). The control group demonstrated an increase in signal intensity 24 hours post- exerc ise, fol lowed by a very slight dec rease at 72 hours post-insult ( A P P E N D I X C - Figure Y) . The experimental group a lso showed a slight increase 24 hours post-exerc ise, fol lowed by a further minimal increase 72 hours after injury ( A P P E N D I X C - Figure Z) [Table 22]. T a b l e 22: Average signal intensity ratio for ST IR image of the vastus lateralis muscle taken before (baseline) and after (days 3, 5) the eccentr ic exerc ise protocol, following hyperbaric/normoxic exposure. Va lues reported as signal intensity ratio - STIR (msec) ± S E M . Day 1 (Baseline) Day 3 (24 hours post- exercise) Day 5 (72 hours post exercise) Control Group (n=8) 0.88 ± 5.99 x 10"2 1.07 ± 6.54 x 10 - 1.06 ± 5.36 x10" 2 Experimental Group (n=8) i . 0 0 ± 3 . 6 3 x 10"2 1.09 ±4.71 x10" 2 1.11 ± 5 . 5 6 x 10"2 I 8 0 F igu re 17: M e a n Shor t T ip Invers ion R e c o v e r y (STIR) Ra t i os before (base l ine) and after hype rba r i c / no rmox i c e x p o s u r e (days 3, 5) for the v a s t u s latera l is m u s c l e . 1.200 -r 1.000 | .800 I .600 -•—Experimental Group -•—Control Group .400 .200 .000 Baseline 24 Hour Post-Ex Day(s) 72 Hours Post-Ex 81 CHAPTER 5: DISCUSSION The use of hyperbaric oxygen as a therapeutic modality is increasing among athletes, trainers, physiotherapists and other medical professionals. However, there is a paucity of scientif ic ev idence to prove the effect iveness of using hyperbaric oxygen as a means of treatment. Prev ious studies examining the use of hyperbaric oxygen to ameliorate exerc ise- induced D O M S did not convincingly prove or refute the eff icacy of hyperbaric oxygen in treating musc le and soft- t issue injuries. The aim of the present study was to investigate the influence of hyperbaric oxygen therapy on perceived musc le so reness , musc le strength, musc le edema and p lasma enzymes . The injury model that was used in this study, delayed-onset muscle so reness , al lowed us to induce a quantifiable musc le injury and then monitor severa l var iables over the course of recovery. Th is "gold-standard" injury model has been investigated in great detail for over 15 years and provides a good representation of musc le injury, damage and compromised force that may occur during sport and recreational activities [101-10, 105-106, 110-113, 127, 131]. However, caution should be appl ied when using this type of soft t issue injury model . It has been suggested by previous investigators [96, 246, 247] that although D O M S was created in the muscle of the subjects, the degree of musc le soreness may be at question, thus negating any beneficial effect the treatment modality may have in the rehabilitation process . This study elongated the musc le length of the quadr icep to 110°-35° to further ensure high tension to the musc le fibres. In addit ion, visual estimation for the presence of e d e m a was made 24 hours post- injury through magnetic resonance imaging. However, although every effort was made to ensure that musc le damage occurred to the quadr icep, the degree of damage for this study is a lso quest ionable. 1 Fema les were used in the study to make the group as homogeneous as possib le and to control for extraneous var iables. Furthermore, sex dif ferences in musc le fatigue have been reported in the literature, with females general ly exhibiting a 82 greater relative fatigue resistance than males [252-253]. Th is phenomenon has been observed in a variety of musc les with the use of var ious fatigue protocols. H icks et al [254] hypothesized that musc le mass , substrate utilization and muscle morphology may be mechan isms involved attributing to dif ferences in fatigue resistance. Estrogen may also be implicated in modulat ing musc le fatigue. Estrogen receptors are found on vascu lar endothelial and smooth musc le cel ls [255]. Est rogens affect vascu lar tone indirectly by modulat ing release of endothel ium-derived vasoact ive factors and directly by modulat ing intracellular ca lc ium in vascular smooth musc le cel ls [255]. Est rogens indirectly affect thrombotic events and inf lammation ( by altering platelet aggregation and leukocyte adherence and migration, respectively [255]. Est rogens also influence production of mitogens which, when re leased at si tes of vascu lar injury, affect vascu lar remodel ing [255]. Therefore, one gender was se lected to control for gender differences assoc ia ted with fatigue resistance. The study, in its entirety, was completed with 16 female participants. Although it was anticipated that the study would recruit 20 subjects, time constraints and the exclusion of a few individuals due to interruptions in treatment hindered this effort. B e c a u s e of this decrease in sample s ize, power of the study was reduced from the pre-calculated 0.76, thereby limiting the ability to detect a treatment effect, given that an effect actually existed. This limitation of a relatively smal l sample s ize could therefore have had an effect on not attaining statistical s igni f icance in the study for the var iables examined. This double-bl ind study examined numerous var iables. Pain and strength parameters were examined to demonstrate perceived musc le so reness and eccentr ic strength decrements, MRI and quadricep circumference were measured to indicate musc le e d e m a that is demonstrable in musc le injury, and blood levels for CK, IL-6, MDA were examined to illustrate skeletal muscle damage, the cytokine response and lipid peroxidation. Through these measurements , the patterns of recovery could be seen between both groups and 83 therefore would allow us to determine whether the experimental group showed a faster course of recovery to basel ine levels over the five day testing period in contrast to the control group. Perceived muscle soreness It was hypothesized that the musc le so reness exper ienced by the D O M S protocol would be reduced by hyperbaric oxygen therapy over the testing period. The results of this study do not support this hypothesis. Hyperbar ic oxygen therapy did not significantly decrease musc le pain or so reness levels over the course of four treatments. T h e s e results contradict the findings of other reports indicating that H B O therapy successfu l ly al leviated muscle soreness in athletes [1, 95]. Bor romeo et al [95] used the visual analog sca le to a s s e s s pain levels for acute ankle sprains over a course of three treatments. He demonstrated pain levels peaking at 3.25 and 2.6 for the H B O and control group, respectively, and decreas ing to near basel ine levels by complet ion of the third treatment. Harr ison et al [246] and Mekjavic et al [225] used the visual analog sca le to a s s e s s pain levels in their sample populat ion. Their studies demonstrated peak ratings as high as 7 for perceived musc le so reness for both groups. Ba lnave et al [147] showed a rating of 6 for peak musc le soreness . Another study by Zhang et al [229] showed peak pain levels of 2.5 and 4.5 for the intervention and placebo, respectively. These studies all show ratings of musc le so reness ranging anywhere between 2.5 and 7. This is similar to the results of this study, which demonstrate that musc le so reness peaked in range between 3.5 and 4.5 for the control and H B O group, respectively. The majority of previous studies cite perceived so reness to peak anywhere between twenty-four and forty-eight hours post-exercise [129, 147, 225, 246]. Similarly, this study supported these finding for both groups. The perception of musc le soreness peaked 24 hours after the high-force eccentr ic exerc ise protocol 84 for the experimental group while the control group peaked 48 hours post- eccentr ic exerc ise. Severa l reasons may be postulated for the failure of the H B O treatments to alleyiate musc le soreness . The f indings of this study may be due to intersubject variability. Pa in or perceived so reness remains a subjective var iable in which the subject is asked to graphically display their perception of pain. Pa in thresholds and tolerance levels are different among individuals [241]. The perception of so reness is very difficult to interpret as perceived muscle so reness is subjective in nature and its perception varies from individual to individual. Stewart [241] has cited that the perceived pain is a product of interpretation by the mind; the intensity felt can be increased or dec reased by consc ious and unconsc ious thought or emotions. The sensory component may be modulated by the subject 's past exper ience where attitudes and psychological var iables may influence descript ion of the sensat ion [129]. Error is easi ly introduced as an individual may report more or less pain that what is actually felt. L e s s pain is usually the direction in which individuals follow in reporting pain [241]. The visual analog sca le has been cited in previous literature as a reliable instrument in measur ing pain or perceived soreness in subjects [15, 195-197, 199, 201]. However, by marking on a 10 cm line the level of pain felt can introduce error in accuracy of what is actually felt versus what is begin reported. Furthermore, f requency in sampl ing may also bias results. Subjects may have been aware of their previous response and perhaps subconsc ious ly bias their following responses . Newham et al [104] reported that pain and tenderness is usually local ized to the distal third portion of the musc le , in the region of the musculotendinous junction where musc le pain receptors are most concentrated. In addit ion, pain assoc ia ted with D O M S appears medially, laterally and then distally, eventually spreading to the center of the muscle belly by 48 hours [104]. General ly , however, pain is 85 evident throughout most of the affected musc le belly while no pain is exper ienced during rest [132]. Al l subjects in the study demonstrated a similar pattern of pain, with the musculotendinous junction to be the primary location of so reness . Furthermore, as expected, all subjects (both control and experimental groups) demonstrated no pain at rest but exper ienced discomfort during movement. Another possib le explanation for the lack of significant dif ferences in pain data was that the st imulus in this study did not cause enough musc le damage and injury. Even though we increased the angle of musc le elongation to an 110° - 3 5 ° musc le range, perhaps there wasn't enough stimulus to induce musc le injury and damage to warrant sufficient D O M S to show significant results in treatment. The exerc ise protocol that was used in this study was one that has been tested by Mclntyre et al [129]. T h e s e investigators have used this protocol on the non- dominant quadr icep musc le to elicit and