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Cytokine and Neurotrophin Response to Acute and Chronic Aerobic Exercise in Individuals with Multiple Sclerosis


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CYTOKINE AND NEUROTROPHIN RESP ONSE TO ACUTE AND CHRONIC AEROBIC EXERCISE IN INDIVI DUALS WITH MULTIPLE SCLEROSIS By VANESSA CASTELLANO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Vanessa Castellano

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This work is dedicated to my parents, Rosa Capdet and Andreu Castellano. Thanks for being amazingly supportive and always be lieving in me. I love you very much. El treball del meu doctorat esta dedicat als meus pares, Rosa Capdet i Andreu Castellano. Gracies per el vostr e support constant i gracies per sempre creure en les meves possibilitats. Us estimo molt.

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iv ACKNOWLEDGMENTS I would like to extend my thanks to my committee chair, Dr. Lesley J. White, for her constant guidance and support throughout my entire doctoral work and my dissertation project. I also would like to thank my committee members Dr. Scott Powers, Dr. John Chow, and Dr. John Petitto for their support throughout the entirety of this project. I am forever indebted to all the me mbers of the Applied Human Physiology Laboratory who helped with the completion of my work (Ashley Blazina, Rachel Canady, Stacey Colon, Jason Drenning, Anna Goodman, Sean McCoy, Darpan Patel, Mai Tran, and Josh Yarrow). I am also indebt ed to Dr. Jessica Staib for her assistance in assay preparation and tro ubleshooting, Kathy Howe for performing the DXA scans and dealing with scheduling conflic ts gracefully, Dr. Mark Tillman and Dr. Chris Hass for statistical assistance, and all the volunteers that participated in the study for their hard work and patience.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Significance..................................................................................................................1 Aims and Hypotheses...................................................................................................5 Aim 1.....................................................................................................................5 Hypothesis 1..........................................................................................................5 Aim 2.....................................................................................................................5 Hypothesis 2..........................................................................................................5 Aim 3.....................................................................................................................6 Hypothesis 3..........................................................................................................6 2 REVIEW OF LITERATURE.......................................................................................7 Significance..................................................................................................................7 Multiple Sclerosis.........................................................................................................8 Cytokine Regulation in Multiple Sclerosis.................................................................10 The Importance of IL-6, TNFand IFNin Individuals with MS...................11 Interleukin-6.................................................................................................12 Tumor necrosis factor...............................................................................13 Interferon...................................................................................................13 Influence of Exercise on Cy tokine Regulation in MS.........................................14 Cytokine response to a singl e bout of exercise in MS.................................15 Cytokine response to chronic exercise training in MS and other diseases...16 Cytokine response to exercise: mechanisms of action.................................18 The Role of Brain-Derived Neurotrophic Factor in Multiple Sclerosis.....................19 Exercise Influence on BDNF Expression............................................................20 Insulin-Like Growth Factor-1 in Multiple Sclerosis..................................................21

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vi Impact of Exercise on IGF-1 in Multiple Sclerosis.............................................22 Exercise Effects on Brain and Peri pheral Concentration of IGF-1.....................23 Muscle IGF-1.......................................................................................................24 Fatigue in Multiple Sclerosis......................................................................................24 Central and Peripheral Fatigue in MS.................................................................26 Exercise May Attenuate Fatigue in Individuals with MS...................................27 General Impact of Regular Exer cise in Multiple Sclerosis........................................28 3 METHODS.................................................................................................................30 Subjects....................................................................................................................... 30 Subject Inclusion/Exclusion Criteria..........................................................................30 Experimental Design..................................................................................................31 Exercise Training Protocol.........................................................................................32 Baseline Measures......................................................................................................33 Graded Exercise Test...........................................................................................33 Single Bout of Aerobic Exercise.........................................................................33 Muscle Fatigue....................................................................................................34 Muscle Strength...................................................................................................34 Quality of Life in Health and Disease.................................................................35 Perceived fatigue..........................................................................................35 Perceived disability......................................................................................35 Quality of life ssessment..............................................................................35 Functional Mobility Assessment.........................................................................35 Six minute walk............................................................................................35 Walking test (25 ft.).....................................................................................35 Walking test (100 ft.)...................................................................................36 Timed up and go test....................................................................................36 Body Composition...............................................................................................36 Post-Exercise Training Measures...............................................................................37 Blood Collection and Processing................................................................................37 Blood Collection Times.......................................................................................37 Cytokine Assessment...........................................................................................38 IGF-1 Assessment...............................................................................................38 BDNF Assessment...............................................................................................39 Plasma Volume Assessment................................................................................39 Statistical Analysis......................................................................................................40 4 RESULTS...................................................................................................................41 Subjects....................................................................................................................... 41 Immune Factors..........................................................................................................42 Chronic Exercise and IL-6...................................................................................42 Single Bout of Exercise and IL-6........................................................................42 Chronic Exercise and TNF...............................................................................43 Single Bout of Exercise and TNF....................................................................44 Chronic Exercise and IFN................................................................................45

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vii Single Bout of Exercise and IFN......................................................................46 Brain-Derived Neur otrophic Factor............................................................................46 Chronic Exercise and BDNF...............................................................................46 Single Bout of Exercise and BDNF.....................................................................47 Insulin-Like Growth Factor-1.....................................................................................51 Plasma Volume...........................................................................................................52 Muscle Function.........................................................................................................52 Knee Extension Endurance of Most Af fected/ Non-Dominant Leg at 180s-1....52 Knee Flexion Endurance of Most Af fected/ Non-Dominant Leg at 180s-1.......52 Knee Extension Endurance of Most Af fected/ Non-Dominant Leg at 90s-1......53 Knee Flexion Endurance of Most Af fected/ Non-Dominant Leg at 90s-1.........53 Isometric Muscle Torque During Extension at 90 of Knee Flexion..................55 Isometric Muscle Torque During Flexion at 90 of Knee Flexion......................55 Isometric Muscle Torque During Exte nsion at 120 of Knee Flexion................55 Isometric Muscle Torque During Fl exion at 120 of Knee Flexion....................55 Isokinetic Muscle Torque at 90s-1......................................................................56 Isokinetic Muscle Torque at 180s-1....................................................................57 Functional Mob ility .....................................................................................................58 Walking Tests......................................................................................................58 Timed Up and Go................................................................................................58 Six Minute Walk..................................................................................................58 Quality of Life in Health and Disease........................................................................59 Perceived Disability.............................................................................................59 Modified Fatigue Impact Scale...........................................................................59 Short Form-36 Quality of Life Questionnaire.....................................................60 5 DISCUSSION.............................................................................................................63 Resting Cytokine Concentration afte r 8 Weeks of Exercise Training........................64 Chronic Exercise May Modul ate Serum BDNF at rest..............................................66 Cytokine and Neurotrophin Response to a Single Bout of Exercise in MS...............70 Cytokine Dynamics after a Single Bout of Exercise...........................................70 Serum BDNF Decreases Following a Single Bout of Exercise..........................72 Muscle Fatigue............................................................................................................74 Functional Mobility....................................................................................................75 Quality of Life in Health and Disease........................................................................76 Perceived Disability.............................................................................................76 Perceived Fatigue................................................................................................76 Short Form-36 Quality of Life Questionnaire.....................................................77 Future Directions........................................................................................................78 LIST OF REFERENCES...................................................................................................80 BIOGRAPHICAL SKETCH.............................................................................................96

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viii LIST OF TABLES Table page 1 Subject characteristics pre and pos t 8 weeks of aerobic exercise............................41 2 Fatigue measures pre and post 8 week s of aerobic exercise during at 180sec-1.....54 3 Fatigue measures pre and post 8 we eks of aerobic exercise at 90sec-1...................54 4 Isometric strength measures pre and post 8 weeks of aerobic exercise at 90 and 120 of knee flexion.................................................................................................57 5 Functional measures pre and pos t 8 weeks of aerobic exercise...............................59 6 Self-Assessed Measures pre and pos t 8 weeks of aerobic exercise.........................60

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ix LIST OF FIGURES Figure page 1 Experimental Design................................................................................................32 2 Blood collection times before and afte r 30 minutes of cycle ergometry at 60%VO2peak...........................................................................................................38 3 IL-6 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST) in both groups.......................................................................................................................42 4 IL-6 plasma concentration during a si ngle bout of exercise in both groups............43 5 TNFplasma concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST).......44 6 TNFplasma concentration during a singl e bout of exercise at 0 (PRE)...............45 7 IFNplasma concentration at rest at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic exercise training...........................................................................................46 8 IFNresponse to a single bout of exer cise in MS and control subjects..................48 9 BDNF concentration at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic exercise training.......................................................................................................49 10 BDNF acute response to exercise in MS subjects at weeks 0 (PRE), 4 (MID) and 8 (POST)..................................................................................................................50 11 BDNF acute response to exercise in cont rol subjects at weeks 0 (PRE), 4 (MID) and 8 (POST)............................................................................................................50 12 IGF-1 concentration at rest at w eek 0 (PRE), 4 (MID) and 8 (POST).....................51

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CYTOKINE AND NEUROTROPHIN RESP ONSE TO ACUTE AND CHRONIC AEROBIC EXERCISE IN INDIVI DUALS WITH MULTIPLE SCLEROSIS By Vanessa Castellano May 2006 Chair: Lesley J. White Cochair: Scott K. Powers Major Department: Applied Physiology and Kinesiology Multiple sclerosis (MS) is an autoimmune di sease that results in progressive neural degeneration. Cytokines and neurotrophic f actors play an important role in the pathogenesis and treatment of MS. Exercise may modulate immune variables known to impact disease progression and neuroprotecti on. We studied the impact of 8 weeks of aerobic exercise on the immune and neurotr ophin response, perceived and muscular fatigue, functional mobility and quality of life measures in MS and matched controls subjects. Subjects performed 30 minute s of cycle ergometry at 60% of VO2peak, three times a week for 8 weeks in a supervis ed environment. Our results revealed improvements in fitness parameters, but no changes in body composition, weight, waist to hip ratio and body mass index. We found d ecreases in resting plasma IL-6 and increases in resting plasma TNFand IFNfollowing 8 weeks of aerobic training in MS subjects. Serum BDNF concentration at re st before the initiation of the exercise

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xi program was significantly reduced in MS subj ects compared to matched controls. Serum BDNF concentration at rest followed a bipha sic response to exercise training and was elevated 4 weeks into the training program and returned to baseline levels following 8 weeks of aerobic training in MS subjects. Serum IGF-1 at rest, muscular fatigue and strength remained unchanged following 8 w eeks of aerobic exercise. A single bout of exercise revealed similar patterns of cytoki ne dynamics between MS and control subjects, while BDNF clearance from the circulation was di fferent at different st ages of training in MS subjects but not in control subjects. Perc eived disability was significantly reduced following 8 weeks of aerobic training while perceived fatigue and quality of life measures had marginal improvements. Our study suggests that exercise may modulate immune and neurotrophin mechanisms in i ndividuals with MS after a short exercise intervention. Although highly specu lative, the impact of exerci se on these parameters is important because it could lead to ne uroprotection in i ndividuals with MS.

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1 CHAPTER 1 INTRODUCTION Significance Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS) and causing axonal demyelinati on that develops into a wide range of debilitating symptoms (1-4). Accumulating evidence suggests that immune mechanisms are involved in the initiation and perpetuation of the patholog ical characteristics of MS (5, 6). In addition, females are more likely to develop MS than males (ratio ~2:1) (7), which is a common trait in autoimmune di seases. Further, most people develop MS between the ages of 18-50 years (7). Because of the heterogeneity of demyelinating lesions across individuals, the symptoms can vary widely, including loss of sensation, optic neuritis, cognitive impairment, pai n, bladder dysfunction, muscle weakness and excess fatigue (4, 7). The most commonly re ported symptoms are muscle weakness and fatigue, which affect 80% of individuals with MS (2). Although the etiology of MS remains unknow n, repeated autoimmune attacks on the CNS are thought to be responsible for infl ammatory damage to axons and subsequent disability in individuals with MS (8). In particular, pro-inflammatory cytokines influence the disease cascade that causes degeneration in MS (9, 10). Pro-inflammatory cytokines such as tumor necrosis factor(TNF) and interferon(IFN) activate nave T cells and other antigen presenting ce lls that disrupt blood brain barrier (BBB) protection (4, 9, 11). High circulating levels of pro-inflammatory cytokines al so stimulate T cell migration through the BBB by upregulating endothelial receptor expr ession such as the VLA-4

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2 receptor (11). These events are critical fo r the initiation of BBB infiltration and the eventual axonal damage in MS (3, 9, 11-13). Therapies that attenuate pro-inflammatory cytokines may reduce BBB disruptions and t hus reduce axonal damage and attenuate disease activity in individuals with MS. Regular exercise has been shown to modul ate proand anti-inflammatory balance, and therefore maintain immune homeostasi s in healthy populations (3, 14, 15). In diseased populations, the imp act of exercise on the immune system is mostly unknown. However, a few studies have shown that exer cise has the potential to reduce systemic inflammation (16, 17). Patients with cardiovas cular disease experien ced a decrease in pro-inflammatory cytokines after an aerobic exercise training pr ogram (17). Further, Castaneda et al. (16) (2004) found that interleukin-6 (IL-6) was reduced in patients with kidney disease subjects undergoing 12 week s of resistance training compared with controls. Furthermore, our laboratory found a significant reduction in plasma concentrations of IFNand TNF(trend) after 8 weeks of resistance training (18). Regular exercise training in MS subject s may have immunomodulatory outcomes that alter systemic inflammation (19). Further studie s to clarify the impact of regular exercise on immune factors such as cytokines are warranted. Recent evidence suggests that in addition to cytokine dysregulation in MS, anomalous signaling of neurotroph ic factors influences neural health (20). Specifically, neurotrophins such as brain-de rived neurotrophic factor (B DNF) and insulin-like growth factor-1 (IGF-1) are critical in preventing cell death, increas ing neural regeneration and stimulating remyelination (21, 22). In dis eases like MS, where neuroprotection is compromised by autoimmune attacks causing neural demyelination and axonal damage,

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3 the maintenance of neuroprotection is important (23). Since elevations in IGF-1 have the potential to aid remyelinati on (24) and serum BDNF concen tration may be lower in MS compared to control subjects (23), therapies that increase IGF-1 and BDNF concentrations may be neuroprotective. Ex ercise stimulates BDNF and IGF-1, both which are known to be neuroprotective (22, 25-27). Exercise may increase circulating IGF-1 in the periphery, which is also an upstream mediator of BDNF (28). IGF-1 administration in the animal model of MS reduces the number of blood brain barrier disruptions and demyelinating lesions (29) Although IGF-1 concentr ation may be normal in MS subjects (30), increases in end ogenous IGF-1 may promote oligodendrocyte development, stimulate axonal sprouting and re pair of damaged axons (31). Exercise also increases BDNF levels in humans and c ontributes to neuroprotection (32). After immobilization stress in animals, Adlard a nd Cotman (2004) (25) found that 3 weeks of running can attenuate muscle atrophy in ra ts through elevations of BDNF secretion. Regular exercise may, therefore, represent a non-pharmacological stimulus to increase IGF-1 and BDNF secretion in individual s with MS where neuronal health is compromised (4, 23, 29). Continuous immune attacks that compromise neural health cont ribute to further disability and increased severi ty of symptoms such as fatigue in individuals with MS. Excessive fatigue is one of the most common debilitating symptoms, affecting approximately 80% of individuals with MS (2). Fatigue contributes to the morbidity associated with MS by limiting endurance and by adversely affecting mood, outlook and ability to cope with accompanying symptoms (2). Fatigue in MS may manifest itself in a variety of forms, including acute fatigue lo calized to specific muscle groups and

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4 persistent central fatigue th at have adverse effects on bot h physical and mental activity (2, 33, 34). The pathophysiology of fatigue is poorly understood in MS. A number of mechanisms have been implicated, includi ng reduced frequency of action potential propagation in partially demyelinated or de generated central moto r axons (central), increased demands for muscle activation, and reduced muscular oxidative capacity due to poor physical fitness (peripheral) (2). Regular exercise has been shown to a ttenuate fatigue in healthy (35, 36) and diseased populations (37, 38). In fact, recent findings suggest that regular exercise may attenuate perceived fatigue in MS subjects (39-41). However, the mechanisms for these effects remain speculative. Further research is warranted to address potential benefits of exercise on MS-related fatigue. Multiple sclerosis is an autoimmune dis ease that results in progressive neural degeneration. Moreover, cytokines play an important role in the pathogenesis and treatment of MS. Therapies that modulate cytokine expression may reduce disease activity. In fact, current dr ug therapies for MS are aimed to modulate inflammatory factors. In theory, exercise may modulate immune variab les known to impact disease progression. Further, exercise may enhan ce neuroprotection through IGF-1 and BDNF production. Lastly, regular exer cise training may attenuate fatigue, preventing a cascade of events that could lead to inactivity, whic h is detrimental for the over all health. The influence of exercise on cyt okine regulation, neurotrophin se cretion and fatigue in MS subjects remains unclear and fu rther investigations are warranted to address these issues.

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5 Aims and Hypotheses The purpose of this investigation is to determine whether aerobic exercise modulates IL-6, TNFand IFN, neurotrophin secretion, and fatigue in MS and matched controls after acute (30 mi nutes of aerobic exercise at 60%VO2max) and chronic aerobic exercise (30 minutes/3 times per week at 60%VO2max for 8 weeks). Aim 1 To determine whether resting levels of circulating cy tokines (IL-6, TNF, and IFN), BDNF and IGF-1 will be altered following eight weeks of aerobic exercise training in MS and matched control subjects. Hypothesis 1 Resting plasma cytokine concentration (IL-6, TNF, and IFN) will be reduced after eight weeks of aerobic exercise, and se rum BDNF and IGF-1 will be elevated in MS and matched control subjects. Aim 2 To determine if an eight week aerobic exercise training program will alter the cytokine (IL-6, TNFand IFN) and BDNF response to an acute bout of aerobic exercise (30 minutes of ae robic exercise at 60% VO2max). Hypothesis 2 The cytokine and neurotrophin response to an acute bout of aerobic exercise (30 minutes of aerobic exercise at 60% VO2max) following an eight week aerobic training program will be normalized in MS subjects. The cytokine and neurotrophin response will be similar between MS and matched healthy co ntrol subjects due to the regulatory impact of chronic exercise on individuals with MS.

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6 Aim 3 To test the effect of eight weeks of aerobic exercise on fatigue in individuals with MS. Hypothesis 3 Muscular fatigue (voluntary muscle fa tigue protocol) and perceived fatigue (Modified Fatigue Impact Scale) will be at tenuated in MS subject s following eight weeks of aerobic exercise training.

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7 CHAPTER 2 REVIEW OF LITERATURE Significance Multiple sclerosis pathophysiology, cytoki ne regulation, neurotrophin secretion, and fatigue in persons with MS will be discussed in this chapter. The impact of acute and chronic aerobic exercise on these variable s will also be adressed. Immune system dysregulation in individuals w ith MS triggers a cascade of events that lead to demyelination and axonal damage (4). Because of these immune attacks, neuroprotection can be compromised and consequently lead to further disability including excessive fatigue and muscle weakness (9, 10). One of the goals of current MS immunomodulatory drug therapies is to reduce inflammation by re ducing pro-inflammatory cytokines. Thus, therapies that minimize excessive infl ammation and enhance neuroprotection may influence disease progression. Regular aerobic exercise has been shown to modulate immune factors such as pro and anti-inflammatory cytokines (19, 42, 43), increasing neuroprotection through elevations of IGF-1 and BDNF secretion (28, 44, 45). Exercise has also been shown to attenuate perceived fatigue in healthy (35, 36) and dis eased populations (37, 38). However, the impact of exercise training on disease variables in persons with MS remains mostly unexplored. Therefore, addi tional investigations are warranted to ascertain the impact of acute and chroni c aerobic exercise on immune factors, neurotrophins and fatigue in individuals with MS.

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8 Multiple Sclerosis Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) causing demyelination and axonal damage that may lead to increasing disability (1). Multiple sclerosi s is the most common inflammatory demyelinating disease in young adults, affecting about 1 of 2000 individuals in the Western Wo rld (4), with females more likely to develop MS than males (ratio ~2:1). The onset of MS is typically between the ages of 18-50 years (7). Much of the permanen t disability results from axonal destruction on long pathways such as the pyramidal tr act supplying the legs and dorsal columns carrying sensory information from the legs (46). Because of the diversity in lesion volume and location across individuals with MS, a variety of symptom expression is possible. In some individuals, symptoms may be minimal, while others may experience extensive loss of sensation, slurred speech, muscle weakness, fatigue, depression, optic neuritis, cognitive impairment, pain and bla dder dysfunction (4, 7). Severe fatigue and muscle weakness are two of the most reported symptoms in MS and have a large impact on the quality of life of these patients (2). In addition, individuals with MS are typically sedentary relatively to hea lthy individuals and often exhi bit decreases in functional capacity (47). Consequently, decreases in physical activity could also in crease the risk of secondary diseases such as cardiovascular disease, diabetes and obesity (47-49). The etiology of MS is currently unkn own. However, genetic predisposition, autoimmune attacks, and environmental factors are thought to be the ca use of disease (1). In addition, MS is characterized by great vari ability and can be divided into different clinical subtypes, including eith er a relapsing course or a mo re severe progressive course (46). Approximately 85% of individuals with MS begin with the relapsing remitting clinical subtype (46), which is characterized as clearly defi ned disease relapses with full

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9 recovery or with sequelae, and with periods between disease relapses characterized by lack of disease progression (50). Those with relapsing remitting MS experience neurologic attacks with variab le recovery but are clinically stable between the attacks. Among this group are a minority of individuals th at will have benign/mild MS with little to no disability into their course (46). I ndividuals with benign/m ild MS make up 10 to 20% of persons with MS (46). Benign MS is characterized by disease in which the patient remains fully functional in all neurologic syst ems fifteen years afte r disease onset (50). Further, there are three progres sive subtypes. Approximately 10% of individuals with MS have the primary progressive clinical subt ype and never experience any attacks, and about 5% have progressing relapsing, whose attacks superimpose a progressive course (46). Primary progressive MS is defined as disease progression from onset with occasional plateaus and temporary minor im provements (50). Secondary progressive is the major progressive form of MS and account s for 30% of the pati ents (46). Secondary progressive MS is defined as initial relapsing remitting disease course followed by progression with or without relapses, minor remissions and plateaus (50). Various degrees of inflammation followe d by axonal degeneration are responsible for the large array of symptoms and different disease courses in individuals with MS. Cytokine research has generated much a ttention and is the focus of therapeutic interventions in MS (51). Th e effects of many therapies for MS are believed to be mediated by changes in cytokine concentr ation. Multiple sclerosis immunomodulatory drugs such as Avonex and Rebif target inhibition of pro-inflammatory cytokines to reduce inflammation (51, 52) For example, Avonex decreases IFNproduction and T cell activation in the periphery of individuals with MS. Ther efore, strategies to reduce

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10 inflammation and minimize axonal damage may influence disease progression and attenuate MS symptoms. Cytokine Regulation in Multiple Sclerosis Cytokines are important initiators and re gulators of the immune system and are produced by leukocytes and other cells that stimulat e proliferation and differentiation of various immune cells (53). Cyt okine concentrations are typica lly low or absent in healthy individuals at rest because th ey are only produced in small quantities in response to local stimuli, such as the presence of antigens, e ndotoxins, or the transduction signals provided by other cytokines (6). Upon en countering an antigen, T cells differentiate into functional dichotomous subsets, depending on the microe nvironment the cell encounters (6). CD4+ helper cells are classified then into T help er 1 (Th1) and T helper 2 (Th2) cells depending on the type of cytokines they produce. Th1 cel ls secrete high levels of tumor necrosis factor(TNF) and interferon(IFN), promoting macrophage activation and antibody-dependent cell cytotoxicity (13). Th2 cells produce interl eukin (IL)-4, IL-6 and IL10, and promote humoral immune responses against extracellula r pathogens (6). Accordingly, cytokines can be classified as Th1 or Th2 cytokines. However, cytokines are also classified according to their e ffects on cell immunity. Cytokines promoting cellular immunity are considered pro-in flammatory cytokines and include IFN, TNF, TNF, IL-2, IL-12, IL-15, IL-17, and IL-18 (6). Cytokines promoting downregulation of cell immunity are considered anti-inflammatory and include IL-4, IL-6, IL-10 and IL-13. However, almost all anti-inflammatory cytoki nes have some pro-inflammatory properties and vice versa (6). The blood brain barrier (BBB) is regarded as a protective system from immune attacks. However, T cell mediated immunological processes may lead to alteration of the

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11 BBB and enable recruitment of other inflammatory cells, such as monocytes (54). Cytokines are critical in the mechanism of T cell activation that can ultimately cause demyelination and tissue damage in MS (55) In the periphery, inflammatory cytokines induce, in a bystander fashion, activation of monocytes, dend rite cells (DCs) and T cells (9). For example, DCs undergo maturation into antigen presenting cells (APCs) after their exposure to pro-inflammatory cytokines and consequently are able to activate T nave cells (54). Dendritic cells th at are activated by inflammato ry cytokines rapidly activate other innate protective cells such as natura l killer (NK) cells to mediate the balance between Th1 and Th2 subsets (56). However, T cel ls may also be in an enhanced state of activation in the periphery of individuals w ith MS, suggesting a breach in the protection of the BBB, and facilitating an overproduction of pro-inflammatory cytokines (Th1 subset) (10). In addition, cytokines produced by activated T cells in MS lesions induce the activation of m acrophages and local microglia effector cells, leading to the increase of axonal destructive activity, which is responsib le for demyelination and tissue damage in MS (1). Therefore, a reduction of pro-infl ammatory cytokine levels in the periphery could attenuate the rate of nave T cell activat ion and interrupt the cascade of events that lead to demyelination and axonal damage in the CNS of individuals with MS. The Importance of IL-6, TNFand IFNin Individuals with MS Preliminary studies revealing the influence of cytokines in MS disease activity has facilitated extensive research to further elucidate mechanisms responsible for immune attacks, and thus potential treatment ther apies. It is also thought that immune dysregulation is the main cause of the demyelination process and axonal damage observed in MS (10). A dysregulation of th e balance between pro-inflammatory (Th1) and anti-inflammatory (Th2) cells with a sh ift to Th1 profile has been reported in

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12 individuals with MS (57). A variety of cytokines modulat e Th1/Th2 balance that is critical to maintain immune homeostasis ( 11, 58). However, the influence of cytokine dysregulation in MS rema ins somewhat unclear. Interleukin-6 (IL-6), Tumor Necrosis factor(TNF) and Interferon(IFN) have a prominent role in the process of de myelination and axonal damage experienced by persons with MS (59). These pro-inflamma tory cytokines may also stimulate T cell activation in the periphery that leads to demyelination (10), and have appeared repeatedly in areas of inflammatory demyelination in the form of perivascular infiltrations within the white matter of the brain of persons with MS (59). The main functions of these cytokines, in relation to MS, are discussed in more detail below. Interleukin-6 Interleukin-6 (IL-6) is produced by many different cells, but the main source in vivo are monocytes, macrophages, fibroblasts and vasc ular endothelial cells (60). Further, IL-6 has been shown to have both pro-inflamma tory and anti-inflamma tory effects, but recently it has become recognized for its an ti-inflammatory properties (42). Abnormally high concentrations of IL-6 in the periphe ry may result in excess inflammation, which may be harmful to the host, and exacerbate auto immune disease activity (6). IL-6 also has immunosupressive effects such as the inhibition of TNFexpression by macrophages and astrocytes. Elevated IL-6 may also disr upt the clearance of mi crobial pathogens (6) and participate in T cell activation, accel erating the MS disease process (5, 6). Circulating IL-6 appears to be the primary inducer of acute phase proteins from the liver, and is also involved in mediating interactions between the endocrine and the immune systems (59). In addition, skeletal muscle is another source of IL-6 production (42). Skeletal muscle contra ctions stimulate IL-6 producti on and may increase circulating

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13 IL-6 concentration via complex sign aling cascades initiated both by Ca2+ dependent and independent stimuli (61). Thus far, it is unclear whether individuals with MS have abnormal levels of IL-6 in skeletal muscle and in the periphery (62), and the impact of this cytokine on regeneration and degenera tion of neurons of individuals with MS warrants further study (63). Tumor necrosis factorTumor necrosis factor(TNF) is produced by macrophages, T cells and B cells (64). Elevated TNFreceptor expression on T cells and in soluble form has also been found in individuals with MS (65). TNFhas been shown to promote demyelination of neurons in the brain and it is al so thought to play a role in muscle wasting that occurs with chronic infections (66). High circulating TNFconcentration in s ubjects with MS is associated with worsening of the disease (67, 68). TNFcan also be expressed in skeletal muscle as a consequence of cach exia (66). However, the impact of TNFin skeletal muscle of MS subjects remain s unknown. It is known however that high TNFplasma concentrations are also associated with low muscle mass and lower muscle strength in frail individuals (69). Given the diverse and potent effect of TNFon the immune and muscle function of MS subjects, stra tegies to reduce TNFmay contribute to reduction in disease severity (64). Since TNFis an important cytokine involved in causing axonal demyelinating lesions in MS subjects, further investigations are warranted. InterferonInterferon(IFN) is secreted mainly by lympho cytes and synthesized primarily by T lymphocytes and natural killer cells following activation of immune and inflammatory stimuli (70). IFNis a major disease promo ting cytokine in MS (64).