induce sufficient musc le so reness . Laurence [247], had suggested that in his study, at 95° - 35° f lexion, the quadr icep musculature may not be elongated enough to induce sufficient musc le soreness . Therefore, this study employed his suggest ion of using 110° - 35° flexion to further elongate the quadr icep musc le , thus ensuring that the st imulus would in fact induce sufficient musc le so reness . Al l subjects were carefully monitored during the exerc ise protocol and were encouraged to exert maximal effort on each contraction. There efforts were a lso observed on the visual display to see that they were not only maximizing their efforts but also maintaining this level consistent ly throughout the protocol. However, even though we ensured these efforts to induce muscle injury, perhaps the level of musc le damage was not sufficient enough to warrant D O M S . Eccentric Strength It was hypothesized that treatment with hyperbaric oxygen would return strength faster than the normal course of recovery. Th is however was not the case as there were non-signif icant f indings between groups in terms of treatment and placebo sess ions . Hyperbaric oxygen did not accelerate the injury heal ing 86 involved in restoring strength as neither the rate or magnitude of recovery of eccentr ic strength differed between groups. Th is contradicts the f indings of Stap les et al [96] who demonstrated significant results when analyzing mean torque data, thus showing a reduction in H B O vs. control group. A n initial decl ine in strength was apparent between both groups in the study immediately after the eccentr ic exerc ise protocol. Th is was fol lowed by a gradual return of torque over four day, although these levels did not return to basel ine (pre-exercise) by day 5. Maclntyre et al [129] have reported similar f indings. In their study, torque measurements did not return to basel ine for 6 and 7 days, respectively. The initial decl ine in strength corroborates other f indings of strength decrements immediately following a high intensity exerc ise protocol [129, 130]. They suggest that the initial decl ine in force may be a function of mechan ica l injury and fatigue (including myofibrillar disruption at the level of the Z-l ine), leading to an acute inflammatory response [129, 130]. A second decl ine in strength (b imodal pattern) was seen with six of the sixteen subjects in this study. Th is corroborates f indings in previous research suggest ing that a bimodal pattern of eccentr ic strength is seen in both an animal model and humans [129, 130]. Faulkner and co l leagues [130] further suggest that the second decl ine in force occurs in response to phagocyt ic activity at the site of the initial damage. Th is deficit in force does not appear to be related to the level of so reness s ince it occurs prior to the so reness and can remain for a greater period [131]. Prev ious studies have reported that there is no relationship between the level of so reness and the decl ine in musc le strength [129, 130, 131, 132]. Strength decrements are seen immediately post-exercise while so reness develops 24-48 hours post- exerc ise. Observ ing a bimodal pattern of eccentr ic torque, lends more ev idence and support to the theory that more than one mechan ism (i.e. mechan ica l , biochemical) is involved in exerc ise- induced muscle damage. Both groups demonstrated gradual recovery over time, although individual variability in eccentr ic torque was evident among all subjects [ A P P E N D I X C - 87 Figures D, E]. To explain this variability, severa l explanat ions may be worth introducing. First, it would have been worthwhile to observe the recovery pattern of all subjects for more than 4 treatment sess ions . By only taking eccentr ic torque measures at post-exerc ise, 24,48 and 72 hours, we weren't able to follow the course of recovery over the previous cited 5-7 days recovery period for D O M S [132]. Furthermore, a learning effect could have played a role in the recovery pattern seen among subjects. Numerous other investigators have reported a training and adaptation effect during eccentr ic exerc ise [131, 145, 172, 173]. N o s a k a et al [173] has suggested that a subsequent single bout of eccentr ic exerc ise may reduce indicators of muscle damage . Newham and co l leagues [104, 110, 131, 143] have suggested that this may b e - c a u s e d by a change in motor unit recruitment pattern, musc le fibre adaptation and/or a regeneration of new mechanical ly resistant fibres resulting from damage and destruction to the original recruited fibres. The subjects could have become more comfortable and more exper ienced day by day as they performed the eccentr ic strength ,test. Al though subjects were encouraged verbally by the investigator to produce maximal effort throughout the exerc ise protocol and isokinetic strength tests, and their efforts were monitored on the visual display, their energy level may have fluctuated daily. This would result in variability in strength levels throughout the course of the testing period. Quadricep Circumference Quadr icep circumference was measured at the 10 and 20 cm point above the superior portion of the patella, over the course of the 5 days. The premise for this measurement was that during the strenuous eccentr ic exerc ise, musc le fatigue and injury would give rise to muscular edema in the quadr icep musc le of the non- dominant leg. E d e m a is a result of the inflammatory process and could lead to increased pain and decreased range of motion. The eccentr ic exerc ise- induced edema , as reflected in quadr icep c i rcumference, did not demonstrate signif icance between groups. Slight increases in 88 circumference were demonstrated between groups at both the 10 and 20 c m mark, however not enough to attain statistical s igni f icance. ! E v a n s et al [150, 165] suggest that ev idence of swell ing has ranged from increased circumference of the exerc ised musc le 24-48 hour post-exercise to ultrastrucfural ev idence of post-exercise edema . Similarly, the results of this study demonstrated that peak swell ing (i.e. increased quadr icep circumference) at the 10 cm point occurred on day 3 (24 hours post-exercise) for both groups. P e a k swell ing at the 20 cm point was evident on day 4 (48 hours post-exercise) for both groups, however the measurements for days 2-5 for the control group remained lower than basel ine. Mekjavic et al [225] a lso demonstrated increases in arm circumference on days 3 and 5, post-exerc ise. E d e m a is a result of either an increase in vascu lar permeabil i ty of smal l blood vesse ls , or leakage of intracellular fluid into the extracellular space [131], resulting in t issue hypoxia, due to the increased oxygen diffusion distance from the capil laries to some cel ls, and the increased interstitial pressure around the capi l lar ies due to the fluid accumulat ion [225]. It has been reported that musc le e d e m a is caused by increased amounts of degraded protein components of the musc le and the release of protein-bound ions in damaged muscle cel ls [42, 165, 154]. A s a result, an increase in intracellular osmotic fluid is present. Taylor et al [242] has suggested that an increase in p lasma proteins produce an imbalance across the vesse l wall as these proteins move into the interstitial space and fluid is drawn out. Free radicals may also play a role in e d e m a as they give rise to proteolytic enzymes when the microcirculation is compromised. Lipid peroxidation initiated by free radicals dec reases the barrier function of cell membranes and may be assoc ia ted with musc le fibre necrosis and enzyme release following damaging exerc ise [112]. The oxygen diffusion distance from the capil laries can be up to four t imes greater than normal with hyperbaric oxygen. This is mainly due to the larger pressure gradient between capil lary and t issue PO2, which increases the number of cel ls that can be oxygenated when 8 9 cel lular oxygenat ion is limited by e d e m a [61]. Therefore, hyperbaric oxygen may be beneficial in reducing e d e m a by ensur ing adequate cel lular oxygenat ion to maintain cellular energy product ion, al lowing the cell to fuel its A T P driven pumps and channels , thus enhancing the reabsorption of fluid from the extracellular s p a c e and decrease e d e m a [83, 225]. Furthermore, secondary vasoconstr ict ion by H B O would reduce blood inflow by 2 0 % without decreas ing oxygen delivery. Th is would a lso reduce e d e m a by decreas ing the, intravascular hydrostatic pressure, and establ ishing a more favorable pressure gradient for fluid movement out of the interstitial space back into the capi l lar ies [225]. In contrast to studies that have demonstrated a reduction in e d e m a by hyperbaric oxygen therapy [3, 5, 72-74, 91], there is inconclusive ev idence that H B O therapy reduces edema with soft t issue athletic injuries. [95]. Our study indicates that this modality of treatment was not effective in minimizing the e d e m a formation assoc ia ted with exerc ise- induced musc le injury. Th is finding is similar to that of Mekjavic et al [225] who suggested that e d e m a in their study was not of sufficient magnitude to establ ish the increased diffusion distances and increased interstitial pressure, which might promote t issue hypoxia. Blood Analysis: Creatine Kinase, Malondialdehyde and Interleukin-6 Creatine Kinase (CK) Creat ine k inase has been studied extensively in relation to musc le damage due to strenuous exerc ise s ince this p lasma enzyme is found exclusively in the skeletal and cardiac musc le [41, 77, 127, 153, 160, 162]. It has been reported that the extent of release of this p lasma enzyme is de layed and is a 1 direct consequence of musc le damage and exerc ise [41, 112, 160, 162]. Furthermore, the magnitude and duration of the increase in C K activity is affected by the type and intensity of activity as well as previous level of activity [115]. It has also been cited that C K has a high degree of variability and its response var ies between 90 individuals [41, 77, 162]. For example, one study in which subjects performed an eccentr ic exerc ise showed increases in C K activity up to 30 000 m-units/ml, while other subjects showed increases of less than 500 m-units/ml [248]. It was hypothesized that hyperbaric oxygen would attenuate the response of creatine k inase over the course of treatment. More specif ical ly, 4-hours post- exerc ise levels would increase from basel ine, but this response would be dampened in contrast to the control group. Ana lys is of the data did not demonstrate significant f indings between groups for treatment with hyperbaric oxygen over the course of one week. Our f indings are consistent with those of previous studies