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14 Administration of IFNto MS subjects worsens diseas e activity (71). In addition, IFNcorrelates with functional impairment in MS (55). Further, IFNcan induce APCs to secrete high levels of other pro-inflammato ry cytokines by virtue of its ability to differentiate nave T cells into Th1 cells (72). These events may therefore accelerate excessive T cell infiltration th rough the blood brain barrier and thus contribute to further demyelination in persons with MS. Elevations of plasma IFNin MS compared to control subjects have also been reported by Link et al. (73) (1999) and in our laboratory ( 18). Further, IFNhas been found to promote muscle wasting and could c ontribute to the deteri oration of skeletal muscle in MS subjects (66). Therefore, IFNis integrally involved in the MS disease process, and inhibiting IFNsecretion or antagonizing its actions could have important outcomes preserving skeletal muscle and modul ating the immune system of individuals with MS (74). Influence of Exercise on Cy tokine Regulation in MS Cytokine regulation following acute a nd chronic exercise remains relatively unexplored in MS subjects. Exercise modulates immunological responses through cytokine production in short bouts of exerci se in healthy populations (42, 53, 75-77). Cytokine dysregulation is linked to the in flammatory processes observed in MS (78). Inflammatory cytokines increase T cell infiltrat ion by activating a series of events that lead to disruption of the bl ood brain barrier leading to further axonal damage (8). Therefore, because of its immunomodulatory potential, exercise may impact cytokine dysregulation in individuals with MS.

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15 Cytokine response to a single bout of exercise in MS It has been proposed that short term rel ease of cytokines durin g acute exercise may contribute to the maintenance of an immune homeostatic environment (79). In addition, many of the acute phase proteins released in response to elevated cytokine levels are protease inhibitors or free radical scavenge rs that attenuate th e magnitude of tissue damage associated with release of toxic molecules and free radicals due to activated neutrophils (79). Therefore, a single bout of exercise repr esents a mild physical stressor and has an array of effects on immune parame ters (76, 80). Consequently, the release of cytokines during and after moderate intens ity exercise bouts could contribute to neuroprotection (80). Moreover, studying the immunological response to a single bout of exercise in MS subjects may yield important information re garding the immediate effects of exercise on autoimmune diseases and how MS subjects respond to stress in general. Early work by Le Page et al (81)(1994) investigated the effect of exercise on the inflammatory phase of experimental autoim mune encephalomyelitis (EAE), the animal model of MS. Exercise training during the i nduction phase of EAE did not exacerbate the disease course (81, 82). In a study by Heesen et al. (19) (2003) se dentary MS subjects showed a blunted cytokine response (i.e., TNF) after a single bout of exercise (30 minutes of cycling at 60% of their VO2max). However, the acute re sponse of other pro and anti-inflammatory cytokines such as IL-6 or IFNwere not measured. Further, the impact of exercise on baseline cytokine concen trations was not inves tigated. Schulz et al. (76) (2004) investigated the impact of acu te exercise on IL-6 but did not found any significant differences between MS subjects and controls. Further, both Heesen et al. (19) (2003) and Schulz et al (76) (2004) collected blood samp les immediately after exercise and 30 minutes later with no additional co llection times. The timing of blood sample

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16 acquisition is important because the dynamics of each cytokine vary considerably in response to exercise (53). Therefore, their co llection time may have not been sufficient to identify the maximal responses of these immune parameters. For example, Pedersen et al. (42) (2003) have published results showi ng IL-6 peaks approximately 2-3 following aerobic exercise. Pilot data from our laborat ory shows that following a moderate bout of aerobic exercise, IL-6 concen tration peaks 2-3 hours af ter exercise (unpublished). Further, Heesen et al. (77) (2003) and Schultz et al.(76) (2004) did not correct for plasma volume changes that might had occurred dur ing the 30 minute exercise bout. Plasma volume is typically reduced 3-14% after 30 minu tes of moderate inte nsity exercise (83, 84). Additional studies are needed to provide a more complete and comprehensive understanding of the dynamic cytokine respon se to physical stress in MS. A special attention should be focused on IL-6, IFNand TNFbecause they are known to directly influence the pathogenesis of MS. Cytokine response to chronic exercise training in MS and other diseases Chronic exercise has been found to modul ate the immune system in healthy (53, 80) and some diseased populations such as ca rdiovascular and kidney disease (16, 17). In a study by Smith et al. (17) (1999), six mont hs of aerobic exercise lowered cytokine secretion of IFNin the periphery of subject s with cardiovascular disease. Further, Castaneda et al. (16) (2004) found that resti ng concentration of plas ma IL-6 was reduced in patients with kidney disease subjects undergoing 12 weeks of resistance training compared with controls. Furthermore, our laboratory showed a significant reduction in plasma concentrations of IFNand TNF(trend), as well as no change in IL-6 concentration after 8 weeks of resistance trai ning (18). Whether ex ercise consistently reduces levels of circulating inflammatory cytokines in MS s ubjects remains unclear.

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17 However, these data suggests that chronic exercise may influence baseline concentration of cytokines known to impact infl ammatory processes in MS. Little is known about the e ffect of chronic aerobic exer cise on immune parameters such as cytokines in autoimmune diseases such as MS (85). To date, there only two published reports on the effect of aerobic exercise on immune parameters of MS subjects (19, 76). In a study by Heesen et al. (19) (2003) sedentary MS subjects showed a blunted cytokine response after a single bout of exercise (30 minutes of cycling at 60% of their VO2max) in the untrained state compared to co ntrols. However, after an eight week aerobic exercise training program the blunted response to a si ngle acute bout of exercise was similar to controls. Therefore, Heesen et al. (19) (2003)showed that aerobic exercise trained MS subjects can promote an immune re sponse to physical stress that is similar to healthy individuals. However, Heesen et al (19) (2003) did not report the effect of chronic exercise training on re sting cytokine concentration, a nd therefore, the impact of exercise training on circulating cytokine con centration in MS subjects remains unclear. Schulz et al. (76) (2004 ) investigated the impact of ch ronic exercise on IL-6 and found that aerobic training for 8 w eeks (30 minutes of cycle ergometry, 3 times/week at 60% VO2max) did not affect resti ng concentration of IL-6. Additional information about the influence of exercise on MS disease activity has come from research using the animal mode l of MS. Le Page et al. (81) (1994) investigated the effect of exercise on the inflammatory phase of experimental autoimmune encephalomyelitis (EAE). Exerci se training in EAE rats did not aggravate the disease course (81, 82). Findings after a 10-day exercise-train ing regimen performed immediately following the induction of EA E included a reduction in the duration and

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18 severity of chronic EAE in rats (81). Further investigations on the impact of exercise on EAE and MS are warranted, since Le Page a nd colleagues did not probe mechanisms of the delayed onset, severity and duration of EA E. Their work suggest that exercise can modulate disease pathophysiology (81). Howe ver, the mechanisms of action remain unknown, but likely involve modulation of the immune system. In summary, cytokine dysr egulation appears to cause inflammation and subsequent demyelination and axonal damage in MS s ubjects that may impact disease progression and severity. Acute and chronic aerobic exercise may modulate immune function by reducing circulating levels of cytokines, a nd therefore influence di sease activity in MS subjects. Cytokine response to exercise: mechanisms of action The cytokine response to acute exercise is complex and it is related to exercise intensity, training status, site of cytokine measurement (i.e., tissue, plasma or urine) and method of measurement (53). Cytokines appear in low concentration (<3pg/ml) in plasma of healthy individuals at re st (86, 87). The time course of cytokine elevation or depression in response to exercise differs depending on the cytokine of interest. For example, IFNand TNFhave been shown to increase immediately after exercise (30 minutes) while IL-6 displays a more delayed response (0-3 hours) (42, 75). A complete understanding of mechanisms responsible fo r the cytokine response to exercise are currently unknown. Early reports suggested that the cytokine response may be due to a pro-inflammatory release produced by musc le damage and perhaps inflammation of skeletal muscle (88-91). However, more recen t studies clearly demonstrate that muscle contraction without any muscle damage can i nduce marked elevations of cytokines (i.e., IL-6) (75). The exact mechanisms mediati ng communication between skeletal muscle

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19 and other cells releasing cytoki nes during and after exercise has not been elucidated, but are most likely multifactorial. In fact, a single cytokine rarely acts alone (53). Typically, these actions depend on the level of cytoki ne produced, endogenous inhibitors and regulators, and interactions with other cytokines (53). Fu rther investigat ion of these events will enhance our understa nding of the impact of ex ercise on the immune system. The Role of Brain-Derived Neurotrophi c Factor in Multiple Sclerosis Brain-derived neurotrophic f actor (BDNF), a member of th e neurotrophic family, is a homodimeric protein that has been highly c onserved in structure and function during evolution (92). BDNF is widely expressed th roughout the brain cortex and also in the periphery in many animal species (93-96). BDNF participates in cellular maintenance and protects neurons from injury (97, 98). BDNF also plays a predominant role in neural development and brain health (99) BDNF acts as a s hort-term potent excitatory neurotransmitter leading to rapid depolarizat ion of postsynaptic ne urons (95). Further, BDNF induces long lasting cha nges in synaptic plasticit y, and plays a key role in learning, memory and behavior (100, 101). It has been demonstrated that BDNF can cross the BBB, suggesting that serum BDNF levels may reflect BDNF levels in the brain (102). Potential s ources of circulating BDNF are platelets, vascular endothelial and smooth muscle cells (103). However, since it is known that BDNF can cross the BBB in both directions, a s ubstantial part of circulating BDNF could also originate from ne urons and glia cells of the central nervous system in a bidirectional fashion (104). During an immune attack, BDNF may protect axons from demyelination and also facilitate remyelination afte r injury in MS (105). Since BDNF concentration may be decreased in MS compared to control subjects (23), the neuroprotective potential of the

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20 brain is jeopardized, and stra tegies to increase BDNF in MS subjects could have important therapeutic outcomes. In fact increases in BDNF production has been proposed as a strategy to increa se neuroprotection in individu als with MS (23). Further, increased resting concentrati on of BDNF may attenuate MS deterioration of nerve and muscle function. Thus, further investigations ar e needed to determine if MS patients have lower concentration of BDNF, and to determin e the effect of exercise on BDNF secretion on MS subjects. Exercise Influence on BDNF Expression There is strong support from the literature indicating that exercise provides brain health benefits by increasing neuroprotecti on and possibly inducing axonal repair through neurotrophin action (21, 22, 25, 32, 106-108). Exer cise is increasingl y recognized as an intervention that can reduce cognitive decline and depressi on, possibly th rough elevated BDNF concentration which maintains neurona l health (25). It ha s been previously demonstrated that voluntary exercise i nduces BDNF upregulation in the brain and periphery helping maintain brain health (i .e., plasticity and memory) (20, 21). For example, exercise enhances the effectiven ess of antidepressant treatment, perhaps by augmentation of BDNF levels in humans (32, 106), along with increa ses in neurogenesis and learning (25). Exercise may also influence clinical populations with neurodegeneration such as Alzheimer’s diseas e or clinical depre ssion (22, 45), and may not only contribute to improved motor and cogni tive function, but also provide resistance to the effects of stress-relate d processes that can occur with in the injured CNS (22, 25). Recent data suggest that BDNF is elevated in exercised muscle (109) and can be retrogradely transported into the spinal cord (110). BDNF also ha s effects on skeletal muscle tissue by inducing the potentiation of spontaneous twitching in myocytes to

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21 enhance muscle contraction (111). In th e periphery, exercise can upregulate the expression of BDNF and maintain skeletal muscle health (25). For example, after immobilization stress, Adlard and Cotman ( 2004) (25) found that exercise can override the negative effects of muscle atrophy in rats thro ugh elevations of BDNF secretion after 3 weeks of running. In BDNF deficient rats, 2 months of wheel running increased BDNF concentration (107). Exercise may, therefore, represent a non-pharmacological stimulus to increase BDNF in MS subjects. In summary, BDNF is critical to mainta in neuronal health, reduce demyelination and may stimulate remyelination in MS subjects. Previous investigations have shown that regular exercise upregulates BDNF in both the CNS and the periphery (20, 21). Therefore, individuals with MS may benefit from regular exercise training because it may increase BDNF concentration, and subseque ntly protect brain function and promote remyelination. Insulin-Like Growth Factor-1 in Multiple Sclerosis Insulin-like growth factor-1 (IGF-1) promotes the surv ival and regeneration of oligodendrocytes, stimulates sy nthesis of myelin, and promot es skeletal muscle health (30). Thus, the regulation of IGF-1 is im portant in MS, where neurodegeneration and muscle atrophy are primary di sease manifestations (112). Several lines of evidence suggest IGF-1 may be beneficial in treating individuals with MS and other demyelinating diseases (113). In the animal model of MS (EAE), treatment with IGF-1 reduces clinical deficits and lesion severity ( 24). In addition, it is known that myelin content rises in the CNS in transgenic mice over expressing IGF-1 (113). In addition, inj ections of IGF-1 in EAE rats have been shown to reduce disease severity and clinical deficits (24). For example, an IGF-1 subcutaneous injection impr oved clinical deficit scores, stride lengths

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22 and exercise wheel rotations within 48 hours of injection (24). In another animal study, after the administration of exogenous IG F-1, EAE onset was delayed, suggesting a decrease or absence of inflammatory cells in the CNS (114). Therefore, it has been suggested that increases in IG F-1 concentration may be useful as a treatment for MS to maintain neural health and promote remye lination (30). MS subjects appear to have normal levels of serum IGF-1, but possible therapies involving this neurotrophin are attractive due to its effect on remyelin ation (29). However, in one study, IGF-1 administration in MS subjects did not influe nce measures of disease status. Seven MS subjects received 50 mg of rhIGF-1 twi ce a day for 6 months, but there were no significant differences between baseline and treatment periods for MRI lesion load or disease activity (29). Perhaps longer admini stration of rhIGF-1 or the promotion of endogenous IGF-1 (i.e., exercise induced) may have more favorable results regarding neuronal repair and remyelination (29). Ther efore, additional studies are needed to investigate the impact of increased endogenous IGF-1 on MS subjects. Impact of Exercise on IGF-1 in Multiple Sclerosis Insulin-like growth facto r-1 provides neuroprotection and may reduce muscle wasting (113). Regular exerci se training is known to stim ulate the production of IGF-1 (28, 44). In turn, IGF-1 can pr omote skeletal muscle protei n synthesis, oligodendrocyte survival, myelin protein synthesis, and myelin regeneration (113). Since exercise promotes IGF-1 production, which further stimul ates favorable changes in both nerve and muscle, further study of the role of exercise training on individuals with MS may yield important clinical information.

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23 Exercise Effects on Brain and Peripheral Concentration of IGF-1 Following moderate exercise training in rats IGF-1 levels increase in both brain and periphery, which may subsequently prom ote remyelination in rats (28). Neurons influenced by increased IGF-1 show elevated spontaneous firing and increased sensitivity to afferent stimulation (28). Furthermore, a systemic inj ection of IGF-I mimicked the effects of chronic exercise in the brain in th ese rats. When uptake of IGF-I by brain cells stopped, neuroprotection was lost Carro et al. (28) (2000) concluded that serum IGF-I mediates the initiation of events that causes the positive impact of exercise on the brain. Thus, stimulation of the uptake of bloodborne IGF-I by nerve cells may lead to neuroprotection. However, the mechanism wher eby exercise increases contributes to neuroprotection remains unclear. Although central mechanisms are pivotal in exercise-induced neuroprotection of the brain, it is now emerging that periphera l mechanisms may also play a significant neuroprotective role through IGF-1 (44, 115). Several re ports suggest that exerciseinduced elevations in periphera l IGF-1 initiate growth-factor cascades in the brain that could lead to remyelination (28, 44). Circulati ng IGF-1 also acts as an upstream mediator of BDNF regulation, neurogenesis, and the ability of exercise to protect the brain from neuronal injury such as demyelination (28, 44). It has been suggested that exercise training can improve memory and information processing efficiency through mechanisms that include the upregulation of IGF-1 and BDNF, and thus po tentially be beneficial in MS subjects where memory and informati on processing may be compromised (21, 116). According to previous findings, central and peripheral elevations in IGF-1 promote neuronal health, and may also aid in the re pair of demyelinated neurons found in MS

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24 subjects (28, 117). Therefore, exercise-induced increases in IGF-1 may play an important role in protecting the CNS fr om injury in MS subjects. Muscle IGF-1 IGF-1 is recognized for its role in skeletal muscle adaptation to exercise (118). It is well documented that IGF-1 has potent effects on myoblast proliferation and differentiation, leading to muscle hypertr ophy (116). Exercise increases muscle IGF-1 content and causes a net increa se in protein (116). The exer cise-induced elevation in IGF1 may prevent myofiber wasting due to disuse and age-associated m yofiber loss (118) in MS subjects. Ultimately, exercise-induced hypertrophy through IGF-1 mechanisms may be needed to produce sufficient levels by the target muscles to ensure maintenance of healthy innervation (116). Ther efore, individuals with MS could benefit from exerciseinduced secretion of IGF-1 to maintain muscle health. In summary, aerobic exercise training may elevate IGF-1 concentration in a variety of sites (i.e., brain, peripher al circulation, skeletal muscle ) and stimulate remyelination, improvements of brain cognition, and muscle hypertrophy, which may ultimately reduce disease progression in persons with MS (21, 116). Therefore, regular aerobic exercise training could potentially c ontribute to functional improve ments in the MS population. Fatigue in Multiple Sclerosis Excessive fatigue and muscle weakness are the most common and debilitating symptoms of individuals with MS (2). Fatigue in MS is defined as an abnormal sense of tiredness or lack of energy, out of proportion to the degree of e ffort or level of disability that significantly interferes with the routin e physical or intellectual functioning (2). Thus, MS-related fatigue is an unusual and abnormal form of fatigue that differs from the fatigue experienced by healthy i ndividuals after exertion (failu re to maintain the required

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25 or expected force). Approxima tely, 65-95% of MS subjects ha ve significant fatigue (2). Fatigue also contributes to the morbidity associated with MS by limiting energy and endurance and by adversely affecting m ood, outlook and ability to cope with accompanying symptoms. The average level of physical activity in individuals with MS has been shown to be reduced compared to their healthy counterparts (119). The mechanisms suggested for excessive fatigue in MS subjects include psychological factors or brain lesi ons in specific neural pathwa ys that may play a role in MS-related fatigue and depression, or that fa tigue contributes to depression in MS subjects (34). Moreover, MS fatigue may be related to demyelination, inflammation and axonal injury (2, 120). It is unclear whether cons tant fatigue in MS s ubjects is due to the burden of diagnosis, reduced activity due to neurological symptoms and not fatigue, or directly related to ot her physiological factors (2, 34). However, it is well known that physical activity, if reduced, can perpetuate adverse changes in muscle strength and fitness in any population. Fatigue in MS may manifest itself in a variety of fo rms, including acute fatigue localized to specific muscle groups and persiste nt central fatigue that has adverse effects on both physical and mental activ ity (2, 33, 34). The etiology of fatigue in MS is not well understood and appears to be complex and multif actorial (2). Both peripheral and central mechanisms have been postulated, but none has satisfactorily expl ained the development of MS fatigue (33). In addition, fatigue is not well explained by gender, psychomatic mechanisms, physical disability, or sleep dysfunction. A recent study by Bakshi (2000) (34) found a significant relationship between depression and fatigue severity. Moreover, functional brain imaging studies indicate that MS is associated with widespread low

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26 metabolism in the brain (121). Specifically, Baks hi et al. (121) (1998) found MS subjects had a 10% reduction in total brain glucose me tabolism. Another study by Roelcke et al. (122) (1997) showed that MS subjects had re duced glucose metabolism with fatigue but not in those individuals wit hout fatigue. However, other studies have shown a lack of association between MS fatigue and neurode generation (brain atr ophy/lesion load) (123, 124). The etiology of fatigue in MS is comp lex and multifactorial. Further research is needed to find effective strategies to attenuate fatigue in MS. Central and Peripheral Fatigue in MS Demyelination, the product of the inflamma tory process that underlies MS, impairs axonal conduction and eventually produces axonal loss and damage (120). Axonal impairment may contribute to centrally me diated fatigue through several mechanisms. For example, delayed or partial innerv ation of voluntary muscles may require a compensatory increase in central motor ex citatory mechanisms (119), and thus, MS subjects may necessitate increased motor driv e to achieve the same levels of muscular contraction. Consequently, MS subjects ma y experience premature fatigue relative to their healthy counterparts (119). Axonal damage has also been correlated with increased fatigue in MS subjects (120). In addition, individuals with MS may have delayed activation of motor units contributing to abnormal communication be tween the cerebral cortex, basal ganglia and cerebellum, and th e descending motor pathways (2). Finally, both physical and mental fatigue may occur simultaneously or independently of each other. The fact that they occur together, al ong with the high frequency of fatigue in MS, points to a possible critica l role of central fatigue in these individuals. Peripheral fatigue is a type of fatigue localized to skeletal muscle, and although individuals with MS seem to experience it often, it may be the result of physical

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27 inactivity or long-term consequences of centr al fatigue (119). Peri pheral fatigue can be caused by negative effects precipitated by inac tivity such as decreased muscle cross sectional area, decreased skel etal muscle strength, muscle wasting, fiber type shifting from Type IIa to Type IIb (125). These change s may lead to an enzymatic shift from slow glycolytic to fast glycolytic muscle fiber characteristics. Due to a reliance of more glycolytic pathways in any activity, and the higher amount of ADP and Pi in the system, Ca2+ reuptake from the SR may be disrupted ( 126). These events can also increase H+ concentration, which can disrupt cr oss-bridge cycling by disrupting Ca2+ attachment to troponin, and therefore ca use peripheral fatigue. Although there is a strong, pl ausible evidence that fatigue is a centrally-mediated complication in MS, the influence of physic al inactivity, patient perception of their impaired capacity and altered innervation of muscles suggest that MS fatigue is multifactorial in origin and may be expressed differently across individuals with MS. Exercise May Attenuate Fatigue in Individuals with MS It is well known that fatigue can be c ounteracted by exercise training in healthy (35, 36) and diseased (37, 38) populations. Consequently, a regular exercise training program may provide a similar stimulus that can attenuate the symptoms of fatigue in individuals with MS. There is evidence indi cating that regular exercise may attenuate perceived fatigue in MS subjec ts (39-41). After 15 weeks of aerobic exercise at moderate intensity, MS subjects experien ced a significant reduction in fatigue (measured with the profile of mood states questi onnaire) and a negative associat ion between improvement in aerobic fitness and fatigue perception (40). As a result of increases in aerobic capacity, MS subjects were able to perform activities of daily living at lowe r relative intensity, preventing excessive tiredness. Using the fati gue severity scale (FSS) for their fatigue

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28 assessment, Mostert and Kesselring (2002) re ported reductions in fatigue (-14%) of MS subjects after only 4 weeks of aerobic exercise training. MS subjects also increased their activity level by 17% after only 4 weeks of regular aerobic exercise (41). Further, White et al. (39) (2004) reported a 24% decrease in perceived fatigue as indicated by the modified fatigue impact scale (MFIS) after 8 weeks of resistance training in MS subjects. In summary, the etiology of MS fatigue is complex and multifactorial. In addition, regular physical activity may attenuate perceived fatigue in individuals with MS. Therefore, fitness improvements experien ced while undergoing an exercise training program may be responsible for the reductions in fatigue, possibly by counteracting the effects of detraining that ar e secondary to MS fatigue. General Impact of Regular Ex ercise in Multiple Sclerosis Regular aerobic exercise trai ning in individuals with MS may help reduce the rate of decline in functional capacity observed in the MS population. In the past, individuals with MS were advised to avoid physical ac tivity because symptoms may worsen with elevations in body temperature (76). Howeve r, many studies suggest that exercise training in MS subjects is sa fe and can promote many important beneficial outcomes, such as improvements in cardiorespira tory and muscle function (40, 41, 112, 127), decreased incidence of depression (40, 127), decreased perceived fatigue (41, 128), and possible regulatory effects on the imm une system of MS subjects (77). MS subjects that completed a 15 week aerobic exercise training program demonstrated significant improvements in aerobic fitness and strength measures compared to non-exercising controls (40). E ach training session consisted of supervised cycle ergometry for 30 minutes at 60% of VO2max, three times a week for 15 consecutive weeks. MS subjects experience d significant increases in physical capacity and maximal

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29 isometric strength, as well as significant decreases in body fat. Specifically, a 10% increase in VO2max was observed after only 5 weeks and an increase of 25% after 15 weeks of aerobic training. Petajan et al. ( 40) (1996) concluded th at aerobic exercise training resulted in improved fitness and ha d a positive impact on factors related to quality of life. Ponichtera-Mul care at al. (129) (1997) also demonstrated an improvement in aerobic capacity between 5-20% after 6 months of aerobic training using cycle ergometry at 60% VO2max. Mostert and Kesselring (2002) (41) also investigated the effects of aerobic exercise for 4 weeks on MS subjects. They concluded that 30 minutes of supervised cycle ergometry five times a week provided improvements in health perception, improved aerobic fitness, increase s in activity level a nd a tendency to less fatigue (41). These results confirm that individuals w ith MS are capable of making favorable adaptations to aerobic exercise training. Although the effects of exercise training on MS disease progression still remain unknown, the over all health benefits of exercise alone, provide a healthy means to maintain or impr ove quality of life in individuals with MS.

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30 CHAPTER 3 METHODS Subjects Twenty seven subjects were recruited fr om the local community. Each subject had physician clearance and signed a consent form approved by the University of Florida Institutional Review Board. Eleven individuals with MS and eleven non-MS healthy controls completed the entire study. Subject Inclusion/Exclusion Criteria Subjects with relapsing remitting MS, who we re clinically stable and had minimal to moderate disability, were included. Mu ltiple sclerosis was diagnosed by a physician according to the Poser criteria (130). A disabi lity status of 0-5.5 (EDSS 0-5.5: minimalto-moderately disabled with ability to walk at least one city block (100 meters)) was required for MS subject study inclusion. All subjects (MS and contro l subjects) needed physician clearance to participate in the study, a systolic blood pressu re of less than 140 mmHg, and a diastolic blood pre ssure of less than 90 mmHg. Subjects with cardiovascular disease, diabetes, thyroid di sorders, gout, and orthopedic limitations as estab lished by the American College of Sports Medicine (131) were excluded from the study. Additiona lly, individuals using prednisone or antispasmotic drugs were excluded. If a subjec t had a relapse, they were excluded from the study. A relapse (attack, exace rbation) was defined as a separate period of worsening in a neurological symptom lasting 24 hours or more after a preceding month of stationary

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31 or improving status. Subjects that were not able to pedal for 20 minutes at 60% of their VO2peak were also excluded. Experimental Design The study consisted of an eight week aer obic exercise training program wherein subjects exercised on a cycle ergometer 3 times weekly for 30 minutes at 60% VO2peak with pre, mid and post exercise biochemi cal, muscular and functional assessments. Baseline measurements of aerobic fitness, muscle strength/endurance, fatigue, body composition and general health questionnair es were acquired. In addition, baseline resting blood samples were drawn for cytokine and neurotrophin assessment. Prior to the initiation of training, subjects performed a 30 minute bout of exercise (cycle ergometry) at 60% of VO2 peak to assess the cytokine and neurotr ophin response to a single bout of aerobic exercise in the untrained state. Following baseline assessments, subjects participated in a supervised eight week aer obic exercise training program (30 min/3 times a week/60% of VO2 peak). All baseline measures were re -assessed at 4 and 8 weeks. The experimental design is shown in Figure 1.