indicating that the eccentr ic exerc ise protocol, which was used in our investigation, resulted in a quantif iable musc le injury [100, 153, 177, 179]. However the lack of signif icance across both groups suggest that H B O was not effective in treating muscle injury or D O M S . In females, the level of C K ranges anywhere between 45-230 u/L, with exerc ise, t rauma, surgery and other ai lments increasing levels 10-15 t imes normal range va lues [249]. Furthermore, some individuals may have increases up to 50 t imes the upper limit of normal range but this response is seen in condit ions such as muscular dystrophy [249]. Elevation in levels may be seen as early as 2-4 hours post exerc ise, with values returning to normal by 48 and 72 hours post-injury. For the purpose of the present study, blood was withdrawn from subjects 4 hours post-exerc ise, fol lowed by subsequent testing for 3 days (24, 48 and 72 hours) to examine increases in serum C K activity levels. A statistical analys is on the creatine k inase data was performed on two separate occas ions , one with the entire subject pool and a second with the removal of one responder in the experimental group who appeared to have a C K value well beyond the normal limits (>2500 uL). This was done to ensure that this high variability introduced into the data by this subject did not mask statistical 91 significant f indings in the sample . Both ana lyses demonstrated no signif icance between groups for treatment effects. The serum creatine k inase response to the eccentr ic exerc ise protocol in the current investigation showed considerable individual variability, al though most subjects d isplayed an immediate increase in serum C K 4 hours post-exerc ise. Ebbel ing and C larkson [127] suggested that subjects who appear to be quite similar when performing the s a m e amount of eccentr ic exerc ise can have changes in circulating C K activity that are different by orders of magnitude. Many factors influence intersubject variability, including age, gender, body composi t ion and race [147]. In contrast, Ebbel ing and C la rkson [127] point out that the increase in circulating C K is "unrelated to either the development of so reness , the amount of strength loss after exerc ise, f i tness level of the subject, or lean body weight". Other investigators have looked at genetic variation and serum C K activity to understand the variability [112]. It is likely that the post-exerc ise rise in circulating C K activity is a manifestation of skeletal muscle damage but not a direct indicator [127]. Malondialdehyde (MDA) A growing amount of ev idence indicates that free radicals play an important role as mediators of skeletal musc le damage and inflammation after strenuous exerc ise [239, 240, 244]. It has been postulated that the generat ion of oxygen free radicals is increased during exerc ise as a result of mitochondrial oxygen consumpt ion and electron transport flux, inducing lipid peroxidation [239]. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. Recent studies indicate that a) oxygen free-radicals mediate, at least in part, the increased microvascular permeabil i ty produced by reoxygenation, and b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia [244]. The literature supports the notion of the interrelationship between ischemic t issue and inflammatory cel ls and therefore, conc ludes that capil lary plugging by granulocytes and oxygen free 92 radical formation may contribute to the ischemic injury [244]. The role of reactive oxygen spec ies in the mediation of exerc ise- induced oxidative damage to musc le and the protection offered by anti-oxidant defense sys tems have been have a lso been well studied [138, 139]. Malondia ldehyde, a product of lipid peroxidation is a way of estimating free radical generat ion as a result of skeletal musc le damage. It was hypothesized that the eccentr ic exerc ise protocol would induce skeletal musc le damage, thus ultimately giving rise to free radicals and lipid peroxidation. Hyperbaric oxygen therapy would have an effect on lipid peroxidation by enhancing the antioxidative defense mechan isms and increasing the b iochemical defense mechan isms against free radicals [193]. A s a result, H B O therapy would attenuate M D A levels. Ana lys is of the data did not demonstrate significant f indings for treatment with hyperbaric oxygen. Hyperbar ic oxygen had no effect on reducing free radical damage and limiting lipid peroxidation levels as ev idenced by malondia ldehyde. The results of both groups demonstrated very little fluctuation in malondia ldehyde levels, increasing slightly after the eccentr ic exerc ise protocol and decreas ing c lose to basel ine levels by day 5. The mean va lues for M D A in this study ranged between 4.0 and 4.6 nmol/ml. Other studies have reported M D A values in the range of 1.0 - 3.0 nmol/ml [229, 250, 251]. This difference may be due to the intensity and duration of the prescr ibed exerc ise protocol, the variability in sampl ing t imes for blood analys is ^and nature of injury. Chi ld et al [251] only sampled M D A levels pre and immediately post-exerc ise. This , therefore would not allow us to determine what the levels of M D A would be at 24, 48 and 72 hours post-exercise. Furthermore, Novell i et al [250] examined biopsies taken from the right femoral quadricep muscle at three time points during aortic surgery. 93 v O n e plausible explanat ion for not demonstrat ing signi f icance in the present study could be that the eccentr ic exerc ise protocol didn't elicit enough skeletal musc le damage to observe changes post-exerc ise. In other words, the effect was minimal which did not allow us to observe a noticeable change with H B O treatments, as expected. If the exerc ise protocol induced sufficient musc le damage or injury, the levels of M D A would have increased higher than what was observed. Furthermore, if H B O therapy was beneficial in reducing lipid peroxidation, the present study might then have been able to see a not iceable effect and obtain statistical s ignif icance. Another factor that may have played a role in lipid peroxidation detection may be the mode of assay sampl ing (i.e. direct spectrophotometry). M D A assay is the most generally used test in the appreciat ion of the role of oxidative st ress in injury and d isease . M D A is one of several products formed during the radical induced decomposi t ion or breakdown of endoperox ides during the last s tages of the oxidation of polyunsaturated fatty ac ids. Most often, at high temperature and low p H , M D A readily participates in nucleophil ic addition reaction with 2-thiobarbituric acid (TBA), generating a red, f luorescent 1:2 M D A : T B A adduct [191]. B e c a u s e of this fact, and facile and sensit ive methods to quantify M D A (as the free a ldehyde or its T B A derivative), the " T B A test" is used as a routine test to detect and quantify lipid peroxidation in a wide array of sample types [191]. Th is reaction that occurs is very sensit ive but its specificity, even with improvement of pre- analytical (sampling, preservatives), and analytical s tages (f luorescence, H P L C ) is still a matter of debate [243]. M D A itself participates in reactions with molecules other than T B A and is a catabol ic substrate. Only certain lipid peroxidation products generate M D A (invariably with low yields) and M D A is neither the so le end product of fatty peroxide formation and decomposi t ion nor a substance generated exclusively through lipid peroxidation [191]. A n extensive review of the literature has conc luded that M D A determination and the T B A test can offer, at best, a narrow and somewhat empir ical window on the complex process of lipid peroxidation [191, 234]. It is subject to interferences, which if not 9 4 cons idered, may lead to erroneous results. Future studies should perhaps focus on more sensit ive and specif ic (chemical or physical methods) modes of lipid peroxidation assessment . Interleukin 6 (IL-6) Increasing numbers of reports have descr ibed the IL-6 response to injury [187- 188, 231-235]. Eccentr ic exerc ise is assoc ia ted with an increase in serum IL-6 concentrat ions and is significantly correlated with the concentration of C K in subsequent days following injury. The time course of cytokine production, the c lose associat ion with musc le damage and the finding of IL-6 in skeletal musc le b iopsies after intense exerc ise lend support to the idea that during eccentr ic exerc ise myofibers are mechanical ly damaged and that this process st imulates the local production of inflammatory cytokines [197]. IL-6 is an integral cytokine mediator of the acute phase response to injury and infection. It plays an active role in the post-injury immune response, making it an attractive therapeutic target in attempts to control hyperinf lammatory-provoked injury [235]. It was hypothesized that the eccentr ic exerc ise protocol would induce an inflammatory response that would lead to the production of IL-6. This increase in p lasma levels of IL-6 would be al leviated by hyperbaric oxygen treatments over the four days of therapy and the inflammatory response would be reduced by treatment. Statistical analysis of the data demonstrated non-signif icant f indings for both groups. This must be interpreted cautiously, as the sample s ize was quite smal l and standard errors of the means large. A great degree of variability in the analys is was demonstrated. This variability was unexplainable and may largely be attributed to error in laboratory technique. Another limitation we encountered in the study was that the laboratory, in conduct ing the analys is, contaminated and destroyed 8 of the 16 subjects (50%), thus leaving a total of four subjects per group (n=4). This therefore diminished our sample s ize and as a result, was not 9 5 sufficient enough attain s igni f icance to substantiate or refute the involvement of hyperbaric oxygen therapy in reducing IL-6 elevations during musc le damage and injury. Examinat ion of the means of both groups provided perplexing results. The experimental group demonstrated a relatively stable level of IL-6 while the control group showed an irregular pattern [Figure 11]. Overal l , all subjects in both groups had variability in the response of IL-6 to the eccentr ic exerc ise protocol and subsequent treatment sess ions . T h e s e results do not typify the normal course of IL-6 that has been reported in the literature. Biffl et al [235] has suggested that IL-6 concentrat ion rose within 2-4 hours post-trauma. Th is was not evident in our investigation as IL-6 levels elevated 24 hours post-exercise for both groups. The magnitude of elevation of IL-6 is related directly to the degree of t issue injury [235], ranging <50 pg/ml in healthy individuals and rising as high as 1000 ng/ml in certain severe d isease states [233]. Assuming that the results were reliable and val id, it appears that hyperbaric oxygen was doing something to the p lasma levels of IL-6 in the experimental group, making the responses stable and less sporadic than the control group. Th is was evident in the graphical depict ions of the means of both control and experimental groups [Figure 11]. Cou ld administering 100% oxygen at high pressure have some impact on IL-6? A study by Rohde et al [188] at the Copenhagen Musc le Resea rch Centre demonstrated that an increase of 5 7 0 % in IL-6 concentration was seen in the control trial (pre- exerc ise to 2 hours post-exercise) and returned to pre-exercise levels at day 2. Bauer et al [233] reported that elevated levels of IL-6 could be found as early as a few hours or up to a few days and these levels fluctuate depending on the acute inflammatory response. For example, patients undergoing elective surgery reached IL-6 p lasma concentrat ions of 100 pg/ml, while patients with viral meningitis ranged between 10 - 1000 ng/ml [233]. A review of the literature proved unsuccessfu l , as we were unable to support or explain the variable f indings found in this study. Future studies need to further examine this question more in detail by perhaps focussing on the inflammatory response and examining 96 in more detail how hyperbaric oxygen therapy affects the ensuing cytokine response. Magnetic Resonance Imaging Magnet ic resonance imaging, being a powerful non- invasive measurement , a l lowed us to determine whether the eccentr ic protocol did in fact produce exerc ise- induced musc le damage and as well as quantify this level of musc le injury as ev idenced by musc le edema . Skeleta l musc le T 2 relaxation time has been assoc ia ted with changes in the fluid component of injured musc le and used as a marker of edema, inflammation and injury [177, 179, 246]. Th is is consistent with previous f indings that suggest that e d e m a results from high intensity eccentr ic exerc ise [174, 177-179]. In the current study, it was hypothesized that hyperbaric oxygen treatment would decrease muscle e d e m a that was induced by the eccentr ic protocol over the time course of therapy. Statistical analys is for both T2-weighted images and STIR images demonstrated non-signif icant f indings for treatment effect over time. This was evident in all three musc les : rectus femoris, vastus medius, and vastus lateralis. Al though T2 and ST IR images indicated an increase in e d e m a 24- hours post-exercise for the same three musc les , respectively, treatment with hyperbaric oxygen did not induce an effect sufficient enough to reduce edema and achieve statistical s igni f icance. The nuclear magnet ic resonance (NMR) signal from which M R images are constructed ar ises from the magnet ic behavior of the hydrogen nuclei in t issue water and fat molecules when t issue is p laced in a strong magnet ic f ield. Inside a strong magnet, the hydrogen nuclei can be excited by the input of energy, specif ical ly, by a "pulse" of energy at the resonant radiofrequency [226]. This excitation causes a fraction of the nuclei to oscil late together in the magnet ic field in an orientation that generates a detectable magnet ic signal that can be recorded electronically and as a result, form images [226]. However, immediately 97 after excitation, nuclei in different magnet ic environments begin to oscil late differently, the oscil lation begins to breakdown and the observed signal decays away [226]. Decay of the N M R signal is referred to as t ransverse relaxation time (T2 relaxation time) and is the time constant that character izes the exponent ial decay of the signal after the initial excitation [226]. T2 relaxation t ime, however does not suppress fat on the observed images. Short Tip Inversion Recovery (STIR) images al lows one to sort out t issue as this form of imaging only shows fluid (bright signal) and suppresses fat [ A P P E N D I X D]. MRI is the most sensit ive non- invasive imaging method for detection and quantification of muscle edema . T 2 weighted images and ST IR images are particularly sensit ive for detection of e d e m a and increased water content in body t issue. The MRI data in this study is based on signal intensity measurements . MRI s ignal intensity measurements are expressed in arbitrary units, which were variable due to a number of factors. The signal intensity obtained from a specif ic vo lume of t issue depends on the physical properties of the t issue (such as water content, t issue composi t ion, and t issue density), the spatial location of the t issue relative to the signal reception coi l , the performance characterist ics of the radiofrequency sys tem, the performance character ist ics of the reception coi l , and the performance characterist ics of the amplif ication system. Many of these characterist ics are variable from moment to moment and from location to location within the body. To overcome this variability, this study used a ratio of signal intensity in the s t ressed musc le divided by the signal intensity s imultaneously obtained in the corresponding exact anatomic location in the opposite quadr icep muscle. Th is type of internal control overcomes variability assoc ia ted with (for instance) body hydration, body posit ion relative to the coil , and the operating characterist ics of the MRI sys tem. 9 8 This measurement sys tem is suscept ib le to signal intensity variability resulting from asymmetr ic posit ioning of the body within the MRI scanner , however care was taken to ensure that all subjects were posit ioned in a symmetr ic and midline fashion within the scanner sys tem. Another potential source of error is injury (acute or chronic) in the dominant (control) leg, which could affect s ignal intensity measurements . The images were visual ly inspected and any area of abnormality/injury in the control leg was avo ided. Due to the expense involved in conduct ing MRI 's on all subjects, this study had to limit image scans to 3 per subject. A s a result, basel ine images were taken, fol lowed by a scan 24 hours post-exercise and finally 72 hours post-exerc ise. Ideally, images taken throughout the entire week of the study and perhaps even 96 and 120 hours post-injury would have been beneficial to see a pattern of recovery. By only taking images at 0, 24 and 72-hour time points, introduces the opportunity for potentially miss ing what is happening between those periods. Finally, enough musc le injury may not have been demonstrated by the eccentr ic exerc ise protocol to attain statistical s igni f icance in treating this soft-t issue injury with hyperbaric oxygen treatments. < 9 9 CHAPTER 6: SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary Hyperbar ic oxygen is a field of much controversy and skept ic ism. The handful of studies that have been publ ished, differ in results as to whether this form of therapy proves effective in treating sport and exercise-related injuries. The purpose of this study was to determine whether intermittent exposure to hyperbaric oxygen played a beneficial role in the recovery from an acute soft t issue injury. A group of sedentary female students (n=16) were randomly ass igned to one of two groups (control [air] & experimental [hyperbaric oxygen]). The subjects performed a high intensity exerc ise protocol, fol lowed by the administration of four daily treatment sess ions . No significant dif ferences (p<0.05) among any of the dependent var iables were observed between those treated with hyperbaric oxygen and those that received normoxic condit ions. Conclusions From a review and analys is of pain, strength, quadr icep c i rcumference, C K , IL-6, M D A and MRI data, it appears that hyperbaric oxygen treatment did not have any effect on the following: 1. reducing perceived muscle soreness over the five-day testing period. 2. improving eccentr ic muscle strength after the exerc ise protocol over the four-day course of treatment. 3. reducing e d e m a as ev idenced by 10 and 20 cm quadr iceps c i rcumference measurements . 4. reducing the C K response, which, is exacerbated by skeletal musc le damage. 5. reducing IL-6 levels which have recently been demonstrated to increase considerably following eccentr ic exerc ise. 100 6. reduce M D A levels indicative of lipid peroxidat ion, which occurs during eccentr ic exerc ise and subsequent skeletal musc le damage. 7. reducing e d e m a as ev idenced by MRI imaging Recommendations for future studies The present study does not lend support to the eff icacy of using this treatment modality in soft-t issue injuries. However, a more focused study on fewer "key" var iables and a larger sample s ize may add light to this a rea of research. The cost of doing the above study was quite expensive and therefore, modif ications were implemented to reduce costs (e.g. 5 to 3 MRI scans/subject) . Future studies should: 1. focus on certain important var iables such as strength, blood parameters (i.e. M D A & IL-6) and MRI over more time periods. These var iables do not carry the subjective component that pain and quadr iceps circumference measurements employ and the variability component that C K has been cited as having. 2. increase the sample s ize to ensure a higher power and reduction of type 2 error. 3. examine p lasma P G E 2 , s ince prostaglandins are produced by invading macrophage during injury and have been cited to sensi t ize pain receptors. Furthermore, P G E 2 has been implicated in the generation of inflammatory pain [156, 157]. 4. examine whether the D O M S model is appropriate for inducing injury to the muscle. In other words, does this type of soft-t issue injury create an oxygen diffusion distance great enough for hyperbaric oxygen to demonstrate its beneficial effects, increasing the oxygen diffusion gradient. Investigations should perhaps focus on other types of injuries such as l igamentous injuries, where there is an increase in the oxygen diffusion 101 distance and the need for a reduction in the inflammatory process is warranted. It is worth noting that the H B O treatment protocol that was used (60 minute sess ions at 2.0 A T A , once a day for four days) in this study was based on previous research protocols looking at the therapeutic effects of this modality. However, pressure, duration of treatment, f requency and total number of treatments vary for a given indication. Ca re must be taken to ensure that oxygen toxicity levels are c losely monitored. Four treatment sess ions were chosen from a practical perspect ive, as an athlete being treated for an injury should have indication of significant improvement in the rate of recovery within severa l days. Caut ion should be appl ied when negating the effects of hyperbaric oxygen therapy on soft t issue injuries based on the results of this study. Limitations of the study should be carefully cons idered as many factors may affect the therapeutic effects of H B O on musc le injury. Harrison et al [246] suggest that H B O therapy will be effective in the treatment of athletic injuries that involve a greater magnitude of soft t issue damage or for injuries in which oxygen availability may be more of a limiting factor due to the location of the injury or magnitude of local e d e m a . Furthermore, researchers are limited due to i ssues of practicality, expense of treatment, toxicity levels and time. Although this study shows inconclusive results with respect to hyperbaric oxygen therapy, good quality research still needs to be conducted in this area, thus adding to the knowledge base on this issue as to its beneficial or non-beneficial effects on musc le t issue damage and injury. 