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32 Figure 1. Experimental Design Exercise Training Protocol All subjects participated in a supervised 8 week aerobic exerci se training protocol. Each exercise session consisted of a 3 minut e warm up at a self-assessed comfortable speed followed by 30 minutes of cycle ergometry at 60% of VO2peak (3 times per week). Each training session was supervised and trai ning intensity was tailored to accommodate each participant’s functional ability. An eight week aerobic training protoc ol was selected because it has been previously found to provide a sufficient stimul us to alter cardiovascular fitness (40), muscular endurance (41), and im mune function (19) in MS su bjects. The weekly training protocol (30 minutes, 3 times/week at 60% VO2peak) has also been recommended by the American College of Sports Medicine because of the known health benefits and has been used by other investigators in studies with subjects with MS and other clinical populations (40, 41). The cycle ergometer was selected as the training modality to 8 weeks of aerobic exercise training 30 min cycle ergometry/3 times per week/ 60%VO2max Week0 Immune factors (IL-6, TNF, IFN) Muscle fatigue Neurotrophins (BDNF, IGF1) Muscle strength Quality of life measures Perceived fatigue Body composition Acute bout of exercise Week4 Week8 BIOCHEMICAL, MUSCLE AND FUNCTIONAL ASSESSMENTS 11 MS Subjects 11 Control Subjects

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33 accommodate for varying levels of physical im pairment and balance in individuals with MS. All training occurred in a supervised ex ercise environment with staff trained in cardiopulmonary resuscitation a nd emergency procedures. Baseline Measures Graded Exercise Test The subjects were asked to visit the laboratory for testing at the same time of day (8-11am) after abstaining from physical activity for 24 hours, and abstaining from alcohol, caffeine or food for the previous 12 hours. A standard 12-l ead electrocardiogram was used to monitor the cardiovascular re sponse of the subjec t continuously during testing. Following five minutes of rest (sit ting on the cycle ergometer), the subject was asked to pedal at 25 W. Every two minutes the resistance was increased by 10-25 W. Depending on their risk stratific ation for exercise testing, s ubjects were asked to keep pedaling until they were exhausted or until th ey reached their 85% of estimated maximal heart rate. Expired gas concentrations were r ecorded continuously using a metabolic cart (Parvomedics). Maximal and submaximal exerci se tests have been used previously in individuals with MS to measure cardiovascular capacity (19, 40, 76). Single Bout of Aerobic Exercise A single bout of aerobic exercise was a ssessed a minimum of 72 hours after the completion of the maximal exercise test. Befo re the start of the exercise bout, 20 ml of blood was acquired from the antecubital vein at rest in a seated position. Subsequently, the subjects cycled at 60% of their measured VO2peak for 30 minutes. This endurance protocol has also been used previous ly in individuals with MS (19, 76).

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34 Muscle Fatigue Voluntary muscle fatigue of both legs was determined by having the subject perform 30 concentric knee extens ions and flexions at 90 s-1 and 180s-1 as described in Lambert et al. (132) (2001). E ach subject was randomly assign ed to a different speed and leg at the beginning of the muscle fatigue exer cises at each time point. The most affected or non-dominant leg was tested followed by th e least affected or dominant leg after 10 minutes of recovery (or viceversa). Subj ects performed one bout of 30 flexions and extensions at both speeds with 5 min of recovery between bouts. The fatigue index for each bout was calculated (132). This fatigue prot ocol has been used in MS subjects in the past (132). All testing was performed at least 24 hours af ter any other exercise bout. Muscle Strength Lower limb muscle strength was tested us ing an isokinetic dynamometer (Kin-Com, Chattanooga, TN). The subjects were positioned sp ecifically for each exercise with joints stabilized. Subjects were seat ed and stabilized on the dynamo meter with hips flexed to 85 and knees flexed to 90. The axis of th e dynamometer was aligned with the axis of the knee joint and the bottom of the force tr ansducer pad positioned against the anterior aspect of the leg, proximal to the lateral malleolus. The rate of isometric force development was assessed at 90 and 120 of knee flexion for both leg extension (quadriceps) and leg flexion (hamstrings). Subjects performed a standardized warm-up before conducting two trials at each knee angle. Peak torque was recorded for each subject at 90 and 120 for bot h knee flexion and extension.

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35 Quality of Life in Health and Disease Perceived fatigue The modified fatigue impact scale (MFIS) was used to assess perceived fatigue in MS and control subjects. The MFIS has been used to assess fatigue in diseased populations such as MS in past studies (33). Perceived disability Subjects completed self-assess ment of disability (EDSS) (133) at all time points throughout training. Quality of life ssessment The Short Form (SF)-36 health survey was used to assess changes in selfperception of health states. The SF-36 has been used widely and is a reliable and valid measure to detect self-perception of health st atus (134) and has been previously used in studies with MS subjects (7). Functional Mobility Assessment To assess function mobility, a six minute walking test, a 25 foot and 100 foot test, and a timed up and go test were performed in all subjects. Six minute walk The six minute walking test was administered as a measure of exercise tolerance and overall functional limitations (135). Th e test was conducted as described by McGavin (136) and used standardized encouragement. Walking test (25 ft.) The subjects also completed a 25 foot walk test. The timed 25-foot walk is a mobility and leg function performance test and has reported high inter-rater and testretest reliability (137). Subjects completed two trials and were asked to walk as rapidly but

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36 as safely as possible during each trial. The amou nt of time to walk 25 feet was recorded with a light sensitive timing de vice (IRD-T175 Broward Timing Systems, Salt Lake City, Utah). Walking test (100 ft.) The subjects also completed a 100 feet walk test. Subjects completed two trials and were asked to walk as rapidly but as safely as possible during each trial. The amount of time to walk 100 feet was recorded with a light sensitive timi ng device (IRD-T175 Broward Timing Systems, Salt Lake City, Utah). Timed up and go test The timed up and go test was used to a ssess functional mobility skills and was measured by timing subjects as they stand up fr om a chair, walk a distance of 3 meters, turn, walk back to the chair, and sit down (138) The subjects were asked to perform one practice trial followed by 2 timed test trials with 2 minutes rest between trials. The average time of the test trials was used as th e criterion score. The same chair was used for all subjects throughout the intervention. The test has been reported to be reliable (ICC=0.99) (138) and correlate with risk of falls and balance (139). Inter-rater reliability for this test is 0.99. Body Composition Dual x-ray absorptiometry (DXA)(Lunar Prodigy Radiation Corp., Madison, WI) was used to measure whole body and appendi cular lean and soft tissue masses. The procedure was performed by a licensed X-ray technician. Body mass index (kg/m2) and waist to hip ratio (cm) was also calculated.

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37 Post-Exercise Training Measures After 4 and 8 weeks of aerobic training, th e subjects underwent the same testing procedures as before starting the 8 week aer obic training program (d escribed in baseline measurements). Blood Collection and Processing Blood samples (20 mL) were obtained by venipuncture before and following exercise to acquire multiple blood samples. All blood samples were acquired from the antecubital vein after resting for 5 minutes in a seated position. The subjects were asked to visit the laboratory at the same time of day (8-11am) after abstaining from physical activity, alcohol, caffeine or food for 12 hour s. Blood samples were collected a minimum of 48 hours after any MS-related drug administra tion to control for the possible impact of the drug on cytokine regulation ( 140). To control for hormonal shifts in females, samples were collected during the early to midfo llicular phase. Whole blood samples were collected in both EDTA tubes for plasma sa mples and in serum tubes for BDNF and IGF1 measurements (10 ml for plasma assessment and 10 ml for serum assessment). Plasma samples were immediately centrifuged at 3000g fo r 15 minutes at 4C and then stored at 80C for subsequent analyses. Blood Collection Times Venous blood (20 mL) was collected prior to a single bout of exercise, thirty minutes post-exercise, 2 and 3 hours post-exercise in three different occasions (before starting the aerobic exercise training program 4 weeks and 8 weeks after engaging in the aerobic exercise training program) as shown in Figure 2. This blood collection protocol was selected because the time course of cytokine elevation or depression differs depending on the cytokine of interest. For example, IFNand TNFmay increase

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38 immediately after exercise (30 minutes) and IL -6 displays a more delayed response (0-3 hours) (42, 75). Figure 2. Blood collection times before and after 30 minutes of cycle ergometry at 60%VO2peak Cytokine Assessment Cytokines IL-6, TNF, and IFNwere analyzed using a multiplex immunoassay utilizing fluorescently labeled microsphere beads and laser based fluorescent detection (Linco Research Inc., St. Charles, MO) for each acquired blood sample. The intra-assay coefficient of variability for IL-6, TNF, and IFNwere 4.9%, 6.1%, and 6.0% respectively. The individual sens itivities (pg/mL) of IL-6, TNF, IFNwere 1.7, 0.7, and 1.7, respectively. There was no significant cross reactivit y between other cytokine antibodies in this panel. Plasma sa mples were analyzed in duplicate. IGF-1 Assessment Serum concentrations of IGF-1 were assessed by using an IGF-1 Quantikine sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) for each blood sample acquired in resting conditions. Serum samples were pretreated to release IGF-1 from binding proteins and diluted 100-fold with a pretreatment constituent prior to the 3 hr POST 2 hr POST 30 min POST 30 min cycle ergometry 60% VO2m a x PRE exercise BLOODCOLLECTIONTIMESBEFOREANDAF TERANEXERCISEACUTEBOUT

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39 assay. To determine the optical density of each well, the samples were read within 30 minutes of adding 50 l of stop solution. Th e samples were read using a microplate reader (Molecular Devices, California) at 450 nm and 540 nm. The optical density readings at 540 nm were subtracted from th e readings at 450nm to correct for possible optical imperfections in the plate. The minimu m sensitivity of IGF-1 in this kit was less than 0.026 ng/ml. No significant cross reactivity with other IGF-1 bi nding proteins has been observed in this kit. The inter and intr aassay CV’s are 8% and 3% respectively. Serum samples were analyzed in duplicate. BDNF Assessment Serum concentrations of BDNF were analyzed by using a BDNF Quantikine sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) for each acquired blood sample. Serum samples were diluted 20-fo ld with a calibrator diluent prior to the assay. To determine the optical density of each well, the samples were read within 30 minutes of adding 50 l of stop solution. Th e samples were read using a microplate reader (Molecular Devices, California) at 450 nm and 540 nm. The optical density readings at 540 nm were subtracted from th e readings at 450nm to correct for possible optical imperfections in the plate. The minimu m sensitivity of BDNF in this kit was less than 20 pg/ml. No significant cr oss reactivity with other ne urotrophic factors has been observed in this kit. The inte r and intraassay CV’s are 8% and 5% respectively. Serum samples were analyzed in duplicate. Plasma Volume Assessment Plasma volume changes were assessed prior to and following a single bout of exercise at weeks 0, 4 and 8 by assessing hema tocrit and hemoglobin concentration from whole blood samples using the method by Dill and Costill (141).

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40 Statistical Analysis All analysis was performed using SPSS 12.0. A multivariate analysis of variance (MANOVA) with time (pre, mid and. post) as the within factor and group (MS vs. control) entered as th e between-subjects factor was used to assess the effect of the exercise training program on all main rela ted variables. An ANOVA with repeated measures on each blood collection point will be used to assess changes in cytokine and neurotrophin dynamics after a single bout of exercise. When necessary, Tukey’s post hoc analyses was implemented. A power analysis was conducted prior to the beginning of the study and found that 9 subjects per group would produce a power of .80. A value of p<0.05 was considered significant. All valu es are expressed as mean standard deviation.

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41 CHAPTER 4 RESULTS Subjects Eleven MS subjects and eleven healthy controls (8 women and 3 men in both groups) that were matched for age, we ight, height, percent body fat and VO2peak (p>0.05) completed the study. There was a si gnificant increase in absolute VO2peak (l/min) after 8 weeks of aerobic exercise tr aining in both groups (p<0.05). There were no significant changes in weight, BMI, wais t to hip ratio, relative VO2peak (ml/kg/min), and percent body fat after the training program in bot h groups (p>0.05). Table 1 describes the characteristics of the subjects before and af ter 8 weeks of aerobic exercise training in more detail. Table 1. Subject characteristics pre an d post 8 weeks of aerobic exercise. MS CONTROL PRE POST % PRE POST % Age (yrs) 40 10 40 10 0 40 10 40 10 0 Height (m) 1.68 0.1 1.68 0.1 0 1.68 0.1 1.68 0.10 Weight (kg) 72 14 73 15 1 78 14 78 14 0 % body fat 35.6 8 34.6 8 -3 37.6 9 37.4 9 -1 VO2peak (l/min) 2.2 0.4 2.5 0.410*2.4 1 2.8 1 14** BMI (kg/m2) 24 4 26 5 8 27 5 28 4 4 Data are expressed as M ean Standard Deviation. indicates a signif icant difference after 8 weeks of aerobic exercise (p<0.05); ** indicates a significant difference after 8 weeks of aerobic exercise (p<0.001); % = percent change.

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42 Immune Factors Chronic Exercise and IL-6 Plasma interleukin-6 at rest was similar between groups at weeks 0, 4 and 8 (p>0.05) (Figure 3). Following 8 weeks of aerobic exercise plasma IL-6 at rest tended to decrease compared to week 0 in both groups (p=0.075). IL-6 was significantly correlated to percent body fat (r = 0.506, p = 0.016), absolute VO2peak (r = 0.408, p = 0.046), 25 foot walk (r = -0.434, p = 0.036), 100 foot walk (r = -0.427, p = 0.038), and timed up and go test (r = -0.415, p = 0.044). Resting IL-6 MS vs CONTROL PREMIDPOST 0 5 10 15 20 25 30 35MS CONTROL $IL-6 (pg/mL) Figure 3. IL-6 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST) in both groups. $ Indicates tendency to decrease in both groups (p<0.10). Data are expressed as Mean Standard Deviation. Single Bout of Exercise and IL-6 The response of plasma IL-6 after a single bou t of exercise was similar between subjects and remained unchanged following the training program (p>0.05) (Figure 4). Both groups displayed similar significant in creases in plasma IL-6 concentration following 30 minutes of aerobi c exercise at 60% of VO2peak. Specifically, plasma IL-6

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43 concentration at baseline increased significan tly 30 minutes post exercise (p=0.021) and tended to increase 2 hours (p =0.09) post exercise in both groups. Although it was not statistically different, the magn itude of increase of IL-6 at 30 minutes into recovery in MS was 6.3%, while controls increased IL-6 by 20%. Single Bout IL6 baseline 3 0 mi n 2 h r 3 h r 5 10 15 20 25 *#CONTROL MS Exercise RECOVERYIL6 (pg/mL) Figure 4. IL-6 plasma concentration during a single bout of exercise in both groups. (Single bouts at PRE, MID and POST are collapsed within groups because they were not significantly different). *indi cates significant differences between time points within groups (p<0.05); # indicat es trend between time points within groups (p<0.10). Data are expressed as Mean Standard Deviation. Chronic Exercise and TNFTNFplasma concentration at rest tended to be higher in MS compared to control subjects throughout the study (p=0.08) (Fi gure 5). Specifically, Multiple Sclerosis subjects increased TNFplasma concentrations at rest from week 0 to week 8, and from week 4 to week 8 (p=0.04), while TNFplasma concentration in control subjects remained unchanged following 8 week s of aerobic exercise (p>0.05). TNFwas

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44 significantly correlated to percen t body fat (r = -0.399, p = 0.033), IFN(r = 0.932, p = 0.001), and total mental SF-36 (r = 0.454, p = 0.017). PREMIDPOST 0 5 10 15MS CONTROL # # TNF(pg/mL) Figure 5. TNFplasma concentration at rest at w eek 0 (PRE), 4 (MID) and 8 (POST). *indicates p<0.05 between MS and Control subjects; # denotes p<0.05 within MS subjects. Data are expresse d as MeanStandard Deviation. Single Bout of Exercise and TNFThe TNFplasma response to a single bout of exercise was similar between MS and control subjects before th e exercise intervention (p>0.05 ) and it remained unchanged throughout the 8 weeks of aerobic exercise tr aining in MS and cont rol subjects (p>0.05) (Figure 6). Both groups experienced si milar significant decreases in TNFplasma concentration following 30 minutes of aerobic exercise at 60% of VO2peak. Specifically, TNFplasma concentration at baseline decr eased significantly 2 hours (-28%, p=0.045) and 3 hours (-48%, p=0.001) following 30 minutes of aerobic exercise in both groups. In addition, TNFalso decreased significantly from 30 min to 2hr (-25%, p=0.13), and 30 min to 3hr (-46%, p=0.001) following a singl e bout of exercise in both groups.

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45 Single Bout TNFbaseline 3 0 mi n 2 h r 3 h r 0.0 2.5 5.0 7.5 10.0MS CONTROL # # # # Exercise RECOVERYTNF(pg/mL) Figure 6. TNFplasma concentration during a singl e bout of exercise at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic exercise training in MS and control subjects. (Single bouts at PRE, MID a nd POST are collapsed within groups because they were not significantly different). # Indicates p<0.05 within groups. Data are expressed as M ean Standard Deviation. Chronic Exercise and IFNThere was an interaction between the pl asma concentration at rest of IFNbetween groups and week of training (weeks 0, 4 and 8) (p=0.027) (Figure 7). Specifically, IFNconcentration at rest increased significantly in MS subjects from week 0 to week 8, (p=0.008), and from week 4 to week 8 (p=0.01). IFNat rest significantly increased in cont rol subjects from week 0 to week 4 (p=0.015) followed by a tendency for IFNat rest to decrease from week 4 to week 8 (p=0.07). In addition, control subjects had similar IFNconcentration at rest during week 0 and week 8 (p=0.3). IFNwas significantly correlated with percent body fat (r = -0.377, p = 0.042), TNF(r = 0.932, p = 0.001), and total mental SF-36 (r = 0.487, p = 0.011).

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46 Single Bout of Exercise and IFNThe plasma IFNresponse to a single bout of exercise was similar between MS and control subjects (p>0.05) and it remained unchanged throughout the training program (p>0.05) (Figure 8). Both groups experien ced similar significant decreases in IFNplasma concentration following 30 minutes of aerobic exercise at 60% of VO2peak. Specifically, IFNplasma concentration at baselin e decreased significantly 2 hours (34%, p=0.017) and 3 hours (-28%, p=0.015) foll owing 30 minutes of aerobic exercise in both groups. Resting IFNPREMIDPOST 0 10 20 30 40 50 60 70MS CONTROL # # # $IFN(pg/mL) Figure 7. IFNplasma concentration at rest at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic exercise training. # indicates p<0.05 within subjects; $ denotes p<0.10 within subjects. Data are expressed as Mean Standard Deviation. Brain-Derived Neurotrophic Factor Chronic Exercise and BDNF There was a significant interaction of se rum BDNF concentration between groups (MS vs Control) and training effect (0, 4 and 8 weeks of training) (p=0.045)(Figure 9).

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47 Resting serum BDNF was significantly lower in MS compared to control subjects at 0 weeks (p=0.026), and tended to be lower in MS compared to control subjects at 8 weeks (p=0.066). Resting serum BDNF concentratio ns remained unchanged in MS subjects between weeks 0 and 8, but BDNF concentratio ns were significantly elevated between weeks 0 and 4 (p=0.04), and tended to decrea se between weeks 4 and 8 (p=0.10). Resting BDNF concentration in cont rol subjects remained unchange d after 4 and 8 weeks of aerobic exercise training (p>0.05). BDNF wa s significantly correlated to total physical SF-36 (r = 0.465, p = 0.015), fatigue index at 180 /sec during the exte nsion phase (r = 0.444, p = 0.022). Single Bout of Exercise and BDNF The response of serum BDNF to a single bout of exercise be tween MS and controls was significantly different (p=0.01) (Figures 10 a nd 11). Specifically, BDNF concentrations were significantly lower in MS subjects comp ared to control subjects before exercise (baseline), 2 and 3 hours after exercise (p< 0.001) when all the points (PRE, MID, POST) were collapsed together. Additional Post hoc analyses revealed MS subjects had significantly lower concentration of BDNF only at baseline during week 0 (p=0.025). Moreover, during week 4, MS subjects tended to have lower concen tration of BDNF only two hours after a single bout of exercise (p=0.09). During we ek 8, MS subjects tended to have lower BDNF concentrations than cont rol subjects at baselin e (p=0.065) and BDNF concentrations were significantly lower in MS compared to control subjects after a single bout of exercise (p=0.04). The magnitude of clearance was the same for MS and control subjects at weeks 0, 4, and 8 (p>0.05) (ra te of clearance =73% in both groups).

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48 Single Bout IFNbaseline 3 0 mi n 2 h r 3 h r 0 10 20 30 40 # # # #CONTROL MS Exercise RECOVERYIFN(pg/ml) Figure 8. IFNresponse to a single bout of exercise in MS and control subjects. (Single bouts at PRE, MID and POST are collaps ed within groups because they were not significantly different). # indicate s significant difference within groups (p<0.05). Data are expressed as Mean Standard Deviation. Multiple sclerosis subjects had significant decreases of BDNF concentrations between baseline measurements and 2 hour s post-exercise, and between baseline measurements and 3 hours post-exercise at weeks 0, 4 and 8 (p<0.001) as illustrated in figure 10. The response of BDNF concentrations to single bout of exer cise within groups (during weeks 0, 4 and 8) was significantly different only between week 4 and week 8 (p=0.044) in MS subjects. During week 4, the rate of BDNF cleara nce was significantly faster (86%) than during week 8 (59%) (p= 0.044). However, there were no differences between the response of BDNF concentrati ons to a single bout of aerobic exercise between week 0 and at week 8 (p=0.3) or between week 0 and week 4 (p=0.2) in MS subjects.

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49 Figure 9. BDNF concentration at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic exercise training. *Indicate s significant differences between points p<0.05 # Indicates p<0.10 between MS and C ontrol subjects; $ Indicates p<0.10 between week 4 and 8 in MS subjects only. Data are expressed as Mean Standard Deviation. Control subjects had significant decreas es of BDNF concentrations between baseline measurements and 2 hours post-exer cise, and between base line measurements and 3 hours post-exercise at weeks 0, 4 a nd 8 (p<0.001) as illustrated figure 11. The circulating BDNF response to a single bout of exercise within cont rol subjects remained unchanged between weeks 0, 4 and 8 (p> 0.05) (rate of clearance =73%). Resting BDNF Concentration MS vs CONTROL PRE MID POST 0 5000 10000 15000 20000 25000 30000 35000 MS CONTROL #$ BDNF (pg/mL)

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50 Single Bout BDNF MS Group baseline 3 0 min2 hr3 hr 10000 20000 30000PRE MID POST *$ Exercise RECOVERY BDNF (pg/mL) Figure 10. BDNF acute response to exercise in MS subjects at weeks 0 (PRE), 4 (MID) and 8 (POST).*indicates si gnificant difference with in time point (p<0.001).$ indicates significant differences across time (p<0.05). Data expressed as Mean Standard Deviation Single Bout BDNF CONTROL Group baseline 3 0 min2 hr3 hr 0 10000 20000 30000PRE MID POST * Exercise RECOVERY BDNF (pg/mL) Figure 11. BDNF acute response to exercise in control subjects at weeks 0 (PRE), 4 (MID) and 8 (POST).*indicates signif icant difference within time point (p<0.05). Data are expressed as Mean Standard

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51 Insulin-Like Growth Factor-1 The concentration of circulating IGF-1 was similar between groups and remained unchanged after 8 weeks of aerobic exercise tr aining (p>0.05) (Figure 12). Resting levels of IGF-1 in MS subjects were 191 119 ng/ml before the study started, 206 154 ng/ml 4 weeks after the initiati on of training, and 161 199 ng/ml after the exercise intervention. Resting levels of IGF-1 in th e control subjects were 226 144 ng/ml before the study started, 200 110 ng/ml 4 weeks afte r the initiation of training, and 200 117 ng/ml after 8 weeks of aerobic exercise tr aining. IGF-1 was signifi cantly correlated to total physical SF-36 (r = 0.461, p = 0.014), fatigue index at 180/sec during the extension phase (r = -0.412, p = 0.019). Resting IGF-1 Concentration MS vs CONTROL PREMIDPOST 0 50 100 150 200 250 300 350 400 450MS CONTROL IGF-1 (ng/mL) Figure 12. IGF-1 concentration at rest at w eek 0 (PRE), 4 (MID) and 8 (POST). There were no significant effect s of aerobic training on res ting concentration of IGF1 (p>0.05). Data are expressed as MeanStandard Deviation

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52 Plasma Volume Plasma volume decreases after a single bout of exercise were similar between MS and controls subjects (4% a nd 5% respectively, p>0.05). In a ddition, decreases in plasma volume after a single bout of exercise remained unchange d after 8 weeks of aerobic exercise training in both groups (p=0.05). Muscle Function Knee Extension Endurance of Most A ffected/ Non-Dominant Leg at 180s-1 Muscle fatigue (fatigue inde x) was significantly higher in MS compared to control subjects (p=0.006) and remained unchanged af ter 8 weeks of aerobic exercise in both groups (p>0.05) (Table 2). Absolute and re lative (relative to body weight and fat free mass) mean extensor torque during knee ex tension endurance was similar in both groups and remained unchanged after 8 weeks of aerobic exercise training (p>0.05). Total extensor work was significantly lower in the MS compared to cont rol subjects (p=0.004). However, after 8 weeks of aerobic exercise total extensor work remained unchanged in both groups (p=0.14). Knee extension power was significantly lower in the MS compared to control subjects (p=0.001) and remained unchanged after 8 weeks of aerobic exercise in both groups (p=0.12). Table 2 provides fatig ue measurements in more detail before and after 8 weeks of aerobic exercise training. Knee Flexion Endurance of Most A ffected/ Non-Dominant Leg at 180s-1 Muscular fatigue (fatigue index) was similar between groups remained unchanged after 8 weeks of aerobic exerci se (p>0.05). Absolute and rela tive (relative to body weight and fat free mass) mean flexor torque duri ng knee flexion endurance was similar between groups and remained unchanged after 8 week s of aerobic exercise training (p>0.05). Total flexor work was significantly lower in the MS compared to control subjects

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53 (p=0.008). However, after 8 weeks of aerobic exercise total flexor work remained unchanged in both groups (p>0.05). Knee flexio n power was significantly lower in the MS compared to control subjects (p=0.006) but did not change after 8 weeks of aerobic exercise in either gr oups (p>0.05) (Table 2). Knee Extension Endurance of Most A ffected/ Non-Dominant Leg at 90s-1 Muscular fatigue (fatigue i ndex) was significantly lower in MS compared to control subjects (p=0.001). Controls tended to decrea se the rate of fatigue after 8 weeks of aerobic exercise (p=0.09), while MS subjects did not (p>0.05) Absolute mean extensor peak torque during knee extension endurance wa s significantly lower in MS compared to control subjects (p=0.007), as we ll as mean peak extensor to rque relative to fat free mass (p=0.023). Mean extensor peak torque relative to body weight tended to be lower in MS compared to control subjects during knee ex tension endurance (p=0.06). Total extensor work was significantly lower in MS comp ared to control subjects (p=0.002) and remained unchanged after 8 weeks of aerobi c exercise in both groups (p=0.11). Knee extension power was significantly lower in MS compared to cont rol subjects (p=0.003) and remained unchanged after 8 weeks of aer obic exercise in either groups (p=0.15) (Table 3). Knee Flexion Endurance of Most A ffected/ Non-Dominant Leg at 90s-1 Muscular fatigue (fatigue index) was similar between MS subjects and controls (p=0.17) and remained unchanged after 8 w eeks of aerobic exercise in both groups (p=0.12). Absolute mean flexor peak to rque during knee flexion endurance was significantly lower MS compared to control subjects (p=0.006), as well as and relative to body weight (p=0.023) and relative to fat fr ee mass (p=0.019). Total flexor work was significantly lower in MS compared to control subjects (p=0 .013) and remained

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54 unchanged after 8 weeks of aerobic exercise in either groups (p=0.21). Knee flexion power was significantly lower in MS comp ared to control su bjects (p=0.001) and remained unchanged after 8 weeks of aerobic ex ercise in either gr oups (p>0.05) (Table 3). Table 2. Fatigue measures pre and post 8 weeks of aerobic exercise during at 180sec-1 Data are expressed as Mean Standard Deviation; #indicates a signifi cant difference between groups (p<0.001). N= newtons; m=meters; Kg= kilograms BW= body weight; FFM= fat free mass; J=joules; W=watts; % = percent change. Table 3. Fatigue measures pre and post 8 weeks of aerobic exercise at 90sec-1 Data are expressed as Mean Standard De viation; indicates significant differences before and after 8 weeks of aerobic exercise; #indicates a significant difference between groups (p<0.05). $trend of significance be tween groups (p<0.10). MS CONTROL Speed 180sec-1 PRE POST % PRE POST % Ext Fatigue Index (%) 79 23 81 21 3# 95 21 99 15 4# Ext peak Torque (Nm) 190 34 199 28 5 209 57 217 68 4 Ext peak Torque (Nm/Kg) 2.7 0.7 2.8 0.7 4 2.7 0.7 2.8 0.7 4 Ext peak Torque (Nm/Kg FFM) 4.4 0.9 4.5 0.9 2 4.4 0.6 4.6 0.8 4 Ext Total Work (J) 673 273 700 231 4# 945 590 1235 621 26# Ext Power (W) 42 24 46 21 15# 69 38 90 47 23# Flex Fatigue Index (%) 86 23 79 20 -17 87 39 90 45 3 Flex peak Torque (Nm) 205 55 197 53 -4 217 87 213 103 -2 Flex peak Torque (Nm/Kg) 3.0 1.1 2.8 1.1 -6 2.9 1.0 2.7 1.2 -7 Flex peak Torque (Nm/Kg FFM) 4.7 1.3 4.5 1.5 -4 4.5 1.1 4.4 1.5 -2 Flex Total Work (J) 765 367 810 383 6# 1180 677 1250 672 6# Flex Power (W) 54 33 59 34 9# 90 50 93 54 3# MS CONTROL Speed 90sec-1 PRE POST % PRE POST % Ext Fatigue Index (%) 92 24 91 15 -1# 61 23 80 19 24*# Ext peak Torque (Nm) 153 33 154 40 7# 184 66 215 66 14# Ext peak Torque (Nm/Kg BW) 2.1 0.6 2.1 0.7 0$ 2.3 0.8 2.7 0.8 15$ Ext peak Torque (Nm/Kg FFM) 3.5 1.0 3.4 1.1 -3# 4.3 2.3 5 2.4 14# Ext Total Work (J) 929 366 1007 410 8# 1338 702 1712 672 22# Ext Power (W) 35 18 41 23 15# 59 37 73 35 19# Flex Fatigue Index (%) 105 24 86 19 -18 94 11 95 14 1 Flex peak Torque (Nm) 142 34 144 29 1# 196 81 187 66 -6# Flex peak Torque (Nm/Kg BW) 1.9 0.4 1.9 0.4 0# 2.5 1.0 2.4 0.9 -4# Flex peak Torque (Nm/Kg FFM) 3.2 0.9 3.2 0.9 0# 4.5 2.4 4.3 2.2 -4# Flex Total Work (J) 866 343 938 304 8# 1191 512 1394 751 15# Flex Power (W) 35 17 41 16 15# 65 25 63 32 -3#

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55 Isometric Muscle Torque During Ex tension at 90 of Knee Flexion Absolute maximal extensor torque (p=0.017) and maximal extensor torque relative to fat free mass (p=0.03) were significantly lo wer in MS compared to control subjects, while maximal extensor torque relative to body weight tended to be lower in MS compared to control subjects (p =0.07) (Table 4). However, absolute and relative maximal torque remained unchanged after 8 weeks of aerobic exercise in both groups (p>0.05). Table 4 provides detailed informati on on muscle strength of both groups. Isometric Muscle Torque During Flexion at 90 of Knee Flexion Maximal absolute and relative flexor torque was similar between groups and remained unchanged after 8 weeks of aer obic exercise (p>0.05) (Table 4). Isometric Muscle Torque During Extension at 120 of Knee Flexion Absolute maximal extensor torque was si gnificantly lower in MS compared to control subjects (p=0.04). Howe ver, relative maximal extensor torque (body weight and fat free mass) was similar between groups (p> 0.05). Eight weeks of aerobic exercise did not change maximal absolute and relative ex tensor torque in either groups (p>0.05) (Table 4). Isometric Muscle Torque During Fl exion at 120 of Knee Flexion Absolute maximal flexor torque (p=0.014) a nd maximal flexor torque relative to fat free mass (p=0.03) were significantly lower in MS compared to control subjects, while maximal flexor torque relative to body wei ght tended to be lower in MS compared to control subjects (p=0.09). However, absolu te and relative maximal flexor torque remained unchanged after 8 weeks of aerobic exer cise in both groups (p>0.05) (Table 4).

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56 Isokinetic Muscle Torque at 90s-1 During the extension phase, absolute peak extensor torque was significantly lower in MS compared to controls (153 33 Nm vs. 184 66 Nm for MS and controls respectively) (p=0.03). Absolute peak torque remained unchanged in both groups after 8 weeks of aerobic exercise (p>0.05). Peak extens or torque relative to body weight tended to be lower in MS compared to control subjects (2.1 0.6 Nm/Kg vs. 2.3 0.8 Nm/Kg for MS and controls respectively, p<0.10), a nd remained the same after 8 weeks of aerobic exercise (p>0.05). Peak extensor torque relative to fat free mass was significantly lower in MS compared to control subj ects (3.5 1.0 Nm/Kg FFM vs. 4.3 2.3 Nm/Kg FFM for MS and controls respectively, p=0.014), and remained the same after 8 weeks of aerobic exercise (p>0.05). During the flexion phase MS subjects had significant lower absolute peak flexor torque than control subjects (142 34 Nm vs. 196 81 Nm, p=0.048) and remained unchanged after 8 weeks of aerobic exercise tr aining in both groups (p>0.05). Peak flexor torque relative to body wei ght was significantly lower in MS compared to control subjects (1.9 0.4 Nm/Kg vs. 2.5 1.0 Nm /Kg for MS and controls respectively, p=0.02), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak flexor torque relative to fat free mass was signi ficantly lower in MS compared to control subjects (3.2 0.9 Nm/Kg FFM vs. 4.5 2.4 Nm/Kg FFM for MS and controls respectively, p=0.02), and remained uncha nged after 8 weeks of aerobic exercise (p>0.05).

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57 Table 4. Isometric strength measures pre and post 8 weeks of aerobic exercise at 90 and 120 of knee flexion MS CONTROL Isometric Strength PRE POST % PRE POST % Ext Torque at 90 (Nm) 96 35 93 24 -3# 123 51 125 56 2# Ext Torque at 90 (Nm/Kg BW) 1.4 0.5 1.3 0.4 -7# 1.6 0.6 1.6 0.7 0# Ext Torque at 90 (Nm/Kg FFM) 2.2 0.8 2.1 0.6 -5# 2.5 0.6 2.6 0.7 4# Ext Torque at 120 (Nm) 113 30 115 19 2# 142 60 136 57 -4# Ext Torque at 120 (Nm/Kg BW) 1.6 0.4 1.6 0.3 0 1.8 0.6 1.7 0.5 -5 Ext Torque at 120 (Nm/Kg FFM) 2.6 0.7 2.6 0.5 0 2.8 0.7 2.7 0.7 -4 Flex Torque at 90 (Nm) 49 22 43 18 -12 52 28 66 39 21 Flex Torque at 90 (Nm/Kg BW) 0.7 0.3 0.6 0.3 -14 0.7 0.3 0.9 0.7 22 Flex Torque at 90 (Nm/Kg FFM) 1.1 0.5 1.0 0.4 -9 1.1 0.4 1.5 1.1 27 Flex Torque at 120 (Nm) 56 26 53 18 -5# 72 25 71 28 -1# Flex Torque at 120 (Nm/Kg BW) 0.8 0.4 0.7 0.3 -12$0.9 0.3 0.9 0.3 0$ Flex Torque at 120 (Nm/Kg FFM) 1.3 0.6 1.2 0.4 -8# 1.5 0.3 1.5 0.5 0# Data are expressed as Mean Standard De viation; #indicates a significant difference between groups (p<0.05). $ trend of signifi cance between groups (p <0.10). N= newtons; m= meters; Kg= kilograms BW= body weight; FF M= fat free mass; J= joules; W= watts; % = percent change Isokinetic Muscle Torque at 180s-1 During the extension phase, absolute peak extensor torque was similar between groups (190 34 Nm vs. 209 57 Nm for MS and control subjects respectively), and remained unchanged after 8 weeks of aerobic ex ercise (p>0.05). Peak extensor torque relative to body weight was the same for both groups (2.7 0.7 Nm/Kg vs. 2.7 0.7 Nm/Kg for MS and controls respectively), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak extensor torque relative to fat free mass was similar between groups (4.4 0.9 Nm/Kg FFM vs. 4.4 0.6 Nm/Kg FFM for MS and controls respectively), and remained unchanged afte r 8 weeks of aerobic exercise (p>0.05). During the flexion phase, absolute peak fl exor torque was similar between groups (205 55 Nm vs. 217 87 Nm for MS and c ontrols), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak fl exor torque relative to body weight was similar between groups (3.0 1.1 Nm/Kg vs. 2.9 1.0 Nm/Kg for MS and controls

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58 respectively), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak flexor torque relative to fat free mass wa s similar between gro ups (4.7 1.3 Nm/Kg FFM vs. 4.5 1.1 Nm/Kg FFM for MS and c ontrols respectively), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Functional Mobility Walking Tests Multiple sclerosis subjects walked signifi cantly slower than control subjects during the 25 feet and 100 feet walking tests (p=0.001) (Table 5). Moreover, after 8 weeks of aerobic exercise training walking perform ance remained unchanged in both groups (p>0.05). Timed Up and Go During the timed up and go test MS subj ects completed the task significantly slower than control subjects (p=0.001). Moreover, time to co mplete the task remained unchanged after the training interv ention in both groups (p>0.05). Six Minute Walk The number of meters walked during the si x minute walk was significantly shorter in the MS compared to cont rol subjects (p=0.001) and rema ined unchanged after 8 weeks of aerobic exercise in both groups (p> 0.05). Table 5 shows functional mobility assessments before and after 8 w eek of aerobic exercise training.

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59 Table 5. Functional measures pre an d post 8 weeks of aerobic exercise MS CONTROL Functional Mobility PRE POST % PRE POST % 25ft walk (sec) 5.3 3.2 5.3 3.2 0# 3.5 0.5 3.3 0.5 -6# 100ft walk (sec) 20.2 11 19.6 10 -2# 12.7 2.4 12.3 2.2 -3# 6 minute walk (m) 541 215 566 240 4# 709 115 717 112 1# Timed up test (sec) 7.4 5 6.4 5 -14# 4.4 0.8 3.9 0.8 -11# Data are expressed as Mean Standard Deviation; #indicates a signifi cant difference between groups (p<0.05). s ec= seconds; m= meters; % = percent change. Quality of Life in Health and Disease Perceived Disability Perceived disability measured by self-a ssessed EDSS was significantly higher in MS compared to control subjects (p=0.0001) (Table 6). The MS subjects decreased their perceived disability by 24% (p=0.04) after 8 weeks of aerobic exercise training. Control subjects had a score of 0 at the beginning of the study and it remained unchanged after 8 weeks of aerobic exercise training (p>0.05). EDSS was significantly correlated to absolute and relative VO2peak (r = -0.6, p = 0.001), total MFIS (r = 0.64, p = 0.001), phys ical SF-36 (r = -0.564, p = 0.003), 25 foot walk (r = 0.697, p = 0.001), 100 foot walk (r = 0.732, p = 0.001), timed up and go (r = 0.773, p = 0.001), 6 minute walk (r = -0.807, p = 0.001), and absolute and relative isometric torque (r = -0.512, p = 0.005). Modified Fatigue Impact Scale The modified fatigue impact scale (M FIS) was significan tly higher in MS compared to control subjects in all subscales (p=0.001) (Table 6). The total score of the MFIS was significantly higher in MS compar ed to control subjects by 71% (31.7 5 vs. 9.2 5 respectively, p=0.001). The physical fatigue subscale of the MFIS was significantly higher in MS compared to control subjects by 77 % (15 2.4 vs. 3.6 2.4

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60 respectively, p=0.001). The cognitive fatigue subscale was significantly higher in MS compared to control subjects by 65% (14.4 2.6 vs. 5.1 2.6 respectively, p=0.003). The psychosocial fatigue scale of the MFIS was significantly higher in MS compared to control subjects by 75% (2.4 0.4 vs. 0.6 0.4 respectively, p=0.001). However, after 8 weeks of aerobic exercise training all fati gue subscales remained unchanged in both groups (p>0.05). Total MFIS was significantly correlated to absolute and relative VO2peak (r = -0.51, p = 0.008), physical SF-36 (r = -0.643, p = 0.001), 25 foot walk ( r = 0.552, p = 0.004), 100 foot walk (r = 0.580, p = 0.002), timed up and go (r = 0.622, p = 0.001), 6 minute walk (r = -0.553, p=0.004), fatigue index at 90/sec during extens ion phase (r = 0.468, p = 0.019), absolute and relative peak torque at 90/sec during extens ion phase (r = -0.64, p = 0.002). Table 6. Self-Assessed Measures pre a nd post 8 weeks of aerobic exercise. Data are expressed as Mean Standard Deviation. indicates a significant difference after 8 weeks of aerobi c exercise (p<0.05); #indicates a significant difference between groups (p<0.001). % = percent change. Short Form-36 Quality of Life Questionnaire The SF-36 quality of life questionnaire wa s significantly lower in MS subjects compared to control subjects (p =0.002) (Table 6). Specifically the total mental subscale of the SF-36 was significantly lower in MS co mpared to control subjects (43 13 vs. 57 8 respectively, p=0.003) and remained uncha nged after 8 weeks of aerobic exercise (p>0.05). The total physical subscale of the SF-36 was significantly lower in MS MS CONTROL Quality of Life PRE POST % PRE POST % EDSS 3.4 2 2.6 2 -24*# 0 0 0 0 0# MFIS (total) 32 22 26 19 -19# 9 9 8 12 -1# SF-36 (mental) 43 13 47 10 9# 57 8 52 11 -9# SF-36 (physical) 42 15 4512 7# 57 8 55 6 -4#

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61 compared to control subjects (42 15 vs 56 4 respectively, p<0.001) and remained unchanged after 8 weeks of aerobic exercise (p>0.05). The physical functioning subscale of the SF-36 was significantly lower in MS co mpared to control subjects (66 34 vs. 96 7 respectively, p<0.001) and remained uncha nged after 8 weeks of aerobic exercise (p>0.05). The role-physical subscale of the SF-36 was significantly lower in MS compared to control subjects (66 41 vs 100 0 respectively, p<0.001) and remained unchanged after 8 weeks of aerobic exercise (p>0.05). The body pain subscale of the SF36 tended to be lower in MS compared to control subjects (70 28 vs. 87 17 respectively, p=0.06) and remained uncha nged after 8 weeks of aerobic exercise (p>0.05). The general health subscale of the SF-36 was significantly lower in MS compared to control subjects (55 26 vs 84 12 respectively, p<0.001) and remained unchanged after 8 weeks of aerobic exercise (p >0.05). The vitality subscale of the SF-36 was significantly lower in MS compared to control subjects (53 29 vs. 63 13 respectively, p=0.04) and remained uncha nged after 8 weeks of aerobic exercise (p>0.05). The social functioni ng subscale of the SF-36 was significantly lower in MS compared to control subjects (78 24 vs 94 10 respectively, p=0.002) and remained unchanged after 8 weeks of aerobic exercise (p>0.05). The role-emotional subscale of the SF-36 was significantly lower in MS compared to control subjects (73 39 vs. 94 20 respectively, p=0.024) and remained uncha nged after 8 weeks of aerobic exercise (p>0.05). The mental health subscale of th e SF-36 was similar between groups (75 13 vs. 771 respectively, p>0.05) and remained unc hanged after 8 weeks of aerobic exercise (p>0.05).

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62 Total physical SF-36 was significantly correlated to EDSS (r = -0.564, p = 0.003), relative VO2peak (r = 0.584, p = 0.002), total MFIS (r = -0.643, p = 0.001), 6 minute walk (r = 0.414, p = 0.028), BDNF (r = 0.465, p = 0.015) and IGF-1 (r = 0.465, p = 0.015). Total mental SF-36 was significantly correlated to absolute VO2peak (r = 0.398, p=0.033), TNF(r = 0.454, p= 0.017), and IFN(r = 0.487, p = 0.011).

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63 CHAPTER 5 DISCUSSION Regular physical activity repres ents and intrinsic means to maintain health and is recommended to reduce the in cidence of many diseases. Exer cise is also recognized for its potential to protect the cen tral nervous system (CNS) from injury as well as promote restoration of function followi ng insult (142). Thus, regular activity may be an effective countermeasure to minimize deleterious cha nges associated with neurodegenerative diseases such as multiple sclerosis (MS), where immune dysregulation and compromised neuroprotection are associat ed with neurodegeneration of the CNS and disease progression. Early information about the influence of exer cise on MS di sease activity comes from research using the animal m odel of MS. Le Page et al. (81) (1994) investigated the effect of exercise on the inflammatory phase of experimental autoimmune encephalomyelitis (EAE). Ex ercise training reduced the duration and severity of EAE in rats (81). However, the mechanisms associated with reduced disease severity remain unknown, but likely involve m odulation of the immune system. Regular aerobic exercise has been shown to modulat e immune factors such as pro and antiinflammatory cytokines (19, 42, 43), and to in crease neuroprotecti on through elevations of IGF-1 and BDNF secretion in healthy populations (22, 27, 28, 44). However; to date, the influence of exercise on factors known to influence di sease activity in people with MS remains unexplored. Therefore, the purpose of this study was to investigate whether aerobic exercise modulates immune markers, neurotrophins and fati gue in individuals with MS. We hypothesized that aerobic exer cise would modulate cytokines IL-6, TNF

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64 and IFN, neurotrophins BDNF and IGF-1, and would reduce muscular and perceived fatigue in MS and matched control subjects. Resting Cytokine Concentration af ter 8 Weeks of Exercise Training Clinical studies investigating the imp act of chronic exercise on cytokine modulation in individuals with MS are limite d. Our study is one of the first to provide evidence that exercise training may influen ce pro and anti-inflammatory cytokines in individuals with MS. Resting co ncentration of plasma IL-6 tended to decrease, which is consistent with our hypothesis. Schulz et al. (76) (2004) inves tigated the impact of chronic aerobic exercise on IL -6, and in contrast to our findings, found that a similar training regimen did not alter resting concentrat ion of IL-6. Further, Castaneda et al. (16) (2004) found that interleukin-6 (IL-6) was reduced in patien ts with kidney inflammatory disease subjects undergoing 12 weeks of resi stance training compar ed with controls. Since regulatory changes of systemic IL-6 may be pivotal for the development of demyelinating lesions in the CNS (143), decreas es in this cytokine may have beneficial outcomes in persons with MS. Previous obs ervations suggest that abnormally high concentrations of IL-6 in the periphery may result in excess inflammation that may exacerbate autoimmune disease activity in MS (6). Also, elevated IL-6 may also disrupt the clearance of microbial pa thogens (6) and participate in T cell activation, accelerating the MS disease process (5, 6). These results provide preliminary evidence that exercise may modulate immune factors in the periphery of persons with MS. Resting concentrations of TNFand IFNincreased in MS subjects following training, which is contrary to our hypothesis. Our results suggest that aerobic training may also increase concentration of pro-infl ammatory cytokines in the periphery of individuals with MS. However, the cons equence of elevated circulatory TNFand IFN

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65 remain unknown. Previous research suggests that elevated TNFconcentration in blood may have beneficial (144) or detrimental eff ects (143) in people w ith MS. For example, while increased TNFconcentrations in blood and CSF ma y correlate with the degree of blood brain barrier dysfunction ( 143), it may also be associated with favorable decreases in disease relapses while on interferontreatment (144). In our study, perceived disability decreased w ith training suggesting that change s in pro-inflammatory cytokines may not be linked to negative disease out comes. In fact, inflammation may be a prerequisite to activate repair mechanisms su ch as remyelination as evidenced by studies showing that inflammation upregulates neur otrophic factors (i.e., BDNF) involved in neuroprotection (145, 146). The role of TNFin MS is complicated by the observation that TNFhas dual roles (4, 143, 145-147) that may be unique to autoimmune diseases such as MS. Although TNFhas been linked to inflammatory demyelination in MS (11, 148, 149), recent reports show strong evidence that TNFmay also be neuroprotective through enhancement of oligodedryocyte proliferati on and stimulation of remyelination (143, 145, 146). If fact, intravenous anti-TNFtherapy does not work in MS patients, and may even worsen MS symptoms (145, 150). It is therefore difficult to resolve the contradictory roles of TNFon disease activity. One explanation may be the existence of two different signaling pathwa ys mediated by two different TNFreceptors (p55 and p75) (143, 145). It is possibl e that exercise can indu ce activation of the “good” inflammatory TNFp75 receptor pathway that promotes cell growth and proliferation (145). Possible mechanisms of action include neuroprotection of the TNFp75 receptor through the induction of supe roxide dismutase (151, 152), protecting neurons from

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66 reactive oxygen species, and cal bindin stabilization of calci um homeostasis in the CNS (153). Our study provides preliminary data suggesting that exer cise may modulate cytokines associated with diseas e activity as well as evidence suggesting that increases in circulating TNFconcentration in MS patients may not be associated with negative outcomes as evidencing by significantly improve d disability levels in our MS subjects following the exercise program. Similar to TNF, plasma concentration of IFNalso increased following 8 weeks of aerobic exercise in MS subjects. To date, IFNis thought to be present during relapses and it is considered detrimental to the CNS of individuals with MS (55, 86, 154). However, the role of IFNin the periphery remains unknown. In our study, IFNand TNFwere highly correlated and seem to follow similar dynamics throughout the study (r = 0.932, p = 0.001). Work by Moldovan et al. ( 55) (2003) showed that T cell secreting IFNex-vivo correlated with func tional impairments in MS patients. In contrast, Kraus et al. (86) (2002) found that ci rculating pro-inflammatory cyt okines did not correlate with disease activity and severity assessed by lesion load in the brain. As mentioned earlier, it remains to be elucidated whether exercise plays a positive or negative role in the pathophysiology of the disease. However, since disability status improved in our subjects indicating that the obse rved changes in TNFand IFNmay not be linked to any changes in perceived disability. Clearly, furthe r investigations are needed to clarify the roles of exercise-induced proinflammato ry cytokines in individuals with MS. Chronic Exercise May Modulate Serum BDNF at rest Inflammation precedes BDNF production, which is a key factor involved in neuroprotection (21, 147, 155, 156). In addition, exercise has been shown to increase neurotrophin production in both the brain and spinal co rd (21, 22, 27, 155, 156). In our

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67 study, before the initiation of the exerci se training program, we observed lower concentration of serum BDNF in MS compared to control subjects at rest. These results are consistent with Sarchielli et al. (23) (2002), where MS s ubjects also had lower levels of BDNF at rest. However, in Sarchielli et al. (23) (2002), BDNF was stimulated from peripheral blood mononuclear cells and the subjects had seco ndary progressive MS. In contrast to our results, Gold et al. (77) (2003) found that BDNF concentration at rest was similar between MS and control subjects. Our results provide further evidence that serum BDNF may be lower in some individuals w ith MS compared to matched controls. Exercise has been shown to increase ne urotrophin production in the brain and spinal cord (21, 22, 27, 155, 156). Our results al so show that serum BDNF at rest was elevated following 4 weeks of exercise tr aining in MS subjects, which supports our cytokine findings where exercise-induced el evation of pro-inflammatory cytokines may lead to BDNF upregulation in MS subjects ( 147). In our study, resting concentration of BDNF followed a biphasic response to chronic ex ercise with elevations at 4 weeks while returning to baseline levels at week 8. This biphasic respons e of resting BDNF to chronic exercise has also been observed in healthy an imal models (157). During the first weeks of training, exercise may produce novel effect s that can enhance neurogenesis (157). However, once the individual is accustomed to chronic exercise, “novelty” and molecular learning effects may diminish and homeostatic mechanisms take over, bringing resting BDNF back to baseline leve ls (157). Although accumulati ng evidence suggests that exercise provides brain health benefits by increasing neuroprotection (21, 22, 25, 27, 32, 106-108), the mechanisms through which exer cise benefits the brain are poorly understood. The ability of BDNF to cross the blood brain barrier has been demonstrated

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68 (102), suggesting that serum BDNF levels may reflect BDNF levels in the brain (158). However, the source of origin and the mode of transport of exercise-induced BDNF actions on the brain remain unknown. Potentia l sources of BDNF include: 1) Muscle BDNF anterogradely transported to the CNS, 2) Schwann cell synthesis of BDNF, 3) injured fiber attracting BDNF to the area, 4) blood borne circulating BDNF (102, 104, 107, 111, 159, 160). Recent data suggest that BDNF is also elev ated in exercised skeletal muscle (109) and can be transported into the spinal cord (110). BDNF also has effects on skeletal muscle tissue by inducing the potentiation of spontaneous twitching in myocytes to enhance muscle contraction (111). In th e periphery, exercise can upregulate the expression of BDNF and maintain skeletal muscle health (25). For example, after immobilization stress, Adlard and Cotman ( 25) (2004) found that exercise can override the negative effects of muscle atrophy in rats thro ugh elevations of BDNF secretion after 3 weeks of running. In BDNF deficient rats, 2 months of wheel running increased BDNF hippocampus concentration (107). Sarchielli et al. (23) (2002) also suggests that BDNF concentration in the CSF is influenced by its concentration in peripheral blood. BDNF can be produced in the peri pheral circulation and transpor ted by a high capacity, saturable system that suggests that increased peri pheral production of this neurotrophin may increase its entry into the CNS (23, 102). In addition, a positive correlation between cortical and serum BDNF concen tration has been observed in rats (104). Furthermore, the role of circulating BDNF on neuroprotecti on in humans remains to be elucidated. Circulating BDNF may contribute to increases in neural repair and plasticity mechanisms in the brain and spinal cord (102, 160). Additionally, BDNF may cross the BBB after a

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69 single bout of exercise in response to a physic al stress, which is discussed in the next section. If circulating BDNF crossed the BBB or was transported to the CNS through other means (i.e., skeletal muscle), it may positively influence oligodendrocyte survival and proliferation, and therefore stimulate re myelination (23, 27). However, our data only captured a snapshot of circulating BDNF at rest, and therefore all these assumptions are speculative. BDNF is, among others, regulated by circulating IGF-1, which may promote neurogenesis, and the ability of exercise to protect the brain from neuronal injury such as demyelination (28, 44). Aerobic exercise tr aining may elevate IGF-1 concentration rapidly in a variety of site s (i.e., brain, peripheral circul ation, skeletal muscle), and stimulate remyelination, improve cognitive function, and enhance muscle hypertrophy, which may ultimately reduce disease progression in persons with MS (21, 116). In our study, serum IGF-1 concentration at rest wa s similar between MS and control subjects before the initiation of the training progr am and remained unchanged after exercise training in both groups. Our study corroborat es past results where there were no differences in serum IGF-1 between MS and control subjects at rest (30, 161). Additionally, our data also corroborates previous findings suggesting that aerobic exercise training does not alter IGF-1 (162). The potential of IGF-1 to promote remye lination in the CNS makes this growth factor a therapeutic ta rget. Although it may be attractive to speculate that chronic exercise may upregulate resting IGF-1 content in the periphery, our study results suggest that serum IGF-1 concentration at rest did not ch ange after training. In similar fashion, a limited trial of exogenous subcutaneous IGF1 treatment in MS was proven unsuccessful

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70 (29), adding to the contradictory re sults observed by IGF-1 administration on remyelination in the EAE model (24, 114, 163, 164) Therefore, future research involving exercise, IGF-1 and MS subjects may be useful to determine whether exercise has an impact in the production of IGF-1 (acutely and chronically), as we ll as the impact of exercise-induced IGF-1 on MS related neuronal repair. Cytokine and Neurotrophin Response to a Single Bout of Exercise in MS We investigated the respons e of immune and neurotrophic factors following a single bout of aerobic exercise. It has been proposed that the short term release of cytokines during acute exercise may cont ribute to the maintenance of an immune homeostatic environment (79). In addition, many of the acute phase proteins released in response to elevated cytokine le vels are protease inhibitors or free radical scavengers that attenuate the magnitude of tissu e damage associated with re lease of toxic molecules and free radicals due to activated neutrophils (79) Therefore, a single bout of exercise may have an array of effects on immune parameters (76, 80) that could contribute to neuroprotection (80). Cytokine Dynamics after a Single Bout of Exercise Skeletal muscle contractions stimul ate IL-6 production and may increase circulating IL-6 concentration via comple x signaling cascades initiated both by Ca2+ dependent and independent stimuli (61). Plas ma IL-6 increases in exponential fashion with exercise and is intensity and durati on dependent (42, 60, 75). In our study, MS and control subjects experienced similar significan t increases in plasma IL-6 concentration following 30 minutes of aerobi c exercise at 60% of VO2peak as reported in the literature by others (See Review by Pedersen (165)). Sp ecifically, IL-6 increa sed significantly 30 minutes post exercise and tended to stay elev ated for 2 hours while re turned to baseline 3

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71 hours post exercise in both groups. In contrast to our findings, Schul z et al. (76) (2004) found that IL-6 remained unchanged immediatel y after a single bout exercise with no additional post exercise collections acqui red. However, the timing of blood sample acquisition is important because the dynamics of each cytokine can vary considerably in response to exercise (53). Ther efore, our results pr ovide preliminary data suggesting that 2 to 3 hours are needed to capture the exerci se-induced IL-6 respons e in MS and control subjects after a mode rate exercise bout. In addition to IL-6, we also assessed the TNFand IFNresponse following a single bout of exercise in both groups. TNFand IFNplasma concentrations decreased in similar fashion in MS and control subjects after a single bout of exercise (-42% and 38% respectively at 3 hours post exercise). Th e response to exercise of both cytokines did not change after training in eith er group. Our results are in cont rast to Heesen et al. (19) (2003), who reported increased TNFand IFNconcentrations 30 minutes post exercise (30 minutes of cycle ergometry at 60% VO2peak) with no additional post exercise blood evaluation. In our study, TNFand IFNconcentrations 30 minutes post exercise were similar to baseline values, with a marked significant decrease 2 and 3 hours post exercise. The kinetic profile of TNFand IFNfollow the opposite dynamics to IL-6 following a single bout of exer cise and provides further in formation on the impact of exercise on immune markers. In our study, MS subjects had a similar cytokine response (IL-6, TNF, and IFN) compared to control subjects before the in itiation of the exercise training program and it remained unchanged throughout training. Limited information is available on the influence of exercise on immune variables th at are known to impact disease activity in