102 R E F E R E N C E S 1. James PB, Scott B, Allen MW. Hyperbaric oxygen therapy in sports injuries. Physiotherapy 1993;79:571-572. 2. Potera C. Healing under pressure. Phys Sports<Med 1995;25:46-47. 3. Fitzpatrick DT. Hyperbaric oxygen therapy. Missouri Med 1994;91:684-689. 4. Mader JT. Hyperbaric oxygen therapy: a committee report. Undersea and Hyperbaric Medical Society, Bethesda, MD; pp1-90, 1989. 5. Bassett BE , Bennett PB . Introduction to the physical and physiological bases of hyperbaric therapy. In: Davis J C , Hunt TK (editors). Hyperbaric oxygen therapy. Bethesda (MD): Undersea Medical Society, 1977: 11-24. 6. Doctor N, Pandya S, Supe A. Hyperbaric oxygen therapy in the diabetic foot. J Postgrad Med 1992;38:112-114. 7. Hunt TK, Niinikoski J , Zederfeldt J et al. Oxygen in wound healing enhancement: cellular effects of oxygen. In: Davis J C and Hunt TK (eds). Hyperbaric Oxygen Therapy. Undersea Medical Society, Bethesda, MD, pp111-122, 1977. 8. Hunt TK, Pai MP. The effect of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg Gynecol Obstet 1971; 135:561-567. 9. Jain KK. Textbook of hyperbaric medicine. Toronto: Hogrefe and Huber, 1990. 10. Atroshenko ZB. Hyperbaric oxygenation in traumatic tissue edema. Soviet Med 1983;9:59-61. J 11. . Hunt TK, Zederfeldt B, Goldstich T. Oxygen and healing. Am J Surg 1969;118:521. 12. Hohn DC. The effect of Oxygen tensions on the microbial function of leukocytes in wounds and in vitro. Surg Forum 1976;27:18-20. 13. Isakov IV, Atroschenko ZB, Balik IPet al. Hyperbaric oxygenation in the prophylaxis of wound infection in the open trauma of the locomotor system. Undersea Biomed Res 1979;6:57-61. 14. Hart GB . HBO and exceptional blood loss anemia. In: Kindwall EP (Ed.). Hyperbaric Medicine Practice. Flagstaff, AZ: Best Publishing Company. 1994;517- 524. 15. Thorn SR. Leukocytes in carbon monoxide-mediated brain oxidative injury. Toxicol Appl Pharmacol 1993b;123:234-247. 16. Colignon M, Carlier AB, Khuc T et al. Hyperbaric oxygen therapy in acute ischemia and crush injuries. Proceedings of 13?h Annual Meeting of European Undersea Biomedical Society 1987:4-9. 103 17. Davidkin NF. Experience with clinical use of hyperbaric oxygenation in cases of trauma and their complications. Ortop Traumatol Protez 1977;9:33-35. 18. Grossman RA. Hyperbaric oxygen in the treatment of burns. Ann Plast Surg 1978;1:163-171. 19. Mainous E G . Osteogenesis enhancement utilizing hyperbaric oxygen therapy. HBO Review 1982;3:181. 20. Sheffield P J . Tissue oxygen measurements with respect to soft tissue wound healing with normobaric and hyperbaric oxygen. Hyperbaric Oxygen Rev 1985;6:18-46. 21. RA, Gottlieb SF , Pevsner NH. Hyperbaric oxygen for treatment of closed head injury. South Africa Med J1994;87(9):933-6. 22. Neubauer RA, Gottlieb SF . Hyperbaric oxygen for brain injury. J Neurosurg 1993;78(4):687-8. 23. Vorob'eva KP, Dziuba A N , Sorokin IN. Changes in autonomic regulation in . patients with multiple sclerosis during hyperbaric oxygenation. Lik Sprava 1998;7:85-8 [Russian].' 24. Kleijnen J , Knipschild P. Hyperbaric oxygen for multiple sclerosis. Review of controlled trials. Acta Neurol Scand 1995;91:330-4. 25. Kindwall EP , McQuillen MP, Khatri BO, Gruchow HW, Kindwall ML. Treatment of multiple sclerosis with hyperbaric oxygen. Results of a national registry. Arch Neurol 1991;48:195-9. 26. Meneghetti G, Sparta S, Rusca F, Facco E, Martini A et al. Hyperbaric oxygen therapy in the treatment of multiple sclerosis. A clinical and electrophysiological study in a 2-year follow-up. Riv Neurol 1990;60:67-71. 27. Van Bever Donker S C . Hyperbaric oxygen therapy for children with cerebral palsy. S^fr/Wec/J1999;89(4):360-1. 28. Cronje F. Hyperbaric oxygen therapy for children with cerebral palsy. S Afr Med J 1999;89(4):359-60; discussion 361. 29. Venter A, Leary M, Schoeman J , Jacklin L, Rodda J et al. Hyperbaric oxygen therapy for children with cerebral palsy. S Afr Med J 1998;88(11): 1362-3. 30. Leach R E . Hyperbaric oxygen therapy in sports. Am J Sports Med 1998;26:489- 490. 31. Bakker DJ . Hyperbaric oxygen therapy: past, present and future indications. Erdmann W and Bruley DF (eds). Oxygen Transport To Tissue XIV. Plenum Press, New York 1992;95-105. 104 32. Soolsma S. The effects of intermittent hyperbaric oxygen on short term recovery from grade II medial collateral ligament injuries [dissertation]. Vancouver: University of British Columbia, 1996. 33. Staples J , Clement D. Hyperbaric oxygen chambers and the treatment of sports injuries. Sports Med 1996;22:219-227. 34. Anderson LH, Watson B, Herring RF et al. Influence of intermittent hyperoxia on hypoxic fibroblasts. J Hyperbaric Med 1992;7:103-114. 35. Young T. Hyperbaric oxygen therapy in wound management. Brit J Nursing 1995;4:796-803. 36. Brown RB, Sands M. Infectious disease indications for hyperbaric oxygen therapy. Comprehensive Ther 1995;21:663-667. 37. Kindwall E P . Uses of hyperbaric oxygen therapy in the 1990s. Clev Clin J Med 1992;59:517-528. 38. Tibbies PM, Edelsberg J S . Hyperbaric oxygen therapy. NEJM 1996;33:1642- 1648. 39. Grim P S , Gottlieb LJ , Boddie A, Batson E. HBO therapy. JAMA 1990;263:2216- 2225. 40. Leach RM, Rees P J , Wilmshurst P. A B C of oxygen: Hyperbaric oxygen therapy. BMJ 1998;317:1140-3. 41. Clarkson P M , Ebbeling C. Investigation of serum creatine kinase variability after muscle-damaging exercise. Clin Sci 1988;75:257-261. 42. Friden J , Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric exercise in man. Int J Sports Med 1983;4:170-176. 43. Perrins JD, Davis J C . Enhancement of healing in soft tissue wounds. In: In: Davis J C and Hunt TK (eds). Hyperbaric Oxygen Therapy. Undersea Medical Society, Bethesda, MD, pp229-248, 1977. 44. Hohn DC. Oxygen and leukocyte microbial killing. In: Davis J C , Hunt TL (eds), Hyperbaric Oxygen Therapy, Bethesda MD, Undersea Med Soc 1977:101-110. 45. Knighton DR, Fiegel VD, Halverson T et al. Oxygen as an antibiotic. Arch Surg 1990;12:97-100 46. Youn BA. Oxygen and its role in wound healing. Internet review. Geisinger Medical Center. 47. Prockop DJ, Divirikko KI, Tuderman L. Biosynthesis of collagen and its disorders. NEJM 1979;301:13-21. 105 \ 48. Pentinnen R, Niinikoski P, Kulonen E. Hyperbaric oxygenation and fracture healing. Acta Chirigica Scandinavia 1972;138:39-44. 49. Pal MP, Hunt TK. The effect of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg Gynecol Obstetr 1972;135:561-567. 50. Banda Ml, Knighton DR, Hunt TK, Schevenstuhl H et al. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 1983;221:1283-1285. 51. Baboir BM. Oxygen dependent microbial killing by phagocytes. NEJM 1974;298:659-668, 721-726. 52. DeChatelet LR. Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J Infect Dis 1975;131:295-303. 53. Curtis A, Konrad HR, Zamboni WA. Hyperbaric oxygen therapy: an overview. Plastic Surg Nursing 1990; 10:63-68. 54. Bird AD, Telfer AB. Effect of hyperbaric oxygenation oh limb circulation. Lancet 1965;1:355-356. 55. Adameic L. Effect of hyperbaric oxygen therapy on some basic vital functions. Acta Physiol 1977;28:215-224. 56. Tabrah FL, Tanner R, Vega R, Batkin S. Baromedicine Today - rational uses of hyperbaric oxygen therapy. Hawaii Med J 1994;53:112-119. 57. Schaefer S E . Fundamentals of hyperbaric oxygen therapy. Ortho Nursing 1992;11:9-15. 58. Wells C H . Tissue gas measurements during hyperbaric oxygen exposure. In: Proceedings of the &h International Congress on Hyperbaric Medicine. Aberdeen, Scotland: Aberdeen University Press; pp118-124. 59. Weglicki WB, Whalen R E , Thompson HK et al. Effects of hyperbaric oxygenation on excess lactate production in exercising dogs. Am J Physiol 1966;210:473- 477. 60. Melamed Y, Shupak A, Bitterman H. Medical problems associated with underwater diving. NEJM 1992;326:30-35. 61. Davis J C , Hunt TK. Problem wounds: the role of oxygen. New York; Elsevier, 1988. 62. Kindwall EP , Goldman RW. Hyperbaric medicine procedures. 1988, Milwaukee; St. Luke's Medical Center. 63. Clark J M , Fisher A M . Oxygen toxicity and extension of tolerance in oxygen therapy. In: Davis J C , Hunt TK (editors). Hyperbaric oxygen therapy. Bethesda (MD): Undersea Medical Society, 1977: 11-24. 106 64. Clark J M , Gelfard R, Stevens WL et al. Pulmonary function in men after oxygen breathing at 3.0 ATA for 3.5 hours. J Appl Physiol 1991;71:878-885. 65. Clark J M , Gelfard WL, Stevens WL et al. Extension of pulmonary oxygen tolerance in man at 2.0 ATA by intermittent exposure of oxygen on normoxic pattern in predictive studies. Undersea Biomed Res 1990;17:25. 66. Stevens W C , Clark J M , Paolone A M et al. Interacting effects of 2.0 ATA P 0 2 and exercise on cardiopulmonary parameters. Undersea Biomed Res 1991; 18:86. 67. Weiss LD, Keith W, Meter V. The applications of hyperbaric oxygen therapy in emergency medicine. Am J Emerg Med 1992;10:558-564. 68. Hill RK. Is more better? A comparison of different clinical hyperbaric treatment pressures - a preliminary report. Undersea Hyperbaric Med Soc 1993;20:12. 69. Foster J H . Hyperbaric oxygen therapy: contraindications and complications. J Oral Maxillofac Surg 1992;50:1081 -1086. 70. Korn HN, Wheeler E S , Miller TA. Effect of hyperbaric oxygen on second-degree bum wound healing. Arch Surg 1977;12:732-737. 71. Cianci P, Petrone G, Shapiro R et al. Adjunctive hyperbaric oxygen therapy in treatment of severe burns. In: Joint Meeting on Diving and Hyperbaric Medicine, Undersea and Hyperbaric Medical Society, Supplement to Vol 17, Bethesda, MD, Aug 11-18; 1990a: p44. 72. Cianci P, Petrone G, Shapiro R et al. Adjunctive hyperbaric oxygen therapy in treatment of severe burns. In: Joint Meeting on Diving and Hyperbaric Medicine, Undersea and Hyperbaric Medical Society, supplement to vol 17, Bethesda, MD, Aug 11-18; 1990b: p42. 