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72 MS. These findings suggest that individuals with MS may respond to physical stress similarly to matched healthy controls. In f act, stabilized levels of interacting Th1/Th2 cytokines are maintained in the benign course of MS and it is hypothesized that benign MS (EDSS <2) is characterized by a fairly balanced cytokine and neuroendocrine network (166). Perhaps MS subjects with higher disability (ED SS >5) may exhibit a different response due to a stronger immune dys regulation. Our subjects reported slightly higher EDSS score than benign MS (EDSS=3.4), but may still maintain a balanced cytokine and neuroendocrine network when reacting to a physical stress. Additional studies are need ed to provide a more complete and comprehensive understanding of the dynamic cytokine respon se to physical stress in MS and its implications on disease activity. Futu re research focused on IL-6, IFNand TNFmay provide further insight because these cyt okines are known to dire ctly influence MS pathophysiology. Serum BDNF Decreases Following a Single Bout of Exercise Understanding the impact of exercise on neurotrophic factors and neuroprotection may yield important information for futu re therapeutic strategies (26, 167-169). Previously, Gold et al. ( 77) (2003) found increases in serum BDNF concentration immediately after a single bout of aerobic exercise at 60% of VO2peak. However, BDNF may clear from the circulation very rapidly (within minutes) af ter subcutaneous injections of BDNF (102). In our study, BDNF concentra tion significantly decreased after 2 hours and 3 hours post exercise in MS and contro l subjects. Serum BDNF clearance after a single bout of exercise averaged 73% cleara nce (of BDNF at base line) following 2 hours of post-exercise recovery in both groups. The rapid clearance of BD NF post exercise may be indicative of 1) of rapid transport of BDNF into the CNS, or 2) BDNF traveling into

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73 the muscle and ultimately transported into the CNS (102, 159). Our findings do not support the speculation made by Gold that BD NF increases post exercise may be long lasting (77). However, the fate of circulat ing BDNF clearance after a single bout of exercise remains unknown and warrants further study. Previous studies suggest a positive associa tion between neural repair in the central nervous and the peripheral concentration of BDNF (77). Kishino and Nakayana (159) (2003) found that a subcutaneous injection of BDNF enters the blood stream and may be transferred to the spinal cord axons. In addition, Kishino a nd Nakayana (159) (2003) also reported that some circulating BDNF may en ter skeletal muscle from the blood stream, and is transported retrogadely to the moto r neuron cell bodies. Kishino and Nakayana (159) (2003) provide strong evidence that syst emic BDNF can enter the CNS and activate signaling cascades responsible for neural re pair. These events are clearly important because BDNF clearing into the CNS (i.e ., exercise bout) could contribute to remyelination in the CNS and the brain (102). In a demyelinating disease like MS, bloodderived BDNF may have a beneficial effect on neuron survival eith er by transport from a periphe ral receptor binding site or by passage across the BBB (170). Our study provided new evidence that a single bout of exercise possibly stimulates clearance of ci rculating BDNF into other areas (i.e., CNS, skeletal muscle). Whether BDNF crossed th e BBB or is transported into the CNS via skeletal muscle is unclear at this time and further investigations are warranted. Potential benefits through exercise may be related in part to B DNF availability that increases neuronal survival (44), facil itated learning (155), and neur ogenesis (155, 171) In human studies, exercise participation predicts better cognitive function (172, 173), lowers risk of

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74 Alzheimer’s disease and dementia in general (174, 175). However, whether exercise is a potent stimulus to provide e nhancement of neuroprotective mechanisms in individuals with MS remains to be elucidated. Muscle Fatigue Excessive systemic fatigue and muscle weakness are the most common and debilitating symptoms of indi viduals with MS (2). It is known that fatigue can be counteracted by exercise training in healt hy (35, 36) and diseased populations (37, 38). Although there is evidence indi cating that regular exercise may attenuate perceived systemic fatigue in MS subjects (39-41), le ss is known about the impact of chronic exercise training on muscle fatigue in th e MS population. Perceived fatigue in MS subjects is discussed later in this chapter. Few studies have reported the impact of chronic aerobic exercise on muscular fatigue in individuals with MS. Consistent with previous findings, MS subjects have lower strength and more muscular fatigue compared to controls (176, 177). However, contrary to our hypothesis, 8 weeks of aerobic exercise did not alter muscular fatigue in MS subjects as measured in this study. Our results corroborate prev ious investigations reporting no improvements in muscular fatigue after participating in an exercise program (178). Others have observed mild improvements in fatigue after regu lar exercise training (179, 180). For example, Surakka et al. (180) (2004) found th at after an unsupervised combined aerobic and resistance exercise program for 6 months, women but not men with MS reduced extensor fatigue. However, in Surakka et al. (180) (2004), fatigue was measured as a 30 second maximal static contra ction. Patti et al. (179) (2003) also showed mild improvements in muscular fatigue afte r a 6 week outpatient rehabilitation program including mild exercise.

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75 The lack of observed change in muscular endurance may be explained in several ways. First, it has been hypothe sized that individuals with MS are unable to fully activate motor units and consequently hinder possible skeletal adaptations to overload stress (39, 177). Second, our muscle fatigue testing protoc ol may not have been specific to detect actual changes in muscle endurance. Base d on the observed improvements in 6-minute walking distance and increased performan ce during the assessment of maximal oxygen consumption, it can be argued that muscle endurance improved with training. The observed changes in aerobic capacity of our s ubjects is consistent wi th previous reports showing a 10-22% gain in aerobic capacity after engaging in an aerobic exercise training program (40, 41, 181, 182). Functional Mobility As expected, MS subjects walked a shor ter distance during the 6 minute walk, and were slower during the timed up and go te sts, and the 25 and 100 ft walking tests compared to control subjects. Multiple sclerosis and control subjects experienced improvements in functional mobility assessments following training, but did not reach statistical significance. In fact, our study f ound similar outcomes compared to Kileff and Ashburn (183)(2004), where a similar aerobic tr aining program did not translate into 25ft and 6 minute walk improvements in MS subjects with moderate disability (EDSS = 4-6). Additionally, our results also found similar observations to White et al. (39) (2004), where after 8 weeks of resist ance training, the 25 ft walking speed remained unchanged as well. Debolt and McCubbin (184) (2004) also observed no changes in functional mobility following 8 weeks of unsupervised re sistance training. However, the fact that MS subjects in our study were able to walk 25 meters further during the 6 minute walk test and performed the timed up and go test one second faster (15% improvement) after

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76 the training program is of clinical importa nce. These results may also help explain reductions in perceived disability and may in crease their ability to engage in more activities of daily living. In individuals with MS, enhanc ing the ability to improve mobility could have a large impact in their quality of life. In individuals with MS, reducing disability may enhance daily activity and help offset the cycle of declining fitness with inactivity. Quality of Life in Health and Disease Exercise has been shown to improve ps ychological and cognitive functioning in humans (172, 185, 186). Specifically, regular ex ercise has antidepr essant properties (186), has been shown to decrea se anxiety (187) and elevates mood and coping skills in response to stress (188). In our study, MS subjects signifi cantly decreased perceived disability, and had marginal improvements in perceived fatigue and quality of life measures after 8 weeks of exercise training. Perceived Disability The observed significant reduction in perc eived disability (self-assessed EDSS score) following training is an important clinical finding because despite the lack of statistical changes in functional mobility, MS subjects perceived their disability to be reduced. The fact that MS subjects were ab le to improve their aerobic capacity and walking distance likely reflects the change in perceived disability. These findings support the role of exercise as a positive therapeutic strategy to attenuate functional declines often observed in this population. Perceived Fatigue As expected, perceived fatigue was signifi cantly higher in MS compared to control subjects in total MFIS and all MFIS subscal es prior to the beginning of the exercise

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77 program. Total MFIS scores decreased 19% in MS subjects while only decreased 1% in controls. Although it was not statistically significant, exercise training improved perceived fatigue in the MS gr oup. Since fatigue is one the most debilitating symptoms in MS, with up to two-thirds of patients describing fatigue as their main complaint (189), fatigue reduction represents a clinically si gnificant outcome. Using the same perceived fatigue assessment, White et al. (39) ( 2004) found a comparable 24% decrease in perceived fatigue after 8 week s of resistance training in MS subjects. Using the fatigue severity scale (FSS) for their fatigue asse ssment, Mostert and Kesselring (41) reported reductions in fatigue (-14%) of MS subject s after only 4 weeks of aerobic exercise training. MS subjects also increased their activity level by 17% af ter only 4 weeks of regular aerobic exercise. After 15 weeks of aer obic exercise at moderate intensity, MS subjects experienced a significant reduction in fatigue (measured with the profile of mood states questionnaire) and a negative associ ation between improvement in aerobic fitness and fatigue perception (40). As a result of increases in aerobic capacity, MS subjects were able to perform activities of daily living at lower relative intensity, preventing excessive fatigue (40). Conse quently, therapeutic interventi ons involving exercise have the potential to provide a means to contro l fatigue in people with MS and perhaps improve daily activity. Short Form-36 Quality of Life Questionnaire The SF-36 total mental and physical co mponents were significantly different between MS and control subjects and rema ined unchanged following 8 weeks of aerobic exercise training in both gr oups. MS subjects improved the total mental and physical score in the SF-36 questionnaire by 9% and 6% respectively, but these changes were not significant. Our findings are cons istent with previous investig ations reporting no effect of

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78 aerobic exercise training on total physical and mental SF-36 outcomes. Mostert and Kesselring (41) (2002) did not find significant changes in total mental and physical SF-36 scores after 4 weeks of aerobic exercise either. In addition, H eesen et al. (19) (2003) did not find any effects of 8 weeks of aerobic ex ercise training on SF36 outcomes. However, other quality of lif e measurements such as the profile of mood states (POMS) have been shown to be affected by aerobic exercise traini ng (40). In Petajan et al. (40) (1996), MS subjects experienced decreases in fatigue, a nger and depression measured by POMS after 15 weeks of aerobic exercise tr aining. Longer exercise interv entions may have an impact on the quality of life of indivi duals with MS and may provide additional benefits related to their psychological state in ad dition to fitness improvements. Future Directions Due to the neuroprotective potential of ex ercise training in neurodegenerative and autoimmune diseases, further research is warranted in this area. Additionally, studies focusing on the impact of exercise on imm une markers are important because exercise may be used as a model for stress after a si ngle bout or as a long term therapeutic intervention. Further, investig ating the immune response to ch ronic and acute exercise of other immune markers such as IL-4 or IL -10 may add important information regarding the potential of exercise to modulate the T h1/Th2 balance. Since regular exercise has been shown to be immunomodulatory in healt hy populations, further investigations may provide further information regarding the imp act of exercise on autoimmune diseases and their progression. Determining the role of exercise and B DNF regulation may provide further insight involving neuroprotective therap eutic strategies in degenerative diseases such as MS. Additionally, studies focusing on the fate of cleared immune markers and neurotrophins

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79 after exercise are important to understand ex ercise-induced mechanisms affecting neural health. The study of IGF-1 may provide addi tional information regarding the observed neuroprotective effects of exercise in mamm als. Clearly, a combination of both animal and human studies are needed to clarify the role of exercise on pathways associated with disease progression in MS. Since exercise provides an intrinsic mean s to modify functional outcomes in the MS population, further study in this area may compliment other therapeutic strategies designed to attenuate MS dis ease progression. In addition, in creasing activity levels in MS patients is crucial for l ong term health. Enhancing muscle strength and endurance through exercise training may increase daily activity, reduce fatigue and depression and increase quality of life in the MS population. Therefore, therapeutic interventions such as regular exercise training are pivotal because they may stimulate protective mechanisms that not only protect against secondary diseas es, but have the potenti al to impact disease progression in individuals with MS.

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96 BIOGRAPHICAL SKETCH Vanessa Castellano was born in Barcelona, Sp ain. She moved to the United States in 1994 to attend Billings Central Catholic High School in Billings, Montana, as an exchange student. She started her college e ducation at the Univer sity of Montana in Missoula, Montana, in August, 1995. After two years studying cell biology she then moved to Athens, Georgia, where she complete d her bachelor’s degree in cell biology in December 1999 at the University of Georgi a. In May 2002, she received a master’s degree in exercise physiology at the Universi ty of Georgia. In August, 2002, she began her doctorate degree in applied physio logy at the University of Florida.


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Permanent Link: http://ufdc.ufl.edu/UFE0013681/00001

Material Information

Title: Cytokine and Neurotrophin Response to Acute and Chronic Aerobic Exercise in Individuals with Multiple Sclerosis
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013681:00001

Permanent Link: http://ufdc.ufl.edu/UFE0013681/00001

Material Information

Title: Cytokine and Neurotrophin Response to Acute and Chronic Aerobic Exercise in Individuals with Multiple Sclerosis
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013681:00001


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CYTOKINE AND NEUROTROPHIN RESPONSE TO ACUTE AND CHRONIC
AEROBIC EXERCISE IN INDIVIDUALS WITH MULTIPLE SCLEROSIS















By

VANESSA CASTELLANO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Vanessa Castellano



























This work is dedicated to my parents, Rosa Capdet and Andreu Castellano. Thanks for
being amazingly supportive and always believing in me. I love you very much.

El treball del meu doctorate esta dedicat als meus pares, Rosa Capdet i Andreu
Castellano. Gracies per el vostre support constant i gracies per sempre creure en les
meves possibilitats. Us estimo molt.















ACKNOWLEDGMENTS

I would like to extend my thanks to my committee chair, Dr. Lesley J. White, for

her constant guidance and support throughout my entire doctoral work and my

dissertation project. I also would like to thank my committee members Dr. Scott Powers,

Dr. John Chow, and Dr. John Petitto for their support throughout the entirety of this

project.

I am forever indebted to all the members of the Applied Human Physiology

Laboratory who helped with the completion of my work (Ashley Blazina, Rachel

Canady, Stacey Colon, Jason Drenning, Anna Goodman, Sean McCoy, Darpan Patel,

Mai Tran, and Josh Yarrow). I am also indebted to Dr. Jessica Staib for her assistance in

assay preparation and troubleshooting, Kathy Howe for performing the DXA scans and

dealing with scheduling conflicts gracefully, Dr. Mark Tillman and Dr. Chris Hass for

statistical assistance, and all the volunteers that participated in the study for their hard

work and patience.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... ........ .. .............. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

A B STR A C T ................................................. ..................................... .. x

CHAPTER


1 IN TR OD U CTION ............................................... .. ......................... ..

S ig n ifi c a n c e .......................................................... ................. .
Aims and Hypotheses .................................. ............ ................
A im 1 ................................................................. . 5
H y p oth esis 1 ....................................................... 5
A im 2 ............................................................. .5
H y p oth esis 2 ....................................................... 5
A im 3 ............................................................. .6
H y p oth esis 3 ....................................................... 6

2 REVIEW OF LITERATURE .............................................................. .............. 7

S ig n ifican ce ....................................................... 7
M multiple Sclerosis ....................................... ........................................... 8
Cytokine R regulation in M multiple Sclerosis ......................................................... 10
The Importance of IL-6, TNF-a and IFN-y in Individuals with MS ................... 11
Interleukin-6 ...... .. ...................... ...... .............................12
Tumor necrosis factor-a ......................................................13
In te rfe ro n ............ ........... ................. ........ .............................. ...............1 3
Influence of Exercise on Cytokine Regulation in MS ....................................1... 14
Cytokine response to a single bout of exercise in MS .............................. 15
Cytokine response to chronic exercise training in MS and other diseases... 16
Cytokine response to exercise: mechanisms of action .............................18
The Role of Brain-Derived Neurotrophic Factor in Multiple Sclerosis ....................19
Exercise Influence on BDNF Expression................................. ............... 20
Insulin-Like Growth Factor-1 in Multiple Sclerosis ...............................................21









Impact of Exercise on IGF-1 in Multiple Sclerosis........................................ 22
Exercise Effects on Brain and Peripheral Concentration of IGF-1 .................23
M u scle IG F -1......................................................24
Fatigue in M multiple Sclerosis .................................................. ........................ 24
Central and Peripheral Fatigue in M S ...................................... .....................26
Exercise May Attenuate Fatigue in Individuals with MS ...................................27
General Impact of Regular Exercise in Multiple Sclerosis .......................................28

3 M E T H O D S ........................................................................................................... 3 0

Subjects .................. ............ .................. ........ 30
Subject Inclusion/Exclusion Criteria ................................................. ............... 30
Experim mental D design ...................................... .............. ............... 31
E x ercise T raining P rotocol .............................................................. .....................32
B baseline M measures ............................................ .. .. .. .. ......... ......... 33
G raded E exercise T est............. .................................. ........ .... .... ............... 33
Single Bout of Aerobic Exercise ..............................................................33
M uscle Fatigue .................................. .. .. .. ...... .. ............34
M uscle Strength ............... .. .......... ........ .... ......... .... ........34
Quality of Life in Health and Disease ............. .......................35
Perceived fatigue .......................................... .... .. .. .... .. ............
P erceived disability ............................................. ............ .............. 35
Quality of life assessment ........ .. ........ ........ ................. ......... 35
Functional M obility A ssessm ent......... ............... .......................... .............. 35
Six m inute w alk ............. .............................. .... .... ..... .. ... 35
W walking test (25 ft.) .......................................... .................. ......35
W walking test (100 ft.) .......................... .. .. ......... ........ ........ ......36
Tim ed up and go test ................................................ ............ ........ ... 36
B ody C om position .......... ..... ...................................................... ...... .... ... .36
Post-Exercise Training M measures ........................................ .......................... 37
Blood Collection and Processing..................................................... ..... .......... 37
B lood C collection T im es............................................................ .....................37
Cytokine A ssessm ent ............................................. ....... ..................38
IG F -1 A ssessm ent .............................. ........................ .. ...... .... ...... ...... 38
B D N F A ssessm ent.......... ...................................................... .... ...... .... ... .39
Plasm a V olum e A ssessm ent................................................... ............... ... 39
Statistical A n aly sis............ ... .............................................................. ........ .. ....... .. 4 0

4 R E S U L T S .............................................................................4 1

Subjects ...................... ......................................41
Immune Factors .......................... ................. 42
Chronic Exercise and IL-6........................ ...............42
Single Bout of Exercise and IL-6 .................................. ....... 42
Chronic Exercise and TNF-a................................................. 43
Single Bout of Exercise and TNF-a .............................................. 44
Chronic Exercise and IFN -y ........................................................ 45









Single Bout of Exercise and IFN-y........................................................... 46
Brain-D erived N eurotrophic Factor....................................... ......................... 46
Chronic Exercise and BD NF ........................................ .......................... 46
Single Bout of Exercise and BDNF.................................................................. 47
Insulin-Like G row th Factor- ............................................. ............................ 51
P lasm a V olum e ..................................................................................................52
M uscle Function ....................................... .............................................. 52
Knee Extension Endurance of Most Affected/ Non-Dominant Leg at 180s-1 ... 52
Knee Flexion Endurance of Most Affected/ Non-Dominant Leg at 180s-1 .......52
Knee Extension Endurance of Most Affected/ Non-Dominant Leg at 900s ......53
Knee Flexion Endurance of Most Affected/ Non-Dominant Leg at 900s-1 .........53
Isometric Muscle Torque During Extension at 900 of Knee Flexion ..................55
Isometric Muscle Torque During Flexion at 900 of Knee Flexion.................. 55
Isometric Muscle Torque During Extension at 1200 of Knee Flexion ...............55
Isometric Muscle Torque During Flexion at 1200 of Knee Flexion .................55
Isokinetic Muscle Torque at 900s-1 ............ ............. ......................... 56
Isokinetic M uscle Torque at 180 s ......................................... ............... 57
F u national M ob ility ...... ... .... .................................................... ...................... .... 58
W walking Tests ......................................................................... ........ .................... 58
T im ed U p and G o ....................... .. .... .................... ......... ........... 58
Six M inute W alk ............. ... ........................................................... .... .... .... .. 58
Quality of Life in Health and Disease ................................................................. 59
Perceived D isability........... ...................................................... .. .... .... ..... 59
M odified Fatigue Im pact Scale ........................................ ....................... 59
Short Form-36 Quality of Life Questionnaire.............................................60

5 D IS C U S S IO N ...................................... ......... .................. ................ 6 3

Resting Cytokine Concentration after 8 Weeks of Exercise Training......................64
Chronic Exercise May Modulate Serum BDNF at rest ...........................................66
Cytokine and Neurotrophin Response to a Single Bout of Exercise in MS ...............70
Cytokine Dynamics after a Single Bout of Exercise ........................... ........70
Serum BDNF Decreases Following a Single Bout of Exercise ..........................72
M u scle F atigu e......................................................................................... .... 74
Functional Mobility ...................... ..................... ............ 75
Quality of Life in H health and D disease .................................... ................................. 76
Perceived D isability........... ...................................................... .. .... .... .....76
Perceived Fatigue ...................................................................... ... ............ 76
Short Form-36 Quality of Life Questionnaire.............................................77
F future D directions .......................................................................78

LIST OF REFEREN CES ............................................................ ................... 80

BIO GRAPH ICAL SK ETCH .................................................. ............................... 96
















LIST OF TABLES

Table p

1 Subject characteristics pre and post 8 weeks of aerobic exercise. .........................41

2 Fatigue measures pre and post 8 weeks of aerobic exercise during at 1800sec1. .....54

3 Fatigue measures pre and post 8 weeks of aerobic exercise at 900sec1 ...................54

4 Isometric strength measures pre and post 8 weeks of aerobic exercise at 900 and
1200 of knee flexion ......... .............. .. ..... .......... .......... ........ ... .... ...... .. 57

5 Functional measures pre and post 8 weeks of aerobic exercise ............................59

6 Self-Assessed Measures pre and post 8 weeks of aerobic exercise .......................60















LIST OF FIGURES


Figure p

1 E xperim mental D design ......... ............................... ....................................... 32

2 Blood collection times before and after 30 minutes of cycle ergometry at
60% V O 2peak. ................................................................ 38

3 IL-6 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST) in both
g r o u p s ................................................................................................................. 4 2

4 IL-6 plasma concentration during a single bout of exercise in both groups. ...........43

5 TNF-a plasma concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST).......44

6 TNF-a plasma concentration during a single bout of exercise at 0 (PRE) ..............45

7 IFN-y plasma concentration at rest at 0 (PRE), 4 (MID) and 8 (POST) weeks of
aerobic exercise training................................................. .............................. 46

8 IFN-y response to a single bout of exercise in MS and control subjects...............48

9 BDNF concentration at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic
ex ercise train in g .................................................... ................ 4 9

10 BDNF acute response to exercise in MS subjects at weeks 0 (PRE), 4 (MID) and
8 (P O S T ). ......................................................... ................ 5 0

11 BDNF acute response to exercise in control subjects at weeks 0 (PRE), 4 (MID)
an d 8 (P O S T )................................................... ................ 5 0

12 IGF-1 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST).....................51















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CYTOKINE AND NEUROTROPHIN RESPONSE TO ACUTE AND CHRONIC
AEROBIC EXERCISE IN INDIVIDUALS WITH MULTIPLE SCLEROSIS


By

Vanessa Castellano

May 2006

Chair: Lesley J. White
Cochair: Scott K. Powers
Major Department: Applied Physiology and Kinesiology

Multiple sclerosis (MS) is an autoimmune disease that results in progressive neural

degeneration. Cytokines and neurotrophic factors play an important role in the

pathogenesis and treatment of MS. Exercise may modulate immune variables known to

impact disease progression and neuroprotection. We studied the impact of 8 weeks of

aerobic exercise on the immune and neurotrophin response, perceived and muscular

fatigue, functional mobility and quality of life measures in MS and matched controls

subjects. Subjects performed 30 minutes of cycle ergometry at 60% of VO2peak, three

times a week for 8 weeks in a supervised environment. Our results revealed

improvements in fitness parameters, but no changes in body composition, weight, waist

to hip ratio and body mass index. We found decreases in resting plasma IL-6 and

increases in resting plasma TNF-a and IFN-y following 8 weeks of aerobic training in

MS subjects. Serum BDNF concentration at rest before the initiation of the exercise









program was significantly reduced in MS subjects compared to matched controls. Serum

BDNF concentration at rest followed a biphasic response to exercise training and was

elevated 4 weeks into the training program and returned to baseline levels following 8

weeks of aerobic training in MS subjects. Serum IGF-1 at rest, muscular fatigue and

strength remained unchanged following 8 weeks of aerobic exercise. A single bout of

exercise revealed similar patterns of cytokine dynamics between MS and control subjects,

while BDNF clearance from the circulation was different at different stages of training in

MS subjects but not in control subjects. Perceived disability was significantly reduced

following 8 weeks of aerobic training while perceived fatigue and quality of life

measures had marginal improvements. Our study suggests that exercise may modulate

immune and neurotrophin mechanisms in individuals with MS after a short exercise

intervention. Although highly speculative, the impact of exercise on these parameters is

important because it could lead to neuroprotection in individuals with MS.














CHAPTER 1
INTRODUCTION

Significance

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous

system (CNS) and causing axonal demyelination that develops into a wide range of

debilitating symptoms (1-4). Accumulating evidence suggests that immune mechanisms

are involved in the initiation and perpetuation of the pathological characteristics of MS

(5, 6). In addition, females are more likely to develop MS than males (ratio -2:1) (7),

which is a common trait in autoimmune diseases. Further, most people develop MS

between the ages of 18-50 years (7). Because of the heterogeneity of demyelinating

lesions across individuals, the symptoms can vary widely, including loss of sensation,

optic neuritis, cognitive impairment, pain, bladder dysfunction, muscle weakness and

excess fatigue (4, 7). The most commonly reported symptoms are muscle weakness and

fatigue, which affect 80% of individuals with MS (2).

Although the etiology of MS remains unknown, repeated autoimmune attacks on

the CNS are thought to be responsible for inflammatory damage to axons and subsequent

disability in individuals with MS (8). In particular, pro-inflammatory cytokines influence

the disease cascade that causes degeneration in MS (9, 10). Pro-inflammatory cytokines

such as tumor necrosis factor-a (TNF-a) and interferon-y (IFN-y) activate naive T cells

and other antigen presenting cells that disrupt blood brain barrier (BBB) protection (4, 9,

11). High circulating levels of pro-inflammatory cytokines also stimulate T cell migration

through the BBB by upregulating endothelial receptor expression such as the VLA-4









receptor (11). These events are critical for the initiation of BBB infiltration and the

eventual axonal damage in MS (3, 9, 11-13). Therapies that attenuate pro-inflammatory

cytokines may reduce BBB disruptions and thus reduce axonal damage and attenuate

disease activity in individuals with MS.

Regular exercise has been shown to modulate pro- and anti-inflammatory balance,

and therefore maintain immune homeostasis in healthy populations (3, 14, 15). In

diseased populations, the impact of exercise on the immune system is mostly unknown.

However, a few studies have shown that exercise has the potential to reduce systemic

inflammation (16, 17). Patients with cardiovascular disease experienced a decrease in

pro-inflammatory cytokines after an aerobic exercise training program (17). Further,

Castaneda et al. (16) (2004) found that interleukin-6 (IL-6) was reduced in patients with

kidney disease subjects undergoing 12 weeks of resistance training compared with

controls. Furthermore, our laboratory found a significant reduction in plasma

concentrations of IFN-y and TNF-a (trend) after 8 weeks of resistance training (18).

Regular exercise training in MS subjects may have immunomodulatory outcomes that

alter systemic inflammation (19). Further studies to clarify the impact of regular exercise

on immune factors such as cytokines are warranted.

Recent evidence suggests that in addition to cytokine dysregulation in MS,

anomalous signaling of neurotrophic factors influences neural health (20). Specifically,

neurotrophins such as brain-derived neurotrophic factor (BDNF) and insulin-like growth

factor-1 (IGF-1) are critical in preventing cell death, increasing neural regeneration and

stimulating remyelination (21, 22). In diseases like MS, where neuroprotection is

compromised by autoimmune attacks causing neural demyelination and axonal damage,









the maintenance of neuroprotection is important (23). Since elevations in IGF-1 have the

potential to aid remyelination (24) and serum BDNF concentration may be lower in MS

compared to control subjects (23), therapies that increase IGF-1 and BDNF

concentrations may be neuroprotective. Exercise stimulates BDNF and IGF-1, both

which are known to be neuroprotective (22, 25-27). Exercise may increase circulating

IGF-1 in the periphery, which is also an upstream mediator of BDNF (28). IGF-1

administration in the animal model of MS, reduces the number of blood brain barrier

disruptions and demyelinating lesions (29). Although IGF-1 concentration may be normal

in MS subjects (30), increases in endogenous IGF-1 may promote oligodendrocyte

development, stimulate axonal sprouting and repair of damaged axons (31). Exercise also

increases BDNF levels in humans and contributes to neuroprotection (32). After

immobilization stress in animals, Adlard and Cotman (2004) (25) found that 3 weeks of

running can attenuate muscle atrophy in rats through elevations of BDNF secretion.

Regular exercise may, therefore, represent a non-pharmacological stimulus to increase

IGF-1 and BDNF secretion in individuals with MS where neuronal health is

compromised (4, 23, 29).

Continuous immune attacks that compromise neural health contribute to further

disability and increased severity of symptoms such as fatigue in individuals with MS.

Excessive fatigue is one of the most common debilitating symptoms, affecting

approximately 80% of individuals with MS (2). Fatigue contributes to the morbidity

associated with MS by limiting endurance and by adversely affecting mood, outlook and

ability to cope with accompanying symptoms (2). Fatigue in MS may manifest itself in a

variety of forms, including acute fatigue localized to specific muscle groups and









persistent central fatigue that have adverse effects on both physical and mental activity

(2, 33, 34). The pathophysiology of fatigue is poorly understood in MS. A number of

mechanisms have been implicated, including reduced frequency of action potential

propagation in partially demyelinated or degenerated central motor axons (central),

increased demands for muscle activation, and reduced muscular oxidative capacity due to

poor physical fitness (peripheral) (2).

Regular exercise has been shown to attenuate fatigue in healthy (35, 36) and

diseased populations (37, 38). In fact, recent findings suggest that regular exercise may

attenuate perceived fatigue in MS subjects (39-41). However, the mechanisms for these

effects remain speculative. Further research is warranted to address potential benefits of

exercise on MS-related fatigue.

Multiple sclerosis is an autoimmune disease that results in progressive neural

degeneration. Moreover, cytokines play an important role in the pathogenesis and

treatment of MS. Therapies that modulate cytokine expression may reduce disease

activity. In fact, current drug therapies for MS are aimed to modulate inflammatory

factors. In theory, exercise may modulate immune variables known to impact disease

progression. Further, exercise may enhance neuroprotection through IGF-1 and BDNF

production. Lastly, regular exercise training may attenuate fatigue, preventing a cascade

of events that could lead to inactivity, which is detrimental for the over all health. The

influence of exercise on cytokine regulation, neurotrophin secretion and fatigue in MS

subjects remains unclear and further investigations are warranted to address these issues.









Aims and Hypotheses

The purpose of this investigation is to determine whether aerobic exercise

modulates IL-6, TNF-a and IFN-y, neurotrophin secretion, and fatigue in MS and

matched controls after acute (30 minutes of aerobic exercise at 60%VO2max) and chronic

aerobic exercise (30 minutes/3 times per week at 60%VO2max for 8 weeks).

Aim 1

To determine whether resting levels of circulating cytokines (IL-6, TNF-a, and

IFN-y), BDNF and IGF-1 will be altered following eight weeks of aerobic exercise

training in MS and matched control subjects.

Hypothesis 1

Resting plasma cytokine concentration (IL-6, TNF-a, and IFN-y) will be reduced

after eight weeks of aerobic exercise, and serum BDNF and IGF-1 will be elevated in MS

and matched control subjects.

Aim 2

To determine if an eight week aerobic exercise training program will alter the

cytokine (IL-6, TNF-a and IFN-y) and BDNF response to an acute bout of aerobic

exercise (30 minutes of aerobic exercise at 60% VO2max).

Hypothesis 2

The cytokine and neurotrophin response to an acute bout of aerobic exercise (30

minutes of aerobic exercise at 60% VO2max) following an eight week aerobic training

program will be normalized in MS subjects. The cytokine and neurotrophin response will

be similar between MS and matched healthy control subjects due to the regulatory impact

of chronic exercise on individuals with MS.






6


Aim 3

To test the effect of eight weeks of aerobic exercise on fatigue in individuals with

MS.

Hypothesis 3

Muscular fatigue (voluntary muscle fatigue protocol) and perceived fatigue

(Modified Fatigue Impact Scale) will be attenuated in MS subjects following eight weeks

of aerobic exercise training.














CHAPTER 2
REVIEW OF LITERATURE

Significance

Multiple sclerosis pathophysiology, cytokine regulation, neurotrophin secretion,

and fatigue in persons with MS will be discussed in this chapter. The impact of acute and

chronic aerobic exercise on these variables will also be addressed. Immune system

dysregulation in individuals with MS triggers a cascade of events that lead to

demyelination and axonal damage (4). Because of these immune attacks, neuroprotection

can be compromised and consequently lead to further disability including excessive

fatigue and muscle weakness (9, 10). One of the goals of current MS immunomodulatory

drug therapies is to reduce inflammation by reducing pro-inflammatory cytokines. Thus,

therapies that minimize excessive inflammation and enhance neuroprotection may

influence disease progression.

Regular aerobic exercise has been shown to modulate immune factors such as pro

and anti-inflammatory cytokines (19, 42, 43), increasing neuroprotection through

elevations of IGF-1 and BDNF secretion (28, 44, 45). Exercise has also been shown to

attenuate perceived fatigue in healthy (35, 36) and diseased populations (37, 38).

However, the impact of exercise training on disease variables in persons with MS

remains mostly unexplored. Therefore, additional investigations are warranted to

ascertain the impact of acute and chronic aerobic exercise on immune factors,

neurotrophins and fatigue in individuals with MS.









Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system

(CNS) causing demyelination and axonal damage that may lead to increasing disability

(1). Multiple sclerosis is the most common inflammatory demyelinating disease in young

adults, affecting about 1 of 2000 individuals in the Western World (4), with females more

likely to develop MS than males (ratio -2:1). The onset of MS is typically between the

ages of 18-50 years (7). Much of the permanent disability results from axonal destruction

on long pathways such as the pyramidal tract supplying the legs and dorsal columns

carrying sensory information from the legs (46). Because of the diversity in lesion

volume and location across individuals with MS, a variety of symptom expression is

possible. In some individuals, symptoms may be minimal, while others may experience

extensive loss of sensation, slurred speech, muscle weakness, fatigue, depression, optic

neuritis, cognitive impairment, pain and bladder dysfunction (4, 7). Severe fatigue and

muscle weakness are two of the most reported symptoms in MS and have a large impact

on the quality of life of these patients (2). In addition, individuals with MS are typically

sedentary relatively to healthy individuals and often exhibit decreases in functional

capacity (47). Consequently, decreases in physical activity could also increase the risk of

secondary diseases such as cardiovascular disease, diabetes and obesity (47-49).

The etiology of MS is currently unknown. However, genetic predisposition,

autoimmune attacks, and environmental factors are thought to be the cause of disease (1).

In addition, MS is characterized by great variability and can be divided into different

clinical subtypes, including either a relapsing course or a more severe progressive course

(46). Approximately 85% of individuals with MS begin with the relapsing remitting

clinical subtype (46), which is characterized as clearly defined disease relapses with full









recovery or with sequelae, and with periods between disease relapses characterized by

lack of disease progression (50). Those with relapsing remitting MS experience

neurologic attacks with variable recovery but are clinically stable between the attacks.

Among this group are a minority of individuals that will have benign/mild MS with little

to no disability into their course (46). Individuals with benign/mild MS make up 10 to

20% of persons with MS (46). Benign MS is characterized by disease in which the patient

remains fully functional in all neurologic systems fifteen years after disease onset (50).

Further, there are three progressive subtypes. Approximately 10% of individuals with MS

have the primary progressive clinical subtype and never experience any attacks, and

about 5% have progressing relapsing, whose attacks superimpose a progressive course

(46). Primary progressive MS is defined as disease progression from onset with

occasional plateaus and temporary minor improvements (50). Secondary progressive is

the major progressive form of MS and accounts for 30% of the patients (46). Secondary

progressive MS is defined as initial relapsing remitting disease course followed by

progression with or without relapses, minor remissions and plateaus (50).

Various degrees of inflammation followed by axonal degeneration are responsible

for the large array of symptoms and different disease courses in individuals with MS.

Cytokine research has generated much attention and is the focus of therapeutic

interventions in MS (51). The effects of many therapies for MS are believed to be

mediated by changes in cytokine concentration. Multiple sclerosis immunomodulatory

drugs such as Avonex and Rebif target inhibition of pro-inflammatory cytokines to

reduce inflammation (51, 52). For example, Avonex decreases IFN-y production and T

cell activation in the periphery of individuals with MS. Therefore, strategies to reduce









inflammation and minimize axonal damage may influence disease progression and

attenuate MS symptoms.

Cytokine Regulation in Multiple Sclerosis

Cytokines are important initiators and regulators of the immune system and are

produced by leukocytes and other cells that stimulate proliferation and differentiation of

various immune cells (53). Cytokine concentrations are typically low or absent in healthy

individuals at rest because they are only produced in small quantities in response to local

stimuli, such as the presence of antigens, endotoxins, or the transduction signals provided

by other cytokines (6). Upon encountering an antigen, T cells differentiate into functional

dichotomous subsets, depending on the microenvironment the cell encounters (6). CD4+

helper cells are classified then into T helper 1 (Thl) and T helper 2 (Th2) cells depending

on the type of cytokines they produce. Thl cells secrete high levels of tumor necrosis

factor-a (TNF-a) and interferon-y (IFN-y), promoting macrophage activation and anti-

body-dependent cell cytotoxicity (13). Th2 cells produce interleukin (IL)-4, IL-6 and IL-

10, and promote humoral immune responses against extracellular pathogens (6).

Accordingly, cytokines can be classified as Thl or Th2 cytokines. However, cytokines

are also classified according to their effects on cell immunity. Cytokines promoting

cellular immunity are considered pro-inflammatory cytokines and include IFN-y, TNF-a,

TNF-P, IL-2, IL-12, IL-15, IL-17, and IL-18 (6). Cytokines promoting downregulation of

cell immunity are considered anti-inflammatory and include IL-4, IL-6, IL-10 and IL-13.

However, almost all anti-inflammatory cytokines have some pro-inflammatory properties

and vice versa (6).

The blood brain barrier (BBB) is regarded as a protective system from immune

attacks. However, T cell mediated immunological processes may lead to alteration of the









BBB and enable recruitment of other inflammatory cells, such as monocytes (54).

Cytokines are critical in the mechanism of T cell activation that can ultimately cause

demyelination and tissue damage in MS (55). In the periphery, inflammatory cytokines

induce, in a bystander fashion, activation of monocytes, dendrite cells (DCs) and T cells

(9). For example, DCs undergo maturation into antigen presenting cells (APCs) after their

exposure to pro-inflammatory cytokines and consequently are able to activate T naive

cells (54). Dendritic cells that are activated by inflammatory cytokines rapidly activate

other innate protective cells such as natural killer (NK) cells to mediate the balance

between Thl and Th2 subsets (56). However, T cells may also be in an enhanced state of

activation in the periphery of individuals with MS, suggesting a breach in the protection

of the BBB, and facilitating an overproduction of pro-inflammatory cytokines (Thl

subset) (10). In addition, cytokines produced by activated T cells in MS lesions induce

the activation of macrophages and local microglia effector cells, leading to the increase of

axonal destructive activity, which is responsible for demyelination and tissue damage in

MS (1). Therefore, a reduction of pro-inflammatory cytokine levels in the periphery

could attenuate the rate of naive T cell activation and interrupt the cascade of events that

lead to demyelination and axonal damage in the CNS of individuals with MS.

The Importance of IL-6, TNF-a and IFN-y in Individuals with MS

Preliminary studies revealing the influence of cytokines in MS disease activity has

facilitated extensive research to further elucidate mechanisms responsible for immune

attacks, and thus potential treatment therapies. It is also thought that immune

dysregulation is the main cause of the demyelination process and axonal damage

observed in MS (10). A dysregulation of the balance between pro-inflammatory (Thl)

and anti-inflammatory (Th2) cells with a shift to Thl profile has been reported in









individuals with MS (57). A variety of cytokines modulate Thl/Th2 balance that is

critical to maintain immune homeostasis (11, 58). However, the influence of cytokine

dysregulation in MS remains somewhat unclear.

Interleukin-6 (IL-6), Tumor Necrosis factor-a (TNF-a) and Interferon-y (IFN-y)

have a prominent role in the process of demyelination and axonal damage experienced by

persons with MS (59). These pro-inflammatory cytokines may also stimulate T cell

activation in the periphery that leads to demyelination (10), and have appeared repeatedly

in areas of inflammatory demyelination in the form of perivascular infiltrations within the

white matter of the brain of persons with MS (59). The main functions of these cytokines,

in relation to MS, are discussed in more detail below.

Interleukin-6

Interleukin-6 (IL-6) is produced by many different cells, but the main source in vivo

are monocytes, macrophages, fibroblasts and vascular endothelial cells (60). Further, IL-6

has been shown to have both pro-inflammatory and anti-inflammatory effects, but

recently it has become recognized for its anti-inflammatory properties (42). Abnormally

high concentrations of IL-6 in the periphery may result in excess inflammation, which

may be harmful to the host, and exacerbate autoimmune disease activity (6). IL-6 also has

immunosupressive effects such as the inhibition of TNF-a expression by macrophages

and astrocytes. Elevated IL-6 may also disrupt the clearance of microbial pathogens (6)

and participate in T cell activation, accelerating the MS disease process (5, 6).

Circulating IL-6 appears to be the primary inducer of acute phase proteins from the

liver, and is also involved in mediating interactions between the endocrine and the

immune systems (59). In addition, skeletal muscle is another source of IL-6 production

(42). Skeletal muscle contractions stimulate IL-6 production and may increase circulating









IL-6 concentration via complex signaling cascades initiated both by Ca2+ dependent and

independent stimuli (61). Thus far, it is unclear whether individuals with MS have

abnormal levels of IL-6 in skeletal muscle and in the periphery (62), and the impact of

this cytokine on regeneration and degeneration of neurons of individuals with MS

warrants further study (63).

Tumor necrosis factor-a

Tumor necrosis factor-a (TNF-a) is produced by macrophages, T cells and B cells

(64). Elevated TNF-a receptor expression on T cells and in soluble form has also been

found in individuals with MS (65). TNF-a has been shown to promote demyelination of

neurons in the brain and it is also thought to play a role in muscle wasting that occurs

with chronic infections (66). High circulating TNF-a concentration in subjects with MS is

associated with worsening of the disease (67, 68). TNF-a can also be expressed in

skeletal muscle as a consequence of cachexia (66). However, the impact of TNF-a in

skeletal muscle of MS subjects remains unknown. It is known however that high TNF-a

plasma concentrations are also associated with low muscle mass and lower muscle

strength in frail individuals (69).

Given the diverse and potent effect of TNF-a on the immune and muscle function

of MS subjects, strategies to reduce TNF-a may contribute to reduction in disease

severity (64). Since TNF-a is an important cytokine involved in causing axonal

demyelinating lesions in MS subjects, further investigations are warranted.

Interferon-y

Interferon-y (IFN-y) is secreted mainly by lymphocytes and synthesized primarily

by T lymphocytes and natural killer cells following activation of immune and

inflammatory stimuli (70). IFN-y is a major disease promoting cytokine in MS (64).









Administration of IFN-y to MS subjects worsens disease activity (71). In addition, IFN-y

correlates with functional impairment in MS (55). Further, IFN-y can induce APCs to

secrete high levels of other pro-inflammatory cytokines by virtue of its ability to

differentiate naive T cells into Thl cells (72). These events may therefore accelerate

excessive T cell infiltration through the blood brain barrier and thus contribute to further

demyelination in persons with MS.

Elevations of plasma IFN-y in MS compared to control subjects have also been

reported by Link et al. (73) (1999) and in our laboratory (18). Further, IFN-y has been

found to promote muscle wasting and could contribute to the deterioration of skeletal

muscle in MS subjects (66). Therefore, IFN-y is integrally involved in the MS disease

process, and inhibiting IFN-y secretion or antagonizing its actions could have important

outcomes preserving skeletal muscle and modulating the immune system of individuals

with MS (74).

Influence of Exercise on Cytokine Regulation in MS

Cytokine regulation following acute and chronic exercise remains relatively

unexplored in MS subjects. Exercise modulates immunological responses through

cytokine production in short bouts of exercise in healthy populations (42, 53, 75-77).

Cytokine dysregulation is linked to the inflammatory processes observed in MS (78).

Inflammatory cytokines increase T cell infiltration by activating a series of events that

lead to disruption of the blood brain barrier leading to further axonal damage (8).

Therefore, because of its immunomodulatory potential, exercise may impact cytokine

dysregulation in individuals with MS.









Cytokine response to a single bout of exercise in MS

It has been proposed that short term release of cytokines during acute exercise may

contribute to the maintenance of an immune homeostatic environment (79). In addition,

many of the acute phase proteins released in response to elevated cytokine levels are

protease inhibitors or free radical scavengers that attenuate the magnitude of tissue

damage associated with release of toxic molecules and free radicals due to activated

neutrophils (79). Therefore, a single bout of exercise represents a mild physical stressor

and has an array of effects on immune parameters (76, 80). Consequently, the release of

cytokines during and after moderate intensity exercise bouts could contribute to

neuroprotection (80). Moreover, studying the immunological response to a single bout of

exercise in MS subjects may yield important information regarding the immediate effects

of exercise on autoimmune diseases and how MS subjects respond to stress in general.

Early work by Le Page et al. (81)(1994) investigated the effect of exercise on the

inflammatory phase of experimental autoimmune encephalomyelitis (EAE), the animal

model of MS. Exercise training during the induction phase of EAE did not exacerbate the

disease course (81, 82). In a study by Heesen et al. (19) (2003) sedentary MS subjects

showed a blunted cytokine response (i.e., TNF-a) after a single bout of exercise (30

minutes of cycling at 60% of their VO2max). However, the acute response of other pro and

anti-inflammatory cytokines such as IL-6 or IFN-y were not measured. Further, the

impact of exercise on baseline cytokine concentrations was not investigated. Schulz et al.

(76) (2004) investigated the impact of acute exercise on IL-6 but did not found any

significant differences between MS subjects and controls. Further, both Heesen et al. (19)

(2003) and Schulz et al. (76) (2004) collected blood samples immediately after exercise

and 30 minutes later with no additional collection times. The timing of blood sample









acquisition is important because the dynamics of each cytokine vary considerably in

response to exercise (53). Therefore, their collection time may have not been sufficient to

identify the maximal responses of these immune parameters. For example, Pedersen et al.

(42) (2003) have published results showing IL-6 peaks approximately 2-3 following

aerobic exercise. Pilot data from our laboratory shows that following a moderate bout of

aerobic exercise, IL-6 concentration peaks 2-3 hours after exercise (unpublished).

Further, Heesen et al. (77) (2003) and Schultz et al.(76) (2004) did not correct for plasma

volume changes that might had occurred during the 30 minute exercise bout. Plasma

volume is typically reduced 3-14% after 30 minutes of moderate intensity exercise (83,

84). Additional studies are needed to provide a more complete and comprehensive

understanding of the dynamic cytokine response to physical stress in MS. A special

attention should be focused on IL-6, IFN-y and TNF-a because they are known to directly

influence the pathogenesis of MS.

Cytokine response to chronic exercise training in MS and other diseases

Chronic exercise has been found to modulate the immune system in healthy (53,

80) and some diseased populations such as cardiovascular and kidney disease (16, 17). In

a study by Smith et al. (17) (1999), six months of aerobic exercise lowered cytokine

secretion of IFN-y in the periphery of subjects with cardiovascular disease. Further,

Castaneda et al. (16) (2004) found that resting concentration of plasma IL-6 was reduced

in patients with kidney disease subjects undergoing 12 weeks of resistance training

compared with controls. Furthermore, our laboratory showed a significant reduction in

plasma concentrations of IFN-y and TNF-a (trend), as well as no change in IL-6

concentration after 8 weeks of resistance training (18). Whether exercise consistently

reduces levels of circulating inflammatory cytokines in MS subjects remains unclear.









However, these data suggests that chronic exercise may influence baseline concentration

of cytokines known to impact inflammatory processes in MS.

Little is known about the effect of chronic aerobic exercise on immune parameters

such as cytokines in autoimmune diseases such as MS (85). To date, there only two

published reports on the effect of aerobic exercise on immune parameters of MS subjects

(19, 76). In a study by Heesen et al. (19) (2003) sedentary MS subjects showed a blunted

cytokine response after a single bout of exercise (30 minutes of cycling at 60% of their

VO2max) in the untrained state compared to controls. However, after an eight week

aerobic exercise training program the blunted response to a single acute bout of exercise

was similar to controls. Therefore, Heesen et al. (19) (2003)showed that aerobic exercise

trained MS subjects can promote an immune response to physical stress that is similar to

healthy individuals. However, Heesen et al. (19) (2003) did not report the effect of

chronic exercise training on resting cytokine concentration, and therefore, the impact of

exercise training on circulating cytokine concentration in MS subjects remains unclear.

Schulz et al. (76) (2004) investigated the impact of chronic exercise on IL-6 and found

that aerobic training for 8 weeks (30 minutes of cycle ergometry, 3 times/week at 60%

VO2max) did not affect resting concentration of IL-6.

Additional information about the influence of exercise on MS disease activity has

come from research using the animal model of MS. Le Page et al. (81) (1994)

investigated the effect of exercise on the inflammatory phase of experimental

autoimmune encephalomyelitis (EAE). Exercise training in EAE rats did not aggravate

the disease course (81, 82). Findings after a 10-day exercise-training regimen performed

immediately following the induction of EAE included a reduction in the duration and









severity of chronic EAE in rats (81). Further investigations on the impact of exercise on

EAE and MS are warranted, since Le Page and colleagues did not probe mechanisms of

the delayed onset, severity and duration of EAE. Their work suggest that exercise can

modulate disease pathophysiology (81). However, the mechanisms of action remain

unknown, but likely involve modulation of the immune system.

In summary, cytokine dysregulation appears to cause inflammation and subsequent

demyelination and axonal damage in MS subjects that may impact disease progression

and severity. Acute and chronic aerobic exercise may modulate immune function by

reducing circulating levels of cytokines, and therefore influence disease activity in MS

subj ects.

Cytokine response to exercise: mechanisms of action

The cytokine response to acute exercise is complex and it is related to exercise

intensity, training status, site of cytokine measurement (i.e., tissue, plasma or urine) and

method of measurement (53). Cytokines appear in low concentration (<3pg/ml) in plasma

of healthy individuals at rest (86, 87). The time course of cytokine elevation or

depression in response to exercise differs depending on the cytokine of interest. For

example, IFN-y and TNF-a have been shown to increase immediately after exercise (30

minutes) while IL-6 displays a more delayed response (0-3 hours) (42, 75). A complete

understanding of mechanisms responsible for the cytokine response to exercise are

currently unknown. Early reports suggested that the cytokine response may be due to a

pro-inflammatory release produced by muscle damage and perhaps inflammation of

skeletal muscle (88-91). However, more recent studies clearly demonstrate that muscle

contraction without any muscle damage can induce marked elevations of cytokines (i.e.,

IL-6) (75). The exact mechanisms mediating communication between skeletal muscle









and other cells releasing cytokines during and after exercise has not been elucidated, but

are most likely multifactorial. In fact, a single cytokine rarely acts alone (53). Typically,

these actions depend on the level of cytokine produced, endogenous inhibitors and

regulators, and interactions with other cytokines (53). Further investigation of these

events will enhance our understanding of the impact of exercise on the immune system.

The Role of Brain-Derived Neurotrophic Factor in Multiple Sclerosis

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophic family, is

a homodimeric protein that has been highly conserved in structure and function during

evolution (92). BDNF is widely expressed throughout the brain cortex and also in the

periphery in many animal species (93-96). BDNF participates in cellular maintenance and

protects neurons from injury (97, 98). BDNF also plays a predominant role in neural

development and brain health (99) BDNF acts as a short-term potent excitatory

neurotransmitter leading to rapid depolarization of postsynaptic neurons (95). Further,

BDNF induces long lasting changes in synaptic plasticity, and plays a key role in

learning, memory and behavior (100, 101).

It has been demonstrated that BDNF can cross the BBB, suggesting that serum

BDNF levels may reflect BDNF levels in the brain (102). Potential sources of circulating

BDNF are platelets, vascular endothelial and smooth muscle cells (103). However, since

it is known that BDNF can cross the BBB in both directions, a substantial part of

circulating BDNF could also originate from neurons and glia cells of the central nervous

system in a bidirectional fashion (104).

During an immune attack, BDNF may protect axons from demyelination and also

facilitate remyelination after injury in MS (105). Since BDNF concentration may be

decreased in MS compared to control subjects (23), the neuroprotective potential of the









brain is jeopardized, and strategies to increase BDNF in MS subjects could have

important therapeutic outcomes. In fact, increases in BDNF production has been

proposed as a strategy to increase neuroprotection in individuals with MS (23). Further,

increased resting concentration of BDNF may attenuate MS deterioration of nerve and

muscle function. Thus, further investigations are needed to determine if MS patients have

lower concentration of BDNF, and to determine the effect of exercise on BDNF secretion

on MS subjects.

Exercise Influence on BDNF Expression

There is strong support from the literature indicating that exercise provides brain

health benefits by increasing neuroprotection and possibly inducing axonal repair through

neurotrophin action (21, 22, 25, 32, 106-108). Exercise is increasingly recognized as an

intervention that can reduce cognitive decline and depression, possibly through elevated

BDNF concentration which maintains neuronal health (25). It has been previously

demonstrated that voluntary exercise induces BDNF upregulation in the brain and

periphery helping maintain brain health (i.e., plasticity and memory) (20, 21). For

example, exercise enhances the effectiveness of antidepressant treatment, perhaps by

augmentation of BDNF levels in humans (32, 106), along with increases in neurogenesis

and learning (25). Exercise may also influence clinical populations with

neurodegeneration such as Alzheimer's disease or clinical depression (22, 45), and may

not only contribute to improved motor and cognitive function, but also provide resistance

to the effects of stress-related processes that can occur within the injured CNS (22, 25).

Recent data suggest that BDNF is elevated in exercised muscle (109) and can be

retrogradely transported into the spinal cord (110). BDNF also has effects on skeletal

muscle tissue by inducing the potentiation of spontaneous twitching in myocytes to









enhance muscle contraction (111). In the periphery, exercise can upregulate the

expression of BDNF and maintain skeletal muscle health (25). For example, after

immobilization stress, Adlard and Cotman (2004) (25) found that exercise can override

the negative effects of muscle atrophy in rats through elevations of BDNF secretion after

3 weeks of running. In BDNF deficient rats, 2 months of wheel running increased BDNF

concentration (107). Exercise may, therefore, represent a non-pharmacological stimulus

to increase BDNF in MS subjects.

In summary, BDNF is critical to maintain neuronal health, reduce demyelination

and may stimulate remyelination in MS subjects. Previous investigations have shown that

regular exercise upregulates BDNF in both the CNS and the periphery (20, 21).

Therefore, individuals with MS may benefit from regular exercise training because it may

increase BDNF concentration, and subsequently protect brain function and promote

remyelination.

Insulin-Like Growth Factor-1 in Multiple Sclerosis

Insulin-like growth factor-1 (IGF-1) promotes the survival and regeneration of

oligodendrocytes, stimulates synthesis of myelin, and promotes skeletal muscle health

(30). Thus, the regulation of IGF-1 is important in MS, where neurodegeneration and

muscle atrophy are primary disease manifestations (112). Several lines of evidence

suggest IGF-1 may be beneficial in treating individuals with MS and other demyelinating

diseases (113). In the animal model of MS (EAE), treatment with IGF-1 reduces clinical

deficits and lesion severity (24). In addition, it is known that myelin content rises in the

CNS in transgenic mice over expressing IGF-1 (113). In addition, injections of IGF-1 in

EAE rats have been shown to reduce disease severity and clinical deficits (24). For

example, an IGF-1 subcutaneous injection improved clinical deficit scores, stride lengths









and exercise wheel rotations within 48 hours of injection (24). In another animal study,

after the administration of exogenous IGF-1, EAE onset was delayed, suggesting a

decrease or absence of inflammatory cells in the CNS (114). Therefore, it has been

suggested that increases in IGF-1 concentration may be useful as a treatment for MS to

maintain neural health and promote remyelination (30). MS subjects appear to have

normal levels of serum IGF-1, but possible therapies involving this neurotrophin are

attractive due to its effect on remyelination (29). However, in one study, IGF-1

administration in MS subjects did not influence measures of disease status. Seven MS

subjects received 50 mg of rhIGF-1 twice a day for 6 months, but there were no

significant differences between baseline and treatment periods for MRI lesion load or

disease activity (29). Perhaps longer administration of rhIGF-1 or the promotion of

endogenous IGF-1 (i.e., exercise induced) may have more favorable results regarding

neuronal repair and remyelination (29). Therefore, additional studies are needed to

investigate the impact of increased endogenous IGF-1 on MS subjects.

Impact of Exercise on IGF-1 in Multiple Sclerosis

Insulin-like growth factor-1 provides neuroprotection and may reduce muscle

wasting (113). Regular exercise training is known to stimulate the production of IGF-1

(28, 44). In turn, IGF-1 can promote skeletal muscle protein synthesis, oligodendrocyte

survival, myelin protein synthesis, and myelin regeneration (113). Since exercise

promotes IGF-1 production, which further stimulates favorable changes in both nerve and

muscle, further study of the role of exercise training on individuals with MS may yield

important clinical information.









Exercise Effects on Brain and Peripheral Concentration of IGF-1

Following moderate exercise training in rats, IGF-1 levels increase in both brain

and periphery, which may subsequently promote remyelination in rats (28). Neurons

influenced by increased IGF-1 show elevated spontaneous firing and increased sensitivity

to afferent stimulation (28). Furthermore, a systemic injection of IGF-I mimicked the

effects of chronic exercise in the brain in these rats. When uptake of IGF-I by brain cells

stopped, neuroprotection was lost. Carro et al. (28) (2000) concluded that serum IGF-I

mediates the initiation of events that causes the positive impact of exercise on the brain.