73. Cianci P, Sato R. Adjunctive hyperbaric oxygen therapy in the treatment of thermal burns: a review. Burns 1994;20:5-14. 74. Waisbren BA et al. Hyperbaric oxygen in severe burns. Burns Thermal Injury 1982;8:176-179. 75. Stewart R J , Mason SW, Kemp M et al. Hyperbaric oxygen treatment of burn wounds: effect on ATP, phosphocreatine, and collagen synthesis in an animal model. Undersea Hyperbaric Med Soc 1992;19:55. 76. Nylander G, Nordstrom H, Eriksson E. Effects of hyperbaric oxygenation on edema formation after a scald burn. Burns 1984;10:193-196. 77. Smith LL, Keating MN, Holbert D et al. The effects of athletic massage on delayed onset muscle soreness, creatine kinase and neutrophil count: a preliminary report. JOSPT 1994;19:93-99. 78. Gabb G, Robin ED. Hyperbaric oxygen: a therapy in search of diseases. Chest 1987;92:1074-1081. 1 0 7 79. LaVan FB, Hunt TK. Oxygen and wound healing. Clin Plast Surg 1990; 17:463- 472. 80. Mehm W J , Pimsler M, Becker RL et al. The effect of oxygen on in vitro fibroblast proliferation and biosynthesis. J Hyperbaric Med 1988;3:340-346. 81. Skyhar MJ et al. Hyperbaric oxygen reduces edema and necrosis of skeletal muscle in compartment syndromes associated with hemorrhagic hypotension. J Bone Joint Surg 1986;68:1218-1224. 82. Niinikoski JR, Hunt TK. Oxygen tensions in healing bone. Surg Gynecol Obstet 1972;134:746-750. 83. Hammarlund C, Sundberg T. Hyperbaric oxygen reduced size of chronic leg ulcers: a randomized double blind study. Plast Reconstruct Surg 1994;93:829- 834. 84. Strauss MB. Role of hyperbaric oxygen therapy in acute ischemias and crush injuries, an orthopedic perspective. Hyperbaric Oxygen Rev 1981;2:87-106. 85. Vujnovic D. The influence of oxygen on fracture healing. In: Dekleva N (ed). Symposium on Hyperbaric Medicine. Belgrad; pp57-61,1983. 86. Kivisaari J , Niinikoski J . Effects of hyperbaric oxygenation and prolonged hypoxia on healing of open wounds. Acta Chir Scand 1975;141:14-19. 87. Favalli A, Zottola V, Lovisetti G . External fixation and hyperbaric oxygen therapy in the treatment of open fractures of the tibial shaft. Undersea Biomed Res 1990;17:172. 88. Oriani G, Barnini C, Marroni G et al. HBO therapy in treatment of various orthopedic disorders. Minerva Med 1987;73:2983-2988. 89. Bouachour G, Cronier P, Gouello J P et al. Hyperbaric oxygen therapy in the management of crush injuries: a randomized double-blinded placebo-controlled clinical trial. J Trauma: Injury Infect Crit Care 1996;41:333-339. 90. Nylander G. Tissue ischemia and HBO: an experimental study. Acta Chir Scand 1986;533:109-110. 91. Nylander G, Nordstrom H, Larsson J et al. Reduction of post ischemic edema with hyperbaric oxygen. Plast Reconstr Surg 1985;76:596-603. 92. Jones RF, Unsworth, IP, Marosszeky, J E . Hyperbaric oxygen and acute spinal cord injuries in humans. Med J/4usr 1978;2:573-575. 93. Zamboni WA, Roth A C , Bergmann BA et al. Experimental evaluation of oxygen in treatment of ischemic skeletal muscle. Undersea Biomed Soc 1992;19:56. 108 94. Zamboni WA, Roth A C , Russel R C et al. Morphological analysis of microcirculation during reperfusion of ischemic skeletal muscle and the effect of hyperbaric oxygen. Plast Reconstr Surg 1993;91:1110-1123. 95. Borromeo C N , Ryan JL, Marchetto PA, Peterson R, Bove AA. Hyperbaric oxygen therapy for acute ankle sprains. Am J Sports Med 1997;25:619-625. 96. Staples JR. Effects of intermittent hyperbaric oxygen on pain perception and eccentric strength in a human model injury. Am J Sports Med 1999;27:600-605. 97. Staples JR, Clement DB, McKenzie DC et al. The effects of intermittent hyperbaric oxygen on biochemical muscle metabolites of eccentrically-exercised rats [abstract]. Can J Appl Physiol 1995;20 Suppl:49. 98. Webster DA, Horn P, Amin HM et al. Effect of hyperbaric oxygen on ligament healing in a rat model [abstract]. Undersea Hyperb Med 1996;23 Suppl:13. 99. Best TM, Loitz-Ramage B, Corr DT, Vanderby R. Hyperbaric oxygen in the treatment of acute muscle stretch injuries: Results in an animal model. Am J Sports Med 1998;26:367-372. 100. Byrnes W C , Clarkson P M . Delayed onset muscle soreness and training. Clin Sports Med 1986;5:605-614. 101. Armstrong RB. Mechanisms of exercise-induced delayed onset muscular soreness: a brief review. Med Sci Sports Exerc 1984;16:529-538. 102. Armstrong RB. Mechanisms of exercise-induced muscle fibre injury. Sports Med 12;184-207,1991. 103. Crenshaw A G , Thornell LE, Friden J . Intramuscular pressure, torque and swelling for the exercise-induced sore vastus lateralis muscle. Acta Physiol Scand 1994;152:265-277. 104. Newham DJ, Mills KR, Quigley RA, Edwards RHT. Muscle pain and tenderness after exercise. Aust J Sports Med Exerc Sci 14; 129-131: 1982. 105. Cleak MJ, Eston R G . Delayed onset muscle soreness: mechanisms and management. J Sports Sci 1992;10:325-341. 106. Cleak MJ, Eston, R G . Muscle soreness, swelling, stiffness and strength loss after intense eccentric exercise. BrJ Sports Med 1992;26:267-272. 107. Tiidus PM, lanuzzo C D . Effects of intensity and duration of muscle exercise on delayed soreness and serum enzyme activity. Med Sci Sports Exer 15; 161-165: 1983. 108. Cannon J G , Fiatarone MA, Fielding RA, Evans W J . Aging and stress-induced changes in complement activation and neutrophil mobilization. J Appl Physiol 1994;76:2616-2620. 109 109. Duncan PW, Changler J M , Cavanaugh DK, Johnson KR, Buehler A G . Mode and speed specificity of eccentric and concentric exercise training. JOSPT 1989;11:S26-S31. 110. Newham DJ, Mills KR, Quigley DM, Edwards RHT. Pain and fatigue after concentric and eccentric contractions. Clin Sci Cond 64;55-62:1983. 111. Armstrong RB. Initial events in exercise-induced muscular injury. Med Sci Sports Exerc 1990;22:429-435. 112. Ebbeling C B , Clarkson P M . Exercise-induced muscle damage and adaptation. Sports Med 1989;7:207-234. 113. Friden J , Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric exercise in man. Int J Sports Med 1983;4:170-176. 114. Abbott BC, Bigland B. The effect of force & speed changes on the rate of oxygen consumption during negative work. J Physiol 1953;120:319-325. 115. Evans W J . Muscle damage: nutritional considerations. Int J Sports Nutrition 1991;1:214-224. 116. Evans W J , Cannon J G . The metabolic effects of exercised-induced muscle, damage. In: Holloszy JD, editor. Exercise and Sport Science Reviews. Baltimore; Williams & Wilkins, 1991: 99-125. 117. Asmussen E. Positive and negative muscular work. Acta Physiol Scand 1952;28:364-382. 118. Bigland B, Lippold C J . The relation between force, velocity, and integrated electrical activity in human muscles. J Physiol 123;214-224:1965. 119. Infante AA, Klaupiks D, Davies R E . Adenosine triphosphate changes in muscle during negative work. Science 1964;144:1577-1578. 120. Knuttgen HG, Klausen K. Oxygen debt in short-term exercise with concentric and eccentric muscle contraction. J Appl Physiol 1971;30:632-636. 121. Lieber RL, Friden J . Muscle damage is not a function oUorce but active muscle strain. J Appl Physiol 1993;74:520-526. 122. McCully KK, Faulkner JA. Characteristics of lengthening contractions associated with injury to skeletal muscle fibres. J Appl Physiol 1986;61:293-299. 123. Fitzgerald GK, Rothstein J M , Mayhew TP , Lamb RL. Exercise-induced muscle soreness after concentric and eccentric isokinetic contractions. Phys Therapy 1991;71:505-513. 124. Hough T. Ergographic studies in muscle soreness. Am J Physiol 1902;7:76-92. 110 125. Weber MD, Servedio F J , Woodall WR. The effects of three modalities on delayed onset muscle soreness. JOSPT 1994;20:236-242. 126. Appell, HJ , Soares J M C , Duarte JA. Exercise, muscle damage and fatigue. Sports Med 1992;13:108-115. '127. Clarkson P M , Ebbeling C. Investigation of serum creatine kinase variability after muscle-damaging exercise. Clin Sci 1988;75:257-261. 128. Gleeson M, Blannin AK, Zhu B, Brooks S, Cave R. Cardiorespiratory, hormonal and hematological responses to submaximal cycling performed 2 days after eccentric and concentric exercise bouts. J Sports Sci 1995;13:471-479. 129. Maclntyre DL, Reid DW, Lyster DM, Szasz IJ, McKenzie DC. Presence of W B C , decreased strength, and delayed soreness in muscle after eccentric exercise. J Appl Physiol 1996;80:1006- 1013. 130. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during contractions: conditions of occurrence and prevention. Phys Ther 73;911- 921:1993. 131. Newham DJ, Jones DA & Clarkson PM. Repeated high-force eccentric exercise: effects on muscle pain and damage. J Appl Physiol 1987;63:1381-1386. 132. Maclntyre DL, Reid DW, McKenzie DC. Delayed muscle soreness. The inflammatory response to muscle injury and its clinical implications. Sports Med 1995;20:24-40. 133. DeVries HA. Electromyographic observations of the effects of static stretching upon muscular distress. Res Quarterly 1961;32:468-479. 134. Abraham W M . Factors in delayed muscle soreness. Med Sci Sports 1977;9:11- 20. 135. Anderson LH, Watson B, Herring RF et al. Influence of intermittent hyperoxia on hypoxic fibroblasts. J Hyperbaric Med 1992;7:103-114. 136. Stauber WT, Clarkson PM, Fritz VK et al. Extracellular matrix disruption and pain after eccentric muscle action. J Appl Physiol 1990;69:868-74. 137. Jones DA, Newham DJ, Clarkson PM. Skeletal muscle stiffness and pain following eccentric exercise of the elbow flexors. Pain 1988;30:233-242. 138. Pyne DB. Exercise-induced muscle damage and inflammation:, a review. AustJ Sci Med Sport 1994;26:49-58. 139. Pyne DB. Regulation of neutrophil function during exercise. Sports Med 1994;17:245-258. 140. Warhol MJ, Siegel A J , Evans W J , Silverman LM. Skeletal muscle injury and repair in marathon runners after competition. Am J Pathol 1985;118:331-339. 111 141. Waterman-Storer C M . The cytoskeleton of skeletal muscle: is it affected by exercise? A brief review. Med Sci Sports Exerc 1991 ;23:1240-1249. 142. Crenshaw A G , Karlsson S, Styf J et al.- Knee extension torque and intramuscular pressure of the vastus lateralis muscle during eccentric and concentric activities. Eur J Appl Physiol 1995;70:13-19. 143. Newham DJ. The consequences of eccentric contractions and their relationship to delayed onset muscle pain. Eur J Appl Physiol 1988;57:353-359. 144. Hasson S M , Daniels J C , Divine J G et al. Effect of ibuprofen use on muscle soreness, damage, and performance: a preliminary investigation. Med Sci Sports Exerc 1993;25:9-17. 145. Schwane JA, Johnson SR, Vandenakker C B , Armstrong RB. Delayed-onset muscular soreness and plasma C P K and LDH activities after downhill running. Med Sci Sports Exerc 1983;15:51-56. 146. Donnelly A E , Maughan R J , Whiting P H . Effects of ibuprofen on exercise-induced muscle soreness and indices of muscle damage. BrJ Sports Med 1991;24:191- 194. 