Thus, stimulation of the uptake of blood-borne IGF-I by nerve cells may lead to

neuroprotection. However, the mechanism whereby exercise increases contributes to

neuroprotection remains unclear.

Although central mechanisms are pivotal in exercise-induced neuroprotection of

the brain, it is now emerging that peripheral mechanisms may also play a significant

neuroprotective role through IGF-1 (44, 115). Several reports suggest that exercise-

induced elevations in peripheral IGF-1 initiate growth-factor cascades in the brain that

could lead to remyelination (28, 44). Circulating IGF-1 also acts as an upstream mediator

of BDNF regulation, neurogenesis, and the ability of exercise to protect the brain from

neuronal injury such as demyelination (28, 44). It has been suggested that exercise

training can improve memory and information processing efficiency through mechanisms

that include the upregulation of IGF-1 and BDNF, and thus potentially be beneficial in

MS subjects where memory and information processing may be compromised (21, 116).

According to previous findings, central and peripheral elevations in IGF-1 promote

neuronal health, and may also aid in the repair of demyelinated neurons found in MS









subjects (28, 117). Therefore, exercise-induced increases in IGF-1 may play an important

role in protecting the CNS from injury in MS subjects.

Muscle IGF-1

IGF-1 is recognized for its role in skeletal muscle adaptation to exercise (118). It is

well documented that IGF-1 has potent effects on myoblast proliferation and

differentiation, leading to muscle hypertrophy (116). Exercise increases muscle IGF-1

content and causes a net increase in protein (116). The exercise-induced elevation in IGF-

1 may prevent myofiber wasting due to disuse and age-associated myofiber loss (118) in

MS subjects. Ultimately, exercise-induced hypertrophy through IGF-1 mechanisms may

be needed to produce sufficient levels by the target muscles to ensure maintenance of

healthy innervation (116). Therefore, individuals with MS could benefit from exercise-

induced secretion of IGF-1 to maintain muscle health.

In summary, aerobic exercise training may elevate IGF-1 concentration in a variety

of sites (i.e., brain, peripheral circulation, skeletal muscle) and stimulate remyelination,

improvements of brain cognition, and muscle hypertrophy, which may ultimately reduce

disease progression in persons with MS (21, 116). Therefore, regular aerobic exercise

training could potentially contribute to functional improvements in the MS population.

Fatigue in Multiple Sclerosis

Excessive fatigue and muscle weakness are the most common and debilitating

symptoms of individuals with MS (2). Fatigue in MS is defined as an abnormal sense of

tiredness or lack of energy, out of proportion to the degree of effort or level of disability

that significantly interferes with the routine physical or intellectual functioning (2). Thus,

MS-related fatigue is an unusual and abnormal form of fatigue that differs from the

fatigue experienced by healthy individuals after exertion (failure to maintain the required









or expected force). Approximately, 65-95% of MS subjects have significant fatigue (2).

Fatigue also contributes to the morbidity associated with MS by limiting energy and

endurance and by adversely affecting mood, outlook and ability to cope with

accompanying symptoms. The average level of physical activity in individuals with MS

has been shown to be reduced compared to their healthy counterparts (119).

The mechanisms suggested for excessive fatigue in MS subjects include

psychological factors or brain lesions in specific neural pathways that may play a role in

MS-related fatigue and depression, or that fatigue contributes to depression in MS

subjects (34). Moreover, MS fatigue may be related to demyelination, inflammation and

axonal injury (2, 120). It is unclear whether constant fatigue in MS subjects is due to the

burden of diagnosis, reduced activity due to neurological symptoms and not fatigue, or

directly related to other physiological factors (2, 34). However, it is well known that

physical activity, if reduced, can perpetuate adverse changes in muscle strength and

fitness in any population.

Fatigue in MS may manifest itself in a variety of forms, including acute fatigue

localized to specific muscle groups and persistent central fatigue that has adverse effects

on both physical and mental activity (2, 33, 34). The etiology of fatigue in MS is not well

understood and appears to be complex and multifactorial (2). Both peripheral and central

mechanisms have been postulated, but none has satisfactorily explained the development

of MS fatigue (33). In addition, fatigue is not well explained by gender, psychomatic

mechanisms, physical disability, or sleep dysfunction. A recent study by Bakshi (2000)

(34) found a significant relationship between depression and fatigue severity. Moreover,

functional brain imaging studies indicate that MS is associated with widespread low









metabolism in the brain (121). Specifically, Bakshi et al. (121) (1998) found MS subjects

had a 10% reduction in total brain glucose metabolism. Another study by Roelcke et al.

(122) (1997) showed that MS subjects had reduced glucose metabolism with fatigue but

not in those individuals without fatigue. However, other studies have shown a lack of

association between MS fatigue and neurodegeneration (brain atrophy/lesion load) (123,

124). The etiology of fatigue in MS is complex and multifactorial. Further research is

needed to find effective strategies to attenuate fatigue in MS.

Central and Peripheral Fatigue in MS

Demyelination, the product of the inflammatory process that underlies MS, impairs

axonal conduction and eventually produces axonal loss and damage (120). Axonal

impairment may contribute to centrally mediated fatigue through several mechanisms.

For example, delayed or partial innervation of voluntary muscles may require a

compensatory increase in central motor excitatory mechanisms (119), and thus, MS

subjects may necessitate increased motor drive to achieve the same levels of muscular

contraction. Consequently, MS subjects may experience premature fatigue relative to

their healthy counterparts (119). Axonal damage has also been correlated with increased

fatigue in MS subjects (120). In addition, individuals with MS may have delayed

activation of motor units contributing to abnormal communication between the cerebral

cortex, basal ganglia and cerebellum, and the descending motor pathways (2). Finally,

both physical and mental fatigue may occur simultaneously or independently of each

other. The fact that they occur together, along with the high frequency of fatigue in MS,

points to a possible critical role of central fatigue in these individuals.

Peripheral fatigue is a type of fatigue localized to skeletal muscle, and although

individuals with MS seem to experience it often, it may be the result of physical









inactivity or long-term consequences of central fatigue (119). Peripheral fatigue can be

caused by negative effects precipitated by inactivity such as decreased muscle cross

sectional area, decreased skeletal muscle strength, muscle wasting, fiber type shifting

from Type IIa to Type IIb (125). These changes may lead to an enzymatic shift from slow

glycolytic to fast glycolytic muscle fiber characteristics. Due to a reliance of more

glycolytic pathways in any activity, and the higher amount of ADP and Pi in the system,

Ca2+ reuptake from the SR may be disrupted (126). These events can also increase H+

concentration, which can disrupt cross-bridge cycling by disrupting Ca2+ attachment to

troponin, and therefore cause peripheral fatigue.

Although there is a strong, plausible evidence that fatigue is a centrally-mediated

complication in MS, the influence of physical inactivity, patient perception of their

impaired capacity and altered innervation of muscles suggest that MS fatigue is

multifactorial in origin and may be expressed differently across individuals with MS.

Exercise May Attenuate Fatigue in Individuals with MS

It is well known that fatigue can be counteracted by exercise training in healthy

(35, 36) and diseased (37, 38) populations. Consequently, a regular exercise training

program may provide a similar stimulus that can attenuate the symptoms of fatigue in

individuals with MS. There is evidence indicating that regular exercise may attenuate

perceived fatigue in MS subjects (39-41). After 15 weeks of aerobic exercise at moderate

intensity, MS subjects experienced a significant reduction in fatigue (measured with the

profile of mood states questionnaire) and a negative association between improvement in

aerobic fitness and fatigue perception (40). As a result of increases in aerobic capacity,

MS subjects were able to perform activities of daily living at lower relative intensity,

preventing excessive tiredness. Using the fatigue severity scale (FSS) for their fatigue









assessment, Mostert and Kesselring (2002) reported reductions in fatigue (-14%) of MS

subjects after only 4 weeks of aerobic exercise training. MS subjects also increased their

activity level by 17% after only 4 weeks of regular aerobic exercise (41). Further, White

et al. (39) (2004) reported a 24% decrease in perceived fatigue as indicated by the

modified fatigue impact scale (MFIS) after 8 weeks of resistance training in MS subjects.

In summary, the etiology of MS fatigue is complex and multifactorial. In addition,

regular physical activity may attenuate perceived fatigue in individuals with MS.

Therefore, fitness improvements experienced while undergoing an exercise training

program may be responsible for the reductions in fatigue, possibly by counteracting the

effects of detraining that are secondary to MS fatigue.

General Impact of Regular Exercise in Multiple Sclerosis

Regular aerobic exercise training in individuals with MS may help reduce the rate

of decline in functional capacity observed in the MS population. In the past, individuals

with MS were advised to avoid physical activity because symptoms may worsen with

elevations in body temperature (76). However, many studies suggest that exercise

training in MS subjects is safe and can promote many important beneficial outcomes,

such as improvements in cardiorespiratory and muscle function (40, 41, 112, 127),

decreased incidence of depression (40, 127), decreased perceived fatigue (41, 128), and

possible regulatory effects on the immune system of MS subjects (77).

MS subjects that completed a 15 week aerobic exercise training program

demonstrated significant improvements in aerobic fitness and strength measures

compared to non-exercising controls (40). Each training session consisted of supervised

cycle ergometry for 30 minutes at 60% of VO2max, three times a week for 15 consecutive

weeks. MS subjects experienced significant increases in physical capacity and maximal









isometric strength, as well as significant decreases in body fat. Specifically, a 10%

increase in VO2max was observed after only 5 weeks and an increase of 25% after 15

weeks of aerobic training. Petajan et al. (40) (1996) concluded that aerobic exercise

training resulted in improved fitness and had a positive impact on factors related to

quality of life. Ponichtera-Mulcare at al. (129) (1997) also demonstrated an improvement

in aerobic capacity between 5-20% after 6 months of aerobic training using cycle

ergometry at 60% VO2max. Mostert and Kesselring (2002) (41) also investigated the

effects of aerobic exercise for 4 weeks on MS subjects. They concluded that 30 minutes

of supervised cycle ergometry five times a week provided improvements in health

perception, improved aerobic fitness, increases in activity level and a tendency to less

fatigue (41).

These results confirm that individuals with MS are capable of making favorable

adaptations to aerobic exercise training. Although the effects of exercise training on MS

disease progression still remain unknown, the over all health benefits of exercise alone,

provide a healthy means to maintain or improve quality of life in individuals with MS.














CHAPTER 3
METHODS

Subjects

Twenty seven subjects were recruited from the local community. Each subject had

physician clearance and signed a consent form approved by the University of Florida

Institutional Review Board. Eleven individuals with MS and eleven non-MS healthy

controls completed the entire study.

Subject Inclusion/Exclusion Criteria

Subjects with relapsing remitting MS, who were clinically stable and had minimal

to moderate disability, were included. Multiple sclerosis was diagnosed by a physician

according to the Poser criteria (130). A disability status of 0-5.5 (EDSS 0-5.5: minimal-

to-moderately disabled with ability to walk at least one city block (100 meters)) was

required for MS subject study inclusion. All subjects (MS and control subjects) needed

physician clearance to participate in the study, a systolic blood pressure of less than 140

mmHg, and a diastolic blood pressure of less than 90 mmHg.

Subjects with cardiovascular disease, diabetes, thyroid disorders, gout, and

orthopedic limitations as established by the American College of Sports Medicine (131)

were excluded from the study. Additionally, individuals using prednisone or

antispasmotic drugs were excluded. If a subject had a relapse, they were excluded from

the study. A relapse (attack, exacerbation) was defined as a separate period of worsening

in a neurological symptom lasting 24 hours or more after a preceding month of stationary









or improving status. Subjects that were not able to pedal for 20 minutes at 60% of their

VO2peak were also excluded.

Experimental Design

The study consisted of an eight week aerobic exercise training program wherein

subjects exercised on a cycle ergometer 3 times weekly for 30 minutes at 60% VO2peak

with pre, mid and post exercise biochemical, muscular and functional assessments.

Baseline measurements of aerobic fitness, muscle strength/endurance, fatigue, body

composition and general health questionnaires were acquired. In addition, baseline

resting blood samples were drawn for cytokine and neurotrophin assessment. Prior to the

initiation of training, subjects performed a 30 minute bout of exercise (cycle ergometry)

at 60% of V2 peak to assess the cytokine and neurotrophin response to a single bout of

aerobic exercise in the untrained state. Following baseline assessments, subjects

participated in a supervised eight week aerobic exercise training program (30 min/3 times

a week/60% of VO2 peak). All baseline measures were re-assessed at 4 and 8 weeks. The

experimental design is shown in Figure 1.










I I


8 weeks of aerobic exercise training
30 min cycle ergometry/3 times per week/ 60%VOzm,,,

Week 0


BIOCHEMICAL, MUSCLE AND FUNCTIONAL ASSESSMENTS

Immune factors (IL-6, TNF-a, IFN-y) Muscle fatigue
Neurotrophins (BDNF, IGF-1) Muscle strength
Quality of life measures Perceived fatigue




Figure 1. Experimental Design

Exercise Training Protocol

All subjects participated in a supervised 8 week aerobic exercise training protocol.

Each exercise session consisted of a 3 minute warm up at a self-assessed comfortable

speed followed by 30 minutes of cycle ergometry at 60% of VO2peak(3 times per week).

Each training session was supervised and training intensity was tailored to accommodate

each participant's functional ability.

An eight week aerobic training protocol was selected because it has been

previously found to provide a sufficient stimulus to alter cardiovascular fitness (40),

muscular endurance (41), and immune function (19) in MS subjects. The weekly training

protocol (30 minutes, 3 times/week at 60% VO2peak) has also been recommended by the

American College of Sports Medicine because of the known health benefits and has been

used by other investigators in studies with subjects with MS and other clinical

populations (40, 41). The cycle ergometer was selected as the training modality to









accommodate for varying levels of physical impairment and balance in individuals with

MS. All training occurred in a supervised exercise environment with staff trained in

cardiopulmonary resuscitation and emergency procedures.

Baseline Measures

Graded Exercise Test

The subjects were asked to visit the laboratory for testing at the same time of day

(8-1 lam) after abstaining from physical activity for 24 hours, and abstaining from

alcohol, caffeine or food for the previous 12 hours. A standard 12-lead electrocardiogram

was used to monitor the cardiovascular response of the subject continuously during

testing. Following five minutes of rest (sitting on the cycle ergometer), the subject was

asked to pedal at 25 W. Every two minutes, the resistance was increased by 10-25 W.

Depending on their risk stratification for exercise testing, subjects were asked to keep

pedaling until they were exhausted or until they reached their 85% of estimated maximal

heart rate. Expired gas concentrations were recorded continuously using a metabolic cart

(Parvomedics). Maximal and submaximal exercise tests have been used previously in

individuals with MS to measure cardiovascular capacity (19, 40, 76).

Single Bout of Aerobic Exercise

A single bout of aerobic exercise was assessed a minimum of 72 hours after the

completion of the maximal exercise test. Before the start of the exercise bout, 20 ml of

blood was acquired from the antecubital vein at rest in a seated position. Subsequently,

the subjects cycled at 60% of their measured VO2peak for 30 minutes. This endurance

protocol has also been used previously in individuals with MS (19, 76).









Muscle Fatigue

Voluntary muscle fatigue of both legs was determined by having the subject

perform 30 concentric knee extensions and flexions at 900 s-1 and 180s-1 as described in

Lambert et al. (132) (2001). Each subject was randomly assigned to a different speed and

leg at the beginning of the muscle fatigue exercises at each time point. The most affected

or non-dominant leg was tested followed by the least affected or dominant leg after 10

minutes of recovery (or viceversa). Subjects performed one bout of 30 flexions and

extensions at both speeds with 5 min of recovery between bouts. The fatigue index for

each bout was calculated (132). This fatigue protocol has been used in MS subjects in the

past (132). All testing was performed at least 24 hours after any other exercise bout.

Muscle Strength

Lower limb muscle strength was tested using an isokinetic dynamometer (Kin-Con,

Chattanooga, TN). The subjects were positioned specifically for each exercise with joints

stabilized. Subjects were seated and stabilized on the dynamometer with hips flexed to

850 and knees flexed to 900. The axis of the dynamometer was aligned with the axis of

the knee joint and the bottom of the force transducer pad positioned against the anterior

aspect of the leg, proximal to the lateral malleolus. The rate of isometric force

development was assessed at 900 and 1200 of knee flexion for both leg extension

(quadriceps) and leg flexion (hamstrings). Subjects performed a standardized warm-up

before conducting two trials at each knee angle. Peak torque was recorded for each

subject at 900 and 120 for both knee flexion and extension.









Quality of Life in Health and Disease

Perceived fatigue

The modified fatigue impact scale (MFIS) was used to assess perceived fatigue in

MS and control subjects. The MFIS has been used to assess fatigue in diseased

populations such as MS in past studies (33).

Perceived disability

Subjects completed self-assessment of disability (EDSS) (133) at all time points

throughout training.

Quality of life assessment

The Short Form (SF)-36 health survey was used to assess changes in self-

perception of health states. The SF-36 has been used widely and is a reliable and valid

measure to detect self-perception of health status (134) and has been previously used in

studies with MS subjects (7).

Functional Mobility Assessment

To assess function mobility, a six minute walking test, a 25 foot and 100 foot test,

and a timed up and go test were performed in all subjects.

Six minute walk

The six minute walking test was administered as a measure of exercise tolerance

and overall functional limitations (135). The test was conducted as described by

McGavin (136) and used standardized encouragement.

Walking test (25 ft.)

The subjects also completed a 25 foot walk test. The timed 25-foot walk is a

mobility and leg function performance test and has reported high inter-rater and test-

retest reliability (137). Subjects completed two trials and were asked to walk as rapidly but









as safely as possible during each trial. The amount of time to walk 25 feet was recorded

with a light sensitive timing device (IRD-T175 Broward Timing Systems, Salt Lake City,

Utah).

Walking test (100 ft.)

The subjects also completed a 100 feet walk test. Subjects completed two trials and

were asked to walk as rapidly but as safely as possible during each trial. The amount of

time to walk 100 feet was recorded with a light sensitive timing device (IRD-T175

Broward Timing Systems, Salt Lake City, Utah).

Timed up and go test

The timed up and go test was used to assess functional mobility skills and was

measured by timing subjects as they stand up from a chair, walk a distance of 3 meters,

turn, walk back to the chair, and sit down (138). The subjects were asked to perform one

practice trial followed by 2 timed test trials with 2 minutes rest between trials. The

average time of the test trials was used as the criterion score. The same chair was used for

all subjects throughout the intervention. The test has been reported to be reliable

(ICC=0.99) (138) and correlate with risk of falls and balance (139). Inter-rater reliability

for this test is 0.99.

Body Composition

Dual x-ray absorptiometry (DXA)(Lunar Prodigy Radiation Corp., Madison, WI)

was used to measure whole body and appendicular lean and soft tissue masses. The

procedure was performed by a licensed X-ray technician. Body mass index (kg/m2) and

waist to hip ratio (cm) was also calculated.









Post-Exercise Training Measures

After 4 and 8 weeks of aerobic training, the subjects underwent the same testing

procedures as before starting the 8 week aerobic training program (described in baseline

measurements).

Blood Collection and Processing

Blood samples (20 mL) were obtained by venipuncture before and following

exercise to acquire multiple blood samples. All blood samples were acquired from the

antecubital vein after resting for 5 minutes in a seated position. The subjects were asked to

visit the laboratory at the same time of day (8-1 lam) after abstaining from physical

activity, alcohol, caffeine or food for 12 hours. Blood samples were collected a minimum

of 48 hours after any MS-related drug administration to control for the possible impact of

the drug on cytokine regulation (140). To control for hormonal shifts in females, samples

were collected during the early to midfollicular phase. Whole blood samples were

collected in both EDTA tubes for plasma samples and in serum tubes for BDNF and IGF-

1 measurements (10 ml for plasma assessment and 10 ml for serum assessment). Plasma

samples were immediately centrifuged at 3000g for 15 minutes at 40C and then stored at -

800C for subsequent analyses.

Blood Collection Times

Venous blood (20 mL) was collected prior to a single bout of exercise, thirty

minutes post-exercise, 2 and 3 hours post-exercise in three different occasions (before

starting the aerobic exercise training program, 4 weeks and 8 weeks after engaging in the

aerobic exercise training program) as shown in Figure 2. This blood collection protocol

was selected because the time course of cytokine elevation or depression differs

depending on the cytokine of interest. For example, IFN-y and TNF-a may increase









immediately after exercise (30 minutes) and IL-6 displays a more delayed response (0-3

hours) (42, 75).










PRE 30 min 2 hr 3 hr
exercise POST POST POST
BLOOD COLLECTION TIMES BEFORE AND AFTER AN EXERCISE ACUTE BOUT


Figure 2. Blood collection times before and after 30 minutes of cycle ergometry at
60%VO2peak

Cytokine Assessment

Cytokines IL-6, TNF-a, and IFN-y were analyzed using a multiplex immunoassay

utilizing fluorescently labeled microsphere beads and laser based fluorescent detection

(Linco Research Inc., St. Charles, MO) for each acquired blood sample. The intra-assay

coefficient of variability for IL-6, TNF-a, and IFN-y were 4.9%, 6.1%, and 6.0%

respectively. The individual sensitivities (pg/mL) of IL-6, TNF-a, IFN-y were 1.7, 0.7,

and 1.7, respectively. There was no significant cross reactivity between other cytokine

antibodies in this panel. Plasma samples were analyzed in duplicate.

IGF-1 Assessment

Serum concentrations of IGF-1 were assessed by using an IGF-1 Quantikine

sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) for each blood

sample acquired in resting conditions. Serum samples were pretreated to release IGF-1

from binding proteins and diluted 100-fold with a pretreatment constituent prior to the









assay. To determine the optical density of each well, the samples were read within 30

minutes of adding 50 pl of stop solution. The samples were read using a microplate

reader (Molecular Devices, California) at 450 nm and 540 nm. The optical density

readings at 540 nm were subtracted from the readings at 450nm to correct for possible

optical imperfections in the plate. The minimum sensitivity of IGF-1 in this kit was less

than 0.026 ng/ml. No significant cross reactivity with other IGF-1 binding proteins has

been observed in this kit. The inter and intra- assay CV's are 8% and 3% respectively.

Serum samples were analyzed in duplicate.

BDNF Assessment

Serum concentrations of BDNF were analyzed by using a BDNF Quantikine

sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) for each acquired

blood sample. Serum samples were diluted 20-fold with a calibrator diluent prior to the

assay. To determine the optical density of each well, the samples were read within 30

minutes of adding 50 pl of stop solution. The samples were read using a microplate

reader (Molecular Devices, California) at 450 nm and 540 nm. The optical density

readings at 540 nm were subtracted from the readings at 450nm to correct for possible

optical imperfections in the plate. The minimum sensitivity of BDNF in this kit was less

than 20 pg/ml. No significant cross reactivity with other neurotrophic factors has been

observed in this kit. The inter and intra- assay CV's are 8% and 5% respectively. Serum

samples were analyzed in duplicate.

Plasma Volume Assessment

Plasma volume changes were assessed prior to and following a single bout of

exercise at weeks 0, 4 and 8 by assessing hematocrit and hemoglobin concentration from

whole blood samples using the method by Dill and Costill (141).









Statistical Analysis

All analysis was performed using SPSS 12.0. A multivariate analysis of variance

(MANOVA) with time (pre, mid and. post) as the within factor and group (MS vs.

control) entered as the between-subjects factor was used to assess the effect of the

exercise training program on all main related variables. An ANOVA with repeated

measures on each blood collection point will be used to assess changes in cytokine and

neurotrophin dynamics after a single bout of exercise. When necessary, Tukey's post hoc

analyses was implemented. A power analysis was conducted prior to the beginning of the

study and found that 9 subjects per group would produce a power of> .80. A value of

p<0.05 was considered significant. All values are expressed as mean standard

deviation.














CHAPTER 4
RESULTS

Subjects

Eleven MS subjects and eleven healthy controls (8 women and 3 men in both

groups) that were matched for age, weight, height, percent body fat and VO2peak (p>0.05)

completed the study. There was a significant increase in absolute VO2peak (1/min) after 8

weeks of aerobic exercise training in both groups (p<0.05). There were no significant

changes in weight, BMI, waist to hip ratio, relative VO2peak (ml/kg/min), and percent

body fat after the training program in both groups (p>0.05). Table 1 describes the

characteristics of the subjects before and after 8 weeks of aerobic exercise training in

more detail.

Table 1. Subject characteristics pre and post 8 weeks of aerobic exercise.
MS CONTROL
PRE POST %A PRE POST %A
Age (yrs) 40 10 40 10 0 40 10 40 10 0
Height (m) 1.68 0.1 1.68 0.1 0 1.68 0.1 1.68 0.1 0
Weight (kg) 72 14 73 15 1 78 14 78 14 0
% body fat 35.6+ 8 34.6 8 -3 37.6 + 9 37.4 9 -1
VO2peak (1/min) 2.2 + 0.4 2.5 0.4 10* 2.4 + 1 2.8 1 14**
BMI (kg/m2) 24 4 26 5 8 27 5 28 4 4
Data are expressed as Mean Standard Deviation. *indicates a significant difference
after 8 weeks of aerobic exercise (p<0.05); **indicates a significant difference after 8
weeks of aerobic exercise (p<0.001); %A = percent change.






42


Immune Factors

Chronic Exercise and IL-6

Plasma interleukin-6 at rest was similar between groups at weeks 0, 4 and 8

(p>0.05) (Figure 3). Following 8 weeks of aerobic exercise plasma IL-6 at rest tended to

decrease compared to week 0 in both groups (p=0.075). IL-6 was significantly correlated

to percent body fat (r = 0.506, p = 0.016), absolute VO2peak (r = 0.408, p = 0.046), 25 foot

walk (r = -0.434, p = 0.036), 100 foot walk (r = -0.427, p = 0.038), and timed up and go

test (r = -0.415, p= 0.044).


Resting IL-6
MS vs CONTROL
$
I I
35-
MS
30 CONTROL




5-
0-
5- Odd


PRE MID POST

Figure 3. IL-6 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST) in both
groups. $ Indicates tendency to decrease in both groups (p<0.10). Data are
expressed as Mean + Standard Deviation.

Single Bout of Exercise and IL-6

The response of plasma IL-6 after a single bout of exercise was similar between

subjects and remained unchanged following the training program (p>0.05) (Figure 4).

Both groups displayed similar significant increases in plasma IL-6 concentration

following 30 minutes of aerobic exercise at 60% of VO2peak. Specifically, plasma IL-6









concentration at baseline increased significantly 30 minutes post exercise (p=0.021) and

tended to increase 2 hours (p=0.09) post exercise in both groups. Although it was not

statistically different, the magnitude of increase of IL-6 at 30 minutes into recovery in

MS was 6.3%, while controls increased IL-6 by 20%.


Single Bout IL- 6

I*
25- '
-0- MS
-0- CONTROL
a 20-


C. 15-

10-

51
baseline 30 min 2 hr 3 hr
Exercise RECOVERY

Figure 4. IL-6 plasma concentration during a single bout of exercise in both groups.
(Single bouts at PRE, MID and POST are collapsed within groups because they
were not significantly different). *indicates significant differences between time
points within groups (p<0.05); # indicates trend between time points within
groups (p<0.10). Data are expressed as Mean + Standard Deviation.

Chronic Exercise and TNF-a

TNF-a plasma concentration at rest tended to be higher in MS compared to control

subjects throughout the study (p=0.08) (Figure 5). Specifically, Multiple Sclerosis

subjects increased TNF-a plasma concentrations at rest from week 0 to week 8, and from

week 4 to week 8 (p=0.04), while TNF-a plasma concentration in control subjects

remained unchanged following 8 weeks of aerobic exercise (p>0.05). TNF-a was






44


significantly correlated to percent body fat (r = -0.399, p = 0.033), IFN-y (r = 0.932, p =

0.001), and total mental SF-36 (r = 0.454, p = 0.017).


#
#I *
151 I
T MS
CONTROL
S1o-



I-


0
PRE MID POST
Figure 5. TNF-a plasma concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST).
*indicates p<0.05 between MS and Control subjects; # denotes p<0.05 within
MS subjects. Data are expressed as MeanStandard Deviation.

Single Bout of Exercise and TNF-a

The TNF-a plasma response to a single bout of exercise was similar between MS

and control subjects before the exercise intervention (p>0.05) and it remained unchanged

throughout the 8 weeks of aerobic exercise training in MS and control subjects (p>0.05)

(Figure 6). Both groups experienced similar significant decreases in TNF-a plasma

concentration following 30 minutes of aerobic exercise at 60% of VO2peak. Specifically,

TNF-a plasma concentration at baseline decreased significantly 2 hours (-28%, p=0.045)

and 3 hours (-48%, p=0.001) following 30 minutes of aerobic exercise in both groups. In

addition, TNF-a also decreased significantly from 30 min to 2hr (-25%, p=0.13), and 30

min to 3hr (-46%, p=0.001) following a single bout of exercise in both groups.