147. Balnave C D , Thompson MW. Effect of training on eccentric exercise-induced v muscle damage. J Appl Physiol 1993;75:1545-1551 148. DeChatelet LR. Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J Infect Dis 1975;131:295-303. 149. Donnelly A E , McCormick K, Maughan R J , Whiting P H , Clarkson P M . Effects of a non-steroidal anti-inflammatory drug on delayed onset muscle soreness and ' indices of damage. BrJ Sports Med 1988;22:35-38. 150. Evans W J , Meredith C N , Cannon J G , et al. Metabolic changes following eccentric exercise in untrained and trained men. J Appl Physiol 1986;61:1864-1868. 151. Noonan TJ , Garrett W E Jr. Injuries at the myotendinous junction. Clin Sports Med 1992;11:783-806. 152. Mair J , Mayr M, Muller E et al. Rapid adaptation to eccentric exercise-induced muscle damage. Int J Sports Med 1995;16:352-356. 153. Clarkson PM, Byrnes W C , McCormick KM, et al. Muscle soreness and serum creatine kinase activity following isometric, eccentric, and concentric exercise. Int J Sports Med 1986;7:152-155. 154. Clarkson P M , Tremblay I. Rapid adaptation to exercise induced muscle damage. J Appl Physiol 1988;65:1 -6. 155. Flandry F, Hunt J P , Terry G C , et al. Analysis of subjective knee complaints using visual analog scales. Am J Sports Med 1991;19:112-118. 1 1 2 156. Smith LL. Acute inflammation: the underlying mechanism in delayed onset muscle soreness? Med Sci Sports Exerc 1991 ;23:542-551. 157. Smith LL, Wells J M , Houmard JA, et al. Increases in plasma prostaglandin E2 after eccentric exercise. A preliminary report. Hormone & Metabolic Research. '1993;25:451-2. 158. Salminen A, Kihlstrom M. Protective effect Of indomethacin against exercise- induced injuries in mouse skeletal muscle fibers. Int J Sports Med 1987;8:46-49. 159. Kuipers H, Keizer HA, Verstappen FTJ, Costill DL. Influence of a prostaglandin- inhibiting drug on muscle soreness after eccentric work. Int J Sports Med 1985;6:336-339. 160. Smith LL, Fulmer MG, Holbert D et al. The impact of a repeated bout of eccentric exercise on muscular strength, muscle soreness and creatine kinase. BrJ Sports Med 1994;28:267-270. 161. Belcastro A. Skeletal muscle calcium-activated neutral protease (calpain) with exercise. J Appl Physiol 1993;74:1381 -1386. 162. Smith LL, Keating MN, Holbert D et al. The effects of athletic massage on delayed onset muscle soreness, creatine kinase and neutrophil count: a preliminary report. JOSPT 1994;19:93-99. 163. Gallin JI, Goldstein IM, Snyderman R. Inflammation: basic principles and clinical correlates. New York: Raven Press Ltd, 1992. 164. Leadbetter M, Wayne B. An introduction to sports induced soft tissue inflammation. Sports Induced Inflammation, 3-23,1989. 165. Evans W J , Cannon J G . The metabolic effects of exercise-induced muscle damage. Chap 3;99-125. 166. Smith LL. Acute inflammation: the underlying mechanism in delayed onset muscle soreness. Med Sci Sports Exerc 1991 ;23:542-551. 167. Smith LL, McCammon M, Smith S et al. White blood cell response to uphill walking and downhill jogging at similar metabolic loads. Eur J Appl Physiol 1989;58:833-837. 168. Benstrup P. Late edema after muscular exercise. Arch Phys Med Rehab 1962;43:401-405. 169. Asmussen E. Observation on experimental muscle soreness. Acta Rheum Scand 2;109-116:1956. 170. Prockop DJ, Divirikko Kl , Tuderman L. Biosynthesis of collagen and its disorders. NEJM 1979;301:13-21. 113 171. Sjodin B, Hellsten Y, Apple W, Apple FS . Biochemical mechanisms for oxygen free radical formation during exercise. Sports Med 1990;10:236-254. 172. Byrnes W C , Clarkson P M , Spencer White J , et al. Delayed onset muscle soreness following repeated bouts of downhill running. J Appl Physiol 1985;59:710-715. 173. Nosaka K. Clarkson P M . McGuiggin ME. Byrne J M . Time course of muscle adaptation after high force eccentric exercise. Eur J App Physiol Occ Physiol 63;70-6: 1991. i 174. McCully KK et al. The use of magnetic resonance to evaluate muscle injury. Med Sci Sport Exerc 1992;24:537-542. 175. Rodenburg JB et al. Phosphate metabolism of prior eccentrically loaded vastus medialis muscle during exercise in humans. Acta Physiol Scand 1995;153:97- 108. 176. Sapega AA et al. Phosphorous nuclear magnetic resonance: a non-invasive technique for the study of muscle bioenergetics during exercise. Med Sci Sport Exerc 1987;19:410-420. 177. Fisher MJ et al. Direct relationship between proton T2 and exercise intensity in skeletal muscle MR images. Invest Radiol 1990;25:480-485. 178. Fleckenstein JL et al. Acute effects of exercise on MR imaging of skeletal muscle in normal volunteers. AJR 1988;151:231-237. 179. Fleckenstein JL eta l . Sports-related muscle injuries: Evaluation with MR imaging. Radiol 1989;172:793-798. 180. Mair J et al. Effects of exercise on plasma myosin heavy chain fragments and MRI of skeletal muscle. J Appl Physiol 1992;72:656-663. 181. Nosaka K, Clarkson PM. Changes in indicators of inflammation after eccentric exercise of the elbow flexors. Med Sci Sport Exerc 1996;28:953-961. 182. Nurenburg P. MR imaging-guided biopsy for correlation of increased signal intensity with ultrastructural change and delayed-onset muscle soreness after exercise. Radiol 1992;184:865-869. 183. Rodenburg J B et al. Changes in phosphorous compounds and water content in skeletal muscle due t eccentric exercise. Eur J Appl Physiol 1994b;68:205-213. 184. Doctor N, Pandya S, Supe A. Hyperbaric oxygen therapy in the diabetic foot. J Postgrad Med 1992;38:112-114. 185. Gracely RH, Kwilosz DM. The descriptor differential scale: applying psychophysical principles to clinical pain assessment. Pain 1988;35:279-288. 114 186. Perkins, J G . Hyperbaric oxygen therapy: an overview. Can J Rep Therapy \ 1994;30:163-169. 187. Pedersen BK, Ostrowski K, Rohde T et al. The cytokine response to strenuous exercise. Can J Physiol Pharmacol 1998;76:505-511. 188. Rohde T, MacLean DA, Richter EA et al. Prolonged submaximal eccentric exercise is associated with increased levels of plasma IL-6. Am J Physiol 1997;36:E85-E91. 189. Bruunsgaard H, Galbo H, Halkjaer-Kristensen J et al. Exercise induced increase in interleukin-6 is related to muscle damage. J Physiol 1997;499:833-841. 190. Valenzuela A. The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress. Life Sciences 1990;48:301-309. 191. Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biol Med 1990;9:515-540. 192. Frankel EN, Neff W E . Formation of malondialdehyde from lipid oxidation productions. Biochimica et Biophysica Acta 1983;754:264-270. 193. Monstrey S J , Mullick P, Narayanan, K et al. Hyperbaric oxygen therapy and free radical production: an experimental study in doxorubicin extravasation injuries. Annals Plastic Surg 1997;38:163-168. 194. Neubauer RA, Walker M. Hyperbaric Oxygen Therapy. James L (ed.). Avery Publishing, New York 1998. 195. Bond A, Lader M. The use of analogue scales in rating subjective feelings. BrJ Med Psychol 1974;47:211-218. 196. Carlsson A M . Assessment of chronic pain. Aspects of the reliability and validity of the visual analogue scale. Pain 1983;16:87-101. 197. Dixon J J . Agreement between horizontal and vertical visual analog scale. BrJ Rheumatol 1982;25:415-416. 198. Gramling S E , Elliott TR. Efficient pain assessment in clinical settings. Beh Res Ther 1992;30:71-73. 199. Price DD, McGrath PA, Rafii A, Buckingham B. The validation of visual analogue scales as ratio scale measures for chronic experimental pain. Pain 1983; 17:45- 56. 200. Scott J , Huskisson C. Graphic representation of pain. Pain 1976;2:175-184. 201. Zusman M. The absolute visual analogue scale (AVAS) as a measure of pain intensity. Aust J Physiother 1986;32:245-246. 115 202. Money FL. Underwater diving, oxygen poisoning and vitamin E. NZ Med J 1972;75:34-35. 203. Faiman FB. Effects of disulfiram on oxygen toxicity in beagle dogs. Aerospace Med 1974;45:29-32. 204. Torley LW, Weiss HS. Effects of age and magnesium ions on oxygen toxicity. Undersea Biomed Res 1975;2:223-227. 205. Dolmierski K J , Maslowski J , Matousek M et al. E E G changes measured by spectrum analysis under hyperbaric conditions. Aviation Space Environ Med 1990:61 ;33. 206. Van Hoesen KB, Camoresi E M , Moon R E et al. Should hyperbaric oxygen be used to treat pregnant patient for acute carbon monoxide poisoning. J /WA261;1039. 207. Thorn SR, Hyperbaric oxygen therapy. J Intensive Care Med 1989:4;58-74. 208. Surman MW. An introduction to hyperbaric oxygen therapy for the ET nurse. JWOCN 1996;23:80-89. 209. Bakker DJ . Clostridial myonecrosis. In Davis J C and Hunt TK (eds). Problem wounds: The role of oxygen. New York: Elsevier, 1988, pp.153-172. 210. Hart G B , Lamb RC, Strauss MB. Gas gangrene I. A collective review. II. A 15- year experience with hyperbaric oxygen. J Trauma 1983;23:991-1000. 211. Ellis ME, Mandal BK. Hyperbaric oxygen treatment: 10 years' experience of a regional infectious disease unit. J Infect Dis 1971;;6:187-190. 212. Hamblin DL. Hyperbaric oxygen: its effect on experimental staphylococcal osteomyelitis in rats. J Bone Joint Surg 1968;50:1129-1141.. 213. Davis J C , Hunt TK. Refractory osteomyelitis of the extremities and the axial skeleton. In Davis J C and Hunt TK (eds). Hyperbaric oxygen therapy. Bethesda, Maryland: Undersea Medical Society, 1977, pp.217-227. 214. Morrey BF, Dunn J M , Heimbach RD, Davis J C . Hyperbaric oxygen and chronic osteomyelitis. Clin Orthop 1979;144:121-127. 215. Walder DN. Aseptic necrosis in bone. In Strauss RH (ed): Diving medicine. New York: Grune and Stratton, 1976, pp.97-108. 216. Laufer A. Aseptic necrosis of the femoral head. J Mt Sinai Hosp 1957;24:957-987. 217. Neubauer RA, Kagan RL, Gottlieb S F . Use of hyperbaric oxygen for the treatment of aseptic bone necrosis: a case study. J Hyper Med 1989;4:69-76. 116 218. Conti V, Tassy J , Leonardelli M, et al. Limits of hyperbaric oxygen in the treatment of aseptic bone necrosis in the femoral head. Bull Med Sub Hyp 1969;1:3-4. 219. Sainty J M , Conti V, Aubert L et al. Role of hyperbaric oxygen in the treatment of aseptic bone necrosis of the hip. Med Aeronaut Spat Med 1980;19:215-217. 220. Tikhilow R M , Akimov G C , Lotovin A P . Effects of oxygen barotherapy on the regeneration of bone tissue. Orthop Traumatol Protez 1980:12:51-52. 221. Sepnov V N , Uglova MV. Features of sternum regeneration in autoplasty under conditions of hyperbaric oxyen. Orthop Traumatol Protez 1979:5:51 -53. 222. Zavesa PX , Shavab YY , Abduchudonov S S . Effects of local oxygen therapy on bone reparative regeneration of the bone. Orthop Traumatol Protez 1977:1:71- 72. 223. Cianci P, Bove A. Hyperbaric oxygen therapy in the treatment of acute and chronic peripheral ischemia. Int Med 1985;6:117-137. 224. Rodenburg JB , Steenbeek D, Schiereck P et al. Warm-up, stretching and massage diminish harmful effects of eccentric exercise. Int J Sports Med 1994;91:684-689. 225. Mekjavic IB, Exner JA, Tesch PA et al. Hyperbaric oxygen therapy does not affect recovery from delayed onset muscle soreness. Med Sci Sports Exerc 2000;32:558-563. 226. Meyer RA, Prior BM. Functional magnetic resonance imaging of muscle. Exerc Sport Sci Rev 2000;28:89-93. 227. Friden J , Lieber RL. Structural and mechanical basis of exercise - induced muscle injury. Sports Exerc 1992;24:521-530. 228. Stauber WT. Eccentric action of muscles: physiology, injury and adaptation. In Pandolf KB (ed): Exer Sports Sci. Baltimore, MD: Williams and Wilkins, 1989, pp 157-185. 229. Zhang JZ, Clement D, Taunton J . The efficacy of farabloc, an electromagnetic shield in attenuating delayed-onset muscle soreness. Clin J Sport Med 2000;10:15-21. 230. Zerba E, Kamarowski TE, Faulkner JA. Free radical injury to skeletal muscle of young, adult and old mice. Am J Physiol 1990;258:429-435. 231. Tilg H, Dinarello CA, Mier JW. IL-6 and A P P s : anti-inflammatory and immunosuppressive mediators. Immunology Today 1997;18:428-32. 232. Hirano T. Interleukin 6 and its relation to inflammation and disease. Clin Immunol Immunopathol 1992;62:s60-s65. 117 233. Bauer J , Herrmann F. Interleukin 6 in clinical medicine. Ann Hematol 1991;62:203-210. 234. Northoff H, Weinstock C, Berg A. The cytokine response to strenuous exercise. Int J Sports Med 1994;14:S167-S171. 235. Biff I WL, Moore EE , Moore FA, et al. Interleukin 6 in the injured patient. Marker of injury or mediator of inflammation. Ann Surg 1996;224:647-664. 236. Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57(suppl):715s-725s. 237. Brooks GA, FaheyT. Exercise Physiology: Human Bioenergetics and its Application. New York: Macmillan, 1984, pp 338. 238. Keul J , Doll E. Oxidative energy supply. Energy Metabolism of Human Muscle, Jokl E (ed.).Basel: Karer, 1972. 239. Dekkers J C , van Doomen LJ , Kemper HC. The role of antioxidant vitamins and enzymes in the prevention of exercise-induced muscle damage. Sports Med 1996;21:213-238. 240. Clarkson PM, Thompson HS. Antioxidants: what role do they play in physical activity and health. Am J Clin Nutr 2000;72(suppl):637S-46S. 241. Stewart ML. Measurement of clinical pain. In: Jakok AK. Little, Brown and Company. 1997: 107-137. 242. Taylor K, Fish DR, Mendel FC et al. Effect of a single 30 minute treatment of high voltage pulsed current on edema formation in frog hind limbs. Phys Ther 1992;72:63-68. 243. Lefevre G, Beljean-Leymarie M, Beyerle F, et al. Evaluation of lipid peroxidation . by measuring thiobarbituric acid reactive substances. Ann Biol Clin 1998;56:305- 319. 244. Rochette L, Maupoil V. Free radicals, lipid peroxidation and muscular ischemia. Comptes Rendus des Seances de la Societe de Biologie et de Ses Filiales 1992;186:252-262. 245. Bendtzen K, Hansen MB, Ross C, et al. Cytokines and auto-antibodies to cytokines. Stem Cells 1995;13:206-222. 246. Harrison BC, Robinson D, Davison BJ et al. Treatment of exercise-induced muscle injury via hyperbaric oxygen therapy. Med Sci Sports Exerc 2001 ;33:36- 42. 247. Laurence TF. The effect of microcurrent stimulation on exercise-induced muscle soreness. Masters dissertation, University of British Columbia 1999. 118 248. Newham DJ , Jones DA, Edwards RHT. Large delayed plasma creatine kinase changes after stepping exercise. Muscle and Nerve 1983;6:380-385. 249. Black HR, Quallich HD, Garlect C B . Radical differences in serum creatine kinase levels. Am J Med 1986;81:479-487. 250. Novelli G P , Adembri C, Gandini E et al. Vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion. Am J Surg 1997;172:206-209. 251. Child RB, Wilkinson DM, Fallowfield JL et al. Elevated serum antioxidant capacity and plasma malondialdehyde concentration in response to a simulated half- marathon run. Med Sci Sports Exerc 1998;30:1603-1607. 252. Maughan R J , Harmon J , Leiper D et al. Endurance capacityof untrained males and females in isometric and dynamic muscular contractions. Eur J Appl Physiol 1986;55:395-400. 253. Miller A, MacDougall J , Taarnopolsky M et al. Gender differences in strength and muscle fibre characteristics. Eur J Appl Physiol 1993;66:254-262. 254. Hicks A, Kent-Brauri J , Ditor DS. Sex differences in human skeletal muscle fatigue. Exerc Sport Sci Rev 2001;29:109-112. 255. Miller V M . Gender and vascular reactivity. Lupus 1999;8:409-15. 119 APPENDIX 120 Study Procedure: Shou ld I be chosen to participate in this study, I will be asked to attend 4 sess ions of treatment (1 per day for 5 days). In addit ion, I am fully aware that 12 ml of blood (5ml is equivalent to 1 tsp.) will be withdrawn prior to and 6 hours post-exerc ise, as well as each day following treatment and magnetic resonance imaging (MRI) will a lso be taken of my quadr icep muscle. I will a lso be asked to refrain from any form of exerc ise 12 hours post-injury and keep a brief diary of my activities as a reference. I understand that I will be p laced in either a control or experimental group by randomizat ion of names using a computer. In order to avoid bias, neither the subject nor the investigator will know what treatment the subject is receiving. However, in case of an emergency, the code can and will be broken. Exclusion: I understand that I may be exc luded from the study if I play on a team sport, run or weight train as part of my physical regimen, more than 3 hours per week. Individuals whose activities involve jumping and/or squatting will a lso be exc luded from the study. In addit ion, individuals who have exper ienced de layed- onset muscle soreness to their quadr iceps in the last three months, who have had a past history of severe joint injury, arthritis or other chronic i l lnesses and who are taking prescription drugs or ana lges ics will be exc luded. Hyperbaric oxygen contraindications (e.g. d iabetes, lung cysts, epi lepsy, upper respiratory tract infections, pregnancy, fever or conf inement anxiety) will a lso be evaluated. Risks and Benefits: I understand that the risks are minimal in this study, with mild aural barotraumas (ear ache), nausea , tooth and s inus pain and blurred vision occurring rarely. The benefit, if successfu l , will be no pain and a return of strength of the quadricep musc le sooner than the normal course of recovery for this condit ion. Confidentiality: I understand that any information resulting from this study will be kept strictly confidential and all documents will be identified only by a code number and kept in a locked filing cabinet. I will not be identified by name in any report of the complete study. 122 [ Contact: If I have any concerns about my treatment or rights as a subject, I may contact Dr. R.D. Sprat ley, Director of Resea rch Serv ices at the University of British Co lumb ia , at 822-8595. If I have any quest ions or desire further information with respect to the study, I should contact any of the above investigators. New Findings: I will be adv ised of any new information that becomes avai lable that may affect my wi l l ingness to remain in this study. Patient Consent: I understand that participation in this study is entirely voluntary and that I may refuse to participate or withdraw from the study at any time without consequences . I have received my copy of the consent form for my own records. I consent to participate in this study. Patient Signature Date ( Witness Signature Date Investigator's Signature Date 123 APPENDIX B: VISUAL ANALOG SCALE Subject #: Date: Time: Treatment Day #: Test # : No Pain Worst Pain Experienced Location of pain: Measurement #1: GIRTH MEASUREMENT 10 cm measurement = 20 cm measurement = Measurement #2: 10 cm measurement = 20 cm measurement = HEIGHT = WEIGHT = Treatment times = Time from DOMS to Treatment = 124 APPENDIX C: FIGURES A-Z (RAW DATA) 125  127 128 CO LO 0) > o Q 3 CT C a> • If • o U Si ? Ui o — £ fl o o Z •- CO fl) a a > S =5 -a — .2 •I a Q. 2. c (IUN) onbjoi ueoi/y 129 > o o o c o o o o o o o o C 0 C 0 C 0 C 0 C 0 C D C D C 0 !Q JQ !o 5" S " 3" 5" !O t n t o c o t o c o c o t n t o IHHHI O a If 1 2 E 2 <•> i o o CO T3 ** — c ui 1 I i 3 CO ii- m CO 2̂- ro D CM o o d CD o LO O d o d co o CM o d (s)ajieuiuao 130  © o o o o o o o o C D C O C D C D C 0 C D C O C O cocototocotococo T H m t © > o © o c © © E o o a E 2 O o CM o "5 CO CO Q 1 = > © "•5 Q. ~~ _c © © © *- i - > 3 (0 .£?"° E in CM O O O O O O O O O ~ ~ " o o o o o o o o d d * co (s)aii8uij)U33 O O O O O O I s- CO LD T r CO CM 132 t <i 0) ' - W O ' t l f l S I S C O O O O O O O O O Q ) 3 ) 0 ( D ( D O a ) ! D t o t n t o M t o c o c o w } + HHt to CD > O CD o c o 1- o u . i Q : 0 3 Q . 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(I) O) 2. c O) CO o CU !n 3 "|lU/Vai/\l S 9 | O U J U 137 138 o c d CD O o d o o d o o d o o o d co Q O d CD O o d o o d o o |iu/6d 139 co >. >, >. co CO CO C Q Q • • • (O W i- O N * - T - T - 1 CD O O CD (oesui) auiji uoiiexeiay zi 140 ,- CO in >> >> >. co CO ro Q Q Q • • • I O C M L O T— L O O (oasiu) aiuji uouexeiay ZL 141 , - co LO >< >. >- co CO CO Q Q Q • B • (oesLu) aiuji uoiiexeiay zi 142 ,- CO CO >- co CO CO Q Q Q • • • c M c q c D ^ c v i ' i - o o c p ' ^ t c v i o »- •»- v- d o d o (oasui) 3Ui ; i uoj)exe|3y zi 143 CO LO >. >. CO CO CO Q D Q • • • (oasui) auiji uojiexeiay zi 144 CO i n >« >. >, co CO CO Q Q Q • B • q "iZ 0) o. CT) c "•3 CO o •4-* >. CO TJ m CD 5 f ° i 8 a) £ c CO « 1 £ 5 x o CD CO cj) 2 I h- CO "co "co > « CT CO C 3 CO CO h- > CD CD = £ CT) i- ir o CO u cu JD 3 cn T - O O (o9sui) 3LU!i uoi}exe|3u ZL 145 , - co LO >» CO 03 CO Q Q Q • • • C\J 7 - CO CD C\J »-' -r- d d d d (oasiu) sa6eui! ui±S - soijea A;isua;u| |eu6js 146 y- CO to >. >-CO CO CO Q Q D • • • -a o "i- n a c CO >> co TJ 0) 3 £ 2 s s ° c CO m .2 E 4-1 . _ DC O J CC X 00 o §1 ? i •5 » = o > <D CD CO O) U i l 2 o CD In CO (oasiu) sa6euj! HilS - SOIJBH Ajisuaiui |eu6is 147 CO L O >- >, CO CO CO Q Q Q • • • (oasui) saBeiuj ui±S - S O I J B U Ajisuaiui |eu6|S 1 4 8 CO LO >. >> >. CO CO CO Q Q Q • H • C O CD ~r CM o o o o (oasui) saBeuij y us - soijey Aj|suaju| |eu6js 149 co i n >. >, CO CO CO — D Q m B • (oesui) sa6siui mis - soiiey Aiisua;u| |eu6js 150 T— CO m >, >. >, 03 ro ro Q Q Q • O • o TZ CO Q. D) C 33 « a ^ _ io <S £ i r E o °- "•s & CO M CO CO 75 75 3 «2 > JS TJ co C 3 — +-> CO CO N > a> o> \r o t - o o o o (oasui) SSBBLUI ailS - so i jey Ai isua;u| |eu6js 151 APPENDIX D: STIR IMAGES (DAY 1 [4 Hours Post-Ex], 3 [24 Hours Post-Ex], 5 [72 Hours Post-Ex]) ALL SUBJECTS 1 5 2 1 5 3 IO 08 CO (0 TJ 0) 0) O) as E • mmm CO • • CM o a> I B mQ 3 (0 154  156 157 CO TJ 0 O) E • • =fe O CD JQ 3 (0 158 KO CO 0 E CO • • 5ft o 0 3 CO 159 in 06 co tn > O D) E H CO • • 00 =t= o 0) S B f i H j L n co 160 06 CO 0 U) (0 E a: to • • o 3 CO 161 162 06 CO ro 0) 0 G) ro E (0 =tfc o 0 I B 3 CO 163 164 165 166 167 168

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