Single Bout TNF-a


#
'# --- --- MS
10.0- 4 -- CONTROL

7.5-

5.0-
L
2.5-

0.0
baseline 30 min 2 hr 3 hr

Exercie RECOVERY


Figure 6. TNF-a plasma concentration during a single bout of exercise at 0 (PRE), 4
(MID) and 8 (POST) weeks of aerobic exercise training in MS and control
subjects. (Single bouts at PRE, MID and POST are collapsed within groups
because they were not significantly different). # Indicates p<0.05 within
groups. Data are expressed as Mean + Standard Deviation.

Chronic Exercise and IFN-y

There was an interaction between the plasma concentration at rest of IFN-y

between groups and week of training (weeks 0, 4 and 8) (p=0.027) (Figure 7).

Specifically, IFN-y concentration at rest increased significantly in MS subjects from

week 0 to week 8, (p=0.008), and from week 4 to week 8 (p=0.01). IFN-y at rest

significantly increased in control subjects from week 0 to week 4 (p=0.015) followed by

a tendency for IFN-y at rest to decrease from week 4 to week 8 (p=0.07). In addition,

control subjects had similar IFN-y concentration at rest during week 0 and week 8

(p=0.3). IFN-y was significantly correlated with percent body fat (r = -0.377, p = 0.042),

TNF-a (r = 0.932, p = 0.001), and total mental SF-36 (r = 0.487, p = 0.011).









Single Bout of Exercise and IFN-y

The plasma IFN-y response to a single bout of exercise was similar between MS

and control subjects (p>0.05) and it remained unchanged throughout the training program

(p>0.05) (Figure 8). Both groups experienced similar significant decreases in IFN-y

plasma concentration following 30 minutes of aerobic exercise at 60% of VO2peak.

Specifically, IFN-y plasma concentration at baseline decreased significantly 2 hours (-

34%, p=0.017) and 3 hours (-28%, p=0.015) following 30 minutes of aerobic exercise in

both groups.


Resting IFN-y

I # #
#' I
70- I I $
SI MS
60 CONTROL
2 50-
E
0) 40-
0.
30-
u. 20-
10-
0
PRE MID POST
Figure 7. IFN-y plasma concentration at rest at 0 (PRE), 4 (MID) and 8 (POST) weeks of
aerobic exercise training. # indicates p<0.05 within subjects; $ denotes p<0.10
within subjects. Data are expressed as Mean + Standard Deviation.



Brain-Derived Neurotrophic Factor

Chronic Exercise and BDNF

There was a significant interaction of serum BDNF concentration between groups

(MS vs Control) and training effect (0, 4 and 8 weeks of training) (p=0.045)(Figure 9).









Resting serum BDNF was significantly lower in MS compared to control subjects at 0

weeks (p=0.026), and tended to be lower in MS compared to control subjects at 8 weeks

(p=0.066). Resting serum BDNF concentrations remained unchanged in MS subjects

between weeks 0 and 8, but BDNF concentrations were significantly elevated between

weeks 0 and 4 (p=0.04), and tended to decrease between weeks 4 and 8 (p=0.10). Resting

BDNF concentration in control subjects remained unchanged after 4 and 8 weeks of

aerobic exercise training (p>0.05). BDNF was significantly correlated to total physical

SF-36 (r = 0.465, p = 0.015), fatigue index at 1800/sec during the extension phase (r = -

0.444, p = 0.022).

Single Bout of Exercise and BDNF

The response of serum BDNF to a single bout of exercise between MS and controls was

significantly different (p=0.01) (Figures 10 and 11). Specifically, BDNF concentrations

were significantly lower in MS subjects compared to control subjects before exercise

(baseline), 2 and 3 hours after exercise (p<0.001) when all the points (PRE, MID, POST)

were collapsed together. Additional Post hoc analyses revealed MS subjects had

significantly lower concentration of BDNF only at baseline during week 0 (p=0.025).

Moreover, during week 4, MS subjects tended to have lower concentration of BDNF only

two hours after a single bout of exercise (p=0.09). During week 8, MS subjects tended to

have lower BDNF concentrations than control subjects at baseline (p=0.065) and BDNF

concentrations were significantly lower in MS compared to control subjects after a single

bout of exercise (p=0.04). The magnitude of clearance was the same for MS and control

subjects at weeks 0, 4, and 8 (p>0.05) (rate of clearance =73% in both groups).






48



Single Bout IFN-y
#
I # I
I I#
40- I # I MS
I I
-0- CONTROL
S30-

L.
20.

- 10-


01
baseline 30 min 2 hr 3 hr
p ccl RECOVERY I


Figure 8. IFN-y response to a single bout of exercise in MS and control subjects. (Single
bouts at PRE, MID and POST are collapsed within groups because they were
not significantly different). # indicates significant difference within groups
(p<0.05). Data are expressed as Mean + Standard Deviation.



Multiple sclerosis subjects had significant decreases ofBDNF concentrations

between baseline measurements and 2 hours post-exercise, and between baseline

measurements and 3 hours post-exercise at weeks 0, 4 and 8 (p<0.001) as illustrated in

figure 10. The response of BDNF concentrations to single bout of exercise within groups

(during weeks 0, 4 and 8) was significantly different only between week 4 and week 8

(p=0.044) in MS subjects. During week 4, the rate of BDNF clearance was significantly

faster (86%) than during week 8 (59%) (p=0.044). However, there were no differences

between the response of BDNF concentrations to a single bout of aerobic exercise

between week 0 and at week 8 (p=0.3) or between week 0 and week 4 (p=0.2) in MS

subj ects.









Resting BDNF Concentration
MS vs CONTROL


I $





TO Tm


PRE MID


COMS
- CONTROL


POST


Figure 9. BDNF concentration at 0 (PRE), 4 (MID) and 8 (POST) weeks of aerobic
exercise training. *Indicates significant differences between points p<0.05 #
Indicates p<0.10 between MS and Control subjects; $ Indicates p<0.10
between week 4 and 8 in MS subjects only. Data are expressed as Mean +
Standard Deviation.

Control subjects had significant decreases of BDNF concentrations between

baseline measurements and 2 hours post-exercise, and between baseline measurements

and 3 hours post-exercise at weeks 0, 4 and 8 (p<0.001) as illustrated figure 11. The

circulating BDNF response to a single bout of exercise within control subjects remained

unchanged between weeks 0, 4 and 8 (p>0.05) (rate of clearance =73%).


35000
30000
E 25000
C 20000
- 15000
z
m 10000
5000
0






50


Single Bout BDNF MS Group

I *


-E- PRE
-A-MID
-- POST


baseline 30 min 2 hr 3 hr
Exercise RECOVERY


Figure 10. BDNF acute response to exercise in MS subjects at weeks 0 (PRE), 4 (MID)
and 8 (POST).*indicates significant difference within time point (p<0.001).
indicates significant differences across time (p<0.05). Data expressed as Mean
+ Standard Deviation


Single Bout BDNF CONTROL Group



-E- PRE
30000- T MID

S-0- POST


baseline 30 min 2 hr 3 hr
xe RECOVERY

Figure 11. BDNF acute response to exercise in control subjects at weeks 0 (PRE), 4
(MID) and 8 (POST).*indicates significant difference within time point
(p<0.05). Data are expressed as Mean Standard









Insulin-Like Growth Factor-1

The concentration of circulating IGF-1 was similar between groups and remained

unchanged after 8 weeks of aerobic exercise training (p>0.05) (Figure 12). Resting levels

of IGF-1 in MS subjects were 191 + 119 ng/ml before the study started, 206 + 154 ng/ml

4 weeks after the initiation of training, and 161 + 199 ng/ml after the exercise

intervention. Resting levels of IGF-1 in the control subjects were 226 144 ng/ml before

the study started, 200 110 ng/ml 4 weeks after the initiation of training, and 200 117

ng/ml after 8 weeks of aerobic exercise training. IGF-1 was significantly correlated to

total physical SF-36 (r = 0.461, p = 0.014), fatigue index at 1800/sec during the extension

phase (r = -0.412, p = 0.019).




Resting IGF-1 Concentration
MS vs CONTROL

450-
400- 1 MS
350- CONTROL
,--
E 300-
S250-
200-
S150-
100-
50-
0
PRE MID POST

Figure 12. IGF-1 concentration at rest at week 0 (PRE), 4 (MID) and 8 (POST). There
were no significant effects of aerobic training on resting concentration of IGF-
1 (p>0.05). Data are expressed as MeanStandard Deviation









Plasma Volume

Plasma volume decreases after a single bout of exercise were similar between MS

and controls subjects (4% and 5% respectively, p>0.05). In addition, decreases in plasma

volume after a single bout of exercise remained unchanged after 8 weeks of aerobic

exercise training in both groups (p=0.05).

Muscle Function

Knee Extension Endurance of Most Affected/ Non-Dominant Leg at 180os1

Muscle fatigue (fatigue index) was significantly higher in MS compared to control

subjects (p=0.006) and remained unchanged after 8 weeks of aerobic exercise in both

groups (p>0.05) (Table 2). Absolute and relative (relative to body weight and fat free

mass) mean extensor torque during knee extension endurance was similar in both groups

and remained unchanged after 8 weeks of aerobic exercise training (p>0.05). Total

extensor work was significantly lower in the MS compared to control subjects (p=0.004).

However, after 8 weeks of aerobic exercise total extensor work remained unchanged in

both groups (p=0.14). Knee extension power was significantly lower in the MS compared

to control subjects (p=0.001) and remained unchanged after 8 weeks of aerobic exercise

in both groups (p=0.12). Table 2 provides fatigue measurements in more detail before

and after 8 weeks of aerobic exercise training.

Knee Flexion Endurance of Most Affected/ Non-Dominant Leg at 180os1

Muscular fatigue (fatigue index) was similar between groups remained unchanged

after 8 weeks of aerobic exercise (p>0.05). Absolute and relative (relative to body weight

and fat free mass) mean flexor torque during knee flexion endurance was similar between

groups and remained unchanged after 8 weeks of aerobic exercise training (p>0.05).

Total flexor work was significantly lower in the MS compared to control subjects









(p=0.008). However, after 8 weeks of aerobic exercise total flexor work remained

unchanged in both groups (p>0.05). Knee flexion power was significantly lower in the

MS compared to control subjects (p=0.006) but did not change after 8 weeks of aerobic

exercise in either groups (p>0.05) (Table 2).

Knee Extension Endurance of Most Affected/ Non-Dominant Leg at 90o0s-

Muscular fatigue (fatigue index) was significantly lower in MS compared to control

subjects (p=0.001). Controls tended to decrease the rate of fatigue after 8 weeks of

aerobic exercise (p=0.09), while MS subjects did not (p>0.05). Absolute mean extensor

peak torque during knee extension endurance was significantly lower in MS compared to

control subjects (p=0.007), as well as mean peak extensor torque relative to fat free mass

(p=0.023). Mean extensor peak torque relative to body weight tended to be lower in MS

compared to control subjects during knee extension endurance (p=0.06). Total extensor

work was significantly lower in MS compared to control subjects (p=0.002) and

remained unchanged after 8 weeks of aerobic exercise in both groups (p=0.11). Knee

extension power was significantly lower in MS compared to control subjects (p=0.003)

and remained unchanged after 8 weeks of aerobic exercise in either groups (p=0.15)

(Table 3).

Knee Flexion Endurance of Most Affected/ Non-Dominant Leg at 90o0s-

Muscular fatigue (fatigue index) was similar between MS subjects and controls

(p=0.17) and remained unchanged after 8 weeks of aerobic exercise in both groups

(p=0.12). Absolute mean flexor peak torque during knee flexion endurance was

significantly lower MS compared to control subjects (p=0.006), as well as and relative to

body weight (p=0.023) and relative to fat free mass (p=0.019). Total flexor work was

significantly lower in MS compared to control subjects (p=0.013) and remained









unchanged after 8 weeks of aerobic exercise in either groups (p=0.21). Knee flexion

power was significantly lower in MS compared to control subjects (p=0.001) and

remained unchanged after 8 weeks of aerobic exercise in either groups (p>0.05) (Table

3).

Table 2. Fatigue measures pre and post 8 weeks of aerobic exercise during at 1800sec-1

Speed 180sec1 MS CONTROL
O___p______PRE POST %A PRE POST %A
Ext Fatigue Index (%) 79 23 81 21 3# 95 21 99 15 4#
Ext peak Torque (Nm) 190 34 199 28 5 209 57 217 68 4
Ext peak Torque (Nm/Kg) 2.7 0.7 2.8 + 0.7 4 2.7 0.7 2.8 0.7 4
Ext peak Torque (Nm/Kg FFM) 4.4 0.9 4.5 0.9 2 4.4 0.6 4.6 + 0.8 4
Ext Total Work (J) 673 273 700 231 4# 945 590 1235 621 26#
Ext Power (W) 42 24 46 21 15# 69 38 90 47 23#
Flex Fatigue Index (%) 86 + 23 79 20 -17 87 39 90 45 3
Flex peak Torque (Nm) 205 55 197 53 -4 217 87 213 103 -2
Flex peak Torque (Nm/Kg) 3.0 + 1.1 2.8 + 1.1 -6 2.9 + 1.0 2.7 + 1.2 -7
Flex peak Torque (Nm/Kg FFM) 4.7 + 1.3 4.5 + 1.5 -4 4.5 + 1.1 4.4 + 1.5 -2
Flex Total Work (J) 765 367 810 383 6# 1180 677 1250 672 6#
Flex Power (W) 54 33 59 34 9# 90 50 93 54 3#
Data are expressed as Mean Standard Deviation; "indicates a significant difference
between groups (p<0.001). N= newtons; m=meters; Kg= kilograms BW= body weight;
FFM= fat free mass; J=joules; W=watts; %A= percent change.

Table 3. Fatigue measures pre and post 8 weeks of aerobic exercise at 900sec-1

Speed 900sec-1 MS CONTROL
PRE POST %A PRE POST %A
Ext Fatigue Index (%) 92 24 91 + 15 -1# 61 23 80 19 24*#
Ext peak Torque (Nm) 153 33 154 40 7 184 66 215 66 14
Ext peak Torque (Nm/Kg BW) 2.1 0.6 2.1 0.7 0 2.3 0.8 2.7 0.8 15
Ext peak Torque (Nm/Kg FFM) 3.5 + 1.0 3.4 + 1.1 -3 4.3 2.3 5 2.4 14
Ext Total Work (J) 929 366 1007 410 8 1338 702 1712 672 22
Ext Power (W) 35 18 41 23 15 59 37 73 35 19
Flex Fatigue Index (%) 105 24 86 19 -18 94 11 95 14 1
Flex peak Torque (Nm) 142 34 144 29 196 81 187 66 -6
Flex peak Torque (Nm/Kg BW) 1.9 0.4 1.9 0.4 0 2.5 1.0 2.4 0.9 -4
Flex peak Torque (Nm/Kg FFM) 3.2 + 0.9 3.2 0.9 0 4.5 2.4 4.3 2.2 -4
Flex Total Work (J) 866 343 938 304 8 1191 512 1394 751 15
Flex Power (W) 35 17 41 + 16 15 65 25 63 32 -3
Data are expressed as Mean Standard Deviation; indicates significant differences
before and after 8 weeks of aerobic exercise; #indicates a significant difference between
groups (p<0.05). Trend of significance between groups (p<0.10).









Isometric Muscle Torque During Extension at 900 of Knee Flexion

Absolute maximal extensor torque (p=0.017) and maximal extensor torque relative

to fat free mass (p=0.03) were significantly lower in MS compared to control subjects,

while maximal extensor torque relative to body weight tended to be lower in MS

compared to control subjects (p=0.07) (Table 4). However, absolute and relative maximal

torque remained unchanged after 8 weeks of aerobic exercise in both groups (p>0.05).

Table 4 provides detailed information on muscle strength of both groups.

Isometric Muscle Torque During Flexion at 900 of Knee Flexion

Maximal absolute and relative flexor torque was similar between groups and

remained unchanged after 8 weeks of aerobic exercise (p>0.05) (Table 4).

Isometric Muscle Torque During Extension at 1200 of Knee Flexion

Absolute maximal extensor torque was significantly lower in MS compared to

control subjects (p=0.04). However, relative maximal extensor torque (body weight and

fat free mass) was similar between groups (p>0.05). Eight weeks of aerobic exercise did

not change maximal absolute and relative extensor torque in either groups (p>0.05)

(Table 4).

Isometric Muscle Torque During Flexion at 1200 of Knee Flexion

Absolute maximal flexor torque (p=0.014) and maximal flexor torque relative to fat

free mass (p=0.03) were significantly lower in MS compared to control subjects, while

maximal flexor torque relative to body weight tended to be lower in MS compared to

control subjects (p=0.09). However, absolute and relative maximal flexor torque

remained unchanged after 8 weeks of aerobic exercise in both groups (p>0.05) (Table 4).









Isokinetic Muscle Torque at 90os-l

During the extension phase, absolute peak extensor torque was significantly lower

in MS compared to controls (153 33 Nm vs. 184 66 Nm for MS and controls

respectively) (p=0.03). Absolute peak torque remained unchanged in both groups after 8

weeks of aerobic exercise (p>0.05). Peak extensor torque relative to body weight tended

to be lower in MS compared to control subjects (2.1 0.6 Nm/Kg vs. 2.3 0.8 Nm/Kg

for MS and controls respectively, p<0.10), and remained the same after 8 weeks of

aerobic exercise (p>0.05). Peak extensor torque relative to fat free mass was significantly

lower in MS compared to control subjects (3.5 + 1.0 Nm/Kg FFM vs. 4.3 2.3 Nm/Kg

FFM for MS and controls respectively, p=0.014), and remained the same after 8 weeks of

aerobic exercise (p>0.05).

During the flexion phase MS subjects had significant lower absolute peak flexor

torque than control subjects (142 34 Nm vs. 196 + 81 Nm, p=0.048) and remained

unchanged after 8 weeks of aerobic exercise training in both groups (p>0.05). Peak flexor

torque relative to body weight was significantly lower in MS compared to control

subjects (1.9 0.4 Nm/Kg vs. 2.5 1.0 Nm/Kg for MS and controls respectively,

p=0.02), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak flexor

torque relative to fat free mass was significantly lower in MS compared to control

subjects (3.2 0.9 Nm/Kg FFM vs. 4.5 2.4 Nm/Kg FFM for MS and controls

respectively, p=0.02), and remained unchanged after 8 weeks of aerobic exercise

(p>0.05).









Table 4. Isometric strength measures pre and post 8 weeks of aerobic exercise at 900 and
1200 of knee flexion
Isometric Strength MS CONTROL
PRE POST %A PRE POST %A
Ext Torque at 900 (Nm) 96 35 93 24 -3" 123+ 51 125 56 2#
Ext Torque at 900 (Nm/Kg BW) 1.4 + 0.5 1.3 + 0.4 -7# 1.6 + 0.6 1.6 + 0.7 0#
Ext Torque at 900 (Nm/Kg FFM) 2.2 0.8 2.1 0.6 -5" 2.5 0.6 2.6 0.7 4#
Ext Torque at 120 (Nm) 113 + 30 115 + 19 2# 142+ 60 136 57 -4"
Ext Torque at 1200 (Nm/Kg BW) 1.6 + 0.4 1.6 + 0.3 0 1.8 + 0.6 1.7 + 0.5 -5
Ext Torque at 1200 (Nm/Kg FFM) 2.6 0.7 2.6 0.5 0 2.8 + 0.7 2.7 + 0.7 -4
Flex Torque at 90 (Nm) 49 22 43 + 18 -12 52 28 66 39 21
Flex Torque at 900 (Nm/Kg BW) 0.7 0.3 0.6 0.3 -14 0.7 + 0.3 0.9 + 0.7 22
Flex Torque at 90 (Nm/Kg FFM) 1.1 + 0.5 1.0 + 0.4 -9 1.1 + 0.4 1.5 + 1.1 27
Flex Torque at 1200 (Nm) 56 26 53 18 -5# 72 25 71 28 -1#
Flex Torque at 1200 (Nm/Kg BW) 0.8 + 0.4 0.7 0.3 -12' 0.9 + 0.3 0.9 + 0.3 0s
Flex Torque at 1200 (Nm/Kg FFM) 1.3 + 0.6 1.2 + 0.4 -8# 1.5 + 0.3 1.5 + 0.5 0#
Data are expressed as Mean Standard Deviation; #indicates a significant difference
between groups (p<0.05). $ trend of significance between groups (p<0.10). N= newtons;
m= meters; Kg= kilograms BW= body weight; FFM= fat free mass; J= joules; W= watts;
%A= percent change

Isokinetic Muscle Torque at 1800s1

During the extension phase, absolute peak extensor torque was similar between

groups (190 34 Nm vs. 209 57 Nm for MS and control subjects respectively), and

remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak extensor torque

relative to body weight was the same for both groups (2.7 0.7 Nm/Kg vs. 2.7 0.7

Nm/Kg for MS and controls respectively), and remained unchanged after 8 weeks of

aerobic exercise (p>0.05). Peak extensor torque relative to fat free mass was similar

between groups (4.4 0.9 Nm/Kg FFM vs. 4.4 0.6 Nm/Kg FFM for MS and controls

respectively), and remained unchanged after 8 weeks of aerobic exercise (p>0.05).

During the flexion phase, absolute peak flexor torque was similar between groups

(205 55 Nm vs. 217 87 Nm for MS and controls), and remained unchanged after 8

weeks of aerobic exercise (p>0.05). Peak flexor torque relative to body weight was

similar between groups (3.0 1.1 Nm/Kg vs. 2.9 1.0 Nm/Kg for MS and controls









respectively), and remained unchanged after 8 weeks of aerobic exercise (p>0.05). Peak

flexor torque relative to fat free mass was similar between groups (4.7 + 1.3 Nm/Kg

FFM vs. 4.5 1.1 Nm/Kg FFM for MS and controls respectively), and remained

unchanged after 8 weeks of aerobic exercise (p>0.05).

Functional Mobility

Walking Tests

Multiple sclerosis subjects walked significantly slower than control subjects during

the 25 feet and 100 feet walking tests (p=0.001) (Table 5). Moreover, after 8 weeks of

aerobic exercise training walking performance remained unchanged in both groups

(p>0.05).

Timed Up and Go

During the timed up and go test MS subjects completed the task significantly

slower than control subjects (p=0.001). Moreover, time to complete the task remained

unchanged after the training intervention in both groups (p>0.05).

Six Minute Walk

The number of meters walked during the six minute walk was significantly shorter

in the MS compared to control subjects (p=0.001) and remained unchanged after 8 weeks

of aerobic exercise in both groups (p>0.05). Table 5 shows functional mobility

assessments before and after 8 week of aerobic exercise training.









Table 5. Functional measures pre and post 8 weeks of aerobic exercise
Functional Mobility MS _CONTROL
PRE POST %A PRE POST %A
25ft walk (sec) 5.3 3.2 5.3 3.2 0# 3.5 0.5 3.3 0.5 -6#
100ft walk (sec) 20.2 11 19.6 10 -2" 12.7 2.4 12.3 2.2 -3
6 minute walk (m) 541 215 566 240 4" 709 115 717 112 1#
Timed up test (sec) 7.4 5 6.4 5 -14" 4.4 0.8 3.9 0.8 -11


Data are expressed as Mean Standard Deviation; #indicates a significant difference
between groups (p<0.05). sec= seconds; m= meters; %A= percent change.

Quality of Life in Health and Disease

Perceived Disability

Perceived disability measured by self-assessed EDSS was significantly higher in

MS compared to control subjects (p=0.0001) (Table 6). The MS subjects decreased their

perceived disability by 24% (p=0.04) after 8 weeks of aerobic exercise training. Control

subjects had a score of 0 at the beginning of the study and it remained unchanged after 8

weeks of aerobic exercise training (p>0.05).

EDSS was significantly correlated to absolute and relative VO2peak (r = -0.6, p =

0.001), total MFIS (r = 0.64, p = 0.001), physical SF-36 (r = -0.564, p = 0.003), 25 foot

walk (r = 0.697, p = 0.001), 100 foot walk (r = 0.732, p = 0.001), timed up and go (r =

0.773, p = 0.001), 6 minute walk (r = -0.807, p = 0.001), and absolute and relative

isometric torque (r = -0.512, p = 0.005).

Modified Fatigue Impact Scale

The modified fatigue impact scale (MFIS) was significantly higher in MS

compared to control subjects in all subscales (p=0.001) (Table 6). The total score of the

MFIS was significantly higher in MS compared to control subjects by 71% (31.7 5 vs.

9.2 5 respectively, p=0.001). The physical fatigue subscale of the MFIS was

significantly higher in MS compared to control subjects by 77% (15 2.4 vs. 3.6 2.4









respectively, p=0.001). The cognitive fatigue subscale was significantly higher in MS

compared to control subjects by 65% (14.4 2.6 vs. 5.1 2.6 respectively, p=0.003). The

psychosocial fatigue scale of the MFIS was significantly higher in MS compared to

control subjects by 75% (2.4 0.4 vs. 0.6 0.4 respectively, p=0.001). However, after 8

weeks of aerobic exercise training all fatigue subscales remained unchanged in both

groups (p>0.05).

Total MFIS was significantly correlated to absolute and relative VO2peak(r = -0.51,

p = 0.008), physical SF-36 (r = -0.643, p = 0.001), 25 foot walk ( r = 0.552, p = 0.004),

100 foot walk (r = 0.580, p = 0.002), timed up and go (r = 0.622, p = 0.001), 6 minute

walk (r = -0.553, p=0.004), fatigue index at 900/sec during extension phase (r = 0.468, p

= 0.019), absolute and relative peak torque at 900/sec during extension phase (r = -0.64, p

= 0.002).

Table 6. Self-Assessed Measures pre and post 8 weeks of aerobic exercise.
Quality of Life MS CONTROL
PRE POST %A PRE POST %A
EDSS 3.4 2 2.6 2 -24* 0 0 00 0
MFIS (total) 32 22 26 19 -19 ~ 9 9 8 12 -1
SF-36 (mental) 43 13 47 10 9# 57 + 8 52 11 -9#
SF-36 (physical) 42 15 45+12 7# 57 8 55 6 -4
Data are expressed as Mean Standard Deviation. *indicates a significant difference
after 8 weeks of aerobic exercise (p<0.05); #indicates a significant difference between
groups (p<0.001). %A= percent change.

Short Form-36 Quality of Life Questionnaire

The SF-36 quality of life questionnaire was significantly lower in MS subjects

compared to control subjects (p=0.002) (Table 6). Specifically, the total mental subscale

of the SF-36 was significantly lower in MS compared to control subjects (43 13 vs. 57

8 respectively, p=0.003) and remained unchanged after 8 weeks of aerobic exercise

(p>0.05). The total physical subscale of the SF-36 was significantly lower in MS









compared to control subjects (42 15 vs. 56 4 respectively, p<0.001) and remained

unchanged after 8 weeks of aerobic exercise (p>0.05). The physical functioning subscale

of the SF-36 was significantly lower in MS compared to control subjects (66 34 vs. 96

7 respectively, p<0.001) and remained unchanged after 8 weeks of aerobic exercise

(p>0.05). The role-physical subscale of the SF-36 was significantly lower in MS

compared to control subjects (66 41 vs. 100 + 0 respectively, p<0.001) and remained

unchanged after 8 weeks of aerobic exercise (p>0.05). The body pain subscale of the SF-

36 tended to be lower in MS compared to control subjects (70 28 vs. 87 17

respectively, p=0.06) and remained unchanged after 8 weeks of aerobic exercise

(p>0.05). The general health subscale of the SF-36 was significantly lower in MS

compared to control subjects (55 26 vs. 84 12 respectively, p<0.001) and remained

unchanged after 8 weeks of aerobic exercise (p>0.05). The vitality subscale of the SF-36

was significantly lower in MS compared to control subjects (53 29 vs. 63 13

respectively, p=0.04) and remained unchanged after 8 weeks of aerobic exercise

(p>0.05). The social functioning subscale of the SF-36 was significantly lower in MS

compared to control subjects (78 24 vs. 94 10 respectively, p=0.002) and remained

unchanged after 8 weeks of aerobic exercise (p>0.05). The role-emotional subscale of the

SF-36 was significantly lower in MS compared to control subjects (73 39 vs. 94 + 20

respectively, p=0.024) and remained unchanged after 8 weeks of aerobic exercise

(p>0.05). The mental health subscale of the SF-36 was similar between groups (75 + 13

vs. 771 respectively, p>0.05) and remained unchanged after 8 weeks of aerobic exercise

(p>O.05).






62


Total physical SF-36 was significantly correlated to EDSS (r = -0.564, p = 0.003),

relative VO2peak (r = 0.584, p = 0.002), total MFIS (r = -0.643, p = 0.001), 6 minute walk

(r = 0.414, p = 0.028), BDNF (r = 0.465, p = 0.015) and IGF-1 (r = 0.465, p = 0.015).

Total mental SF-36 was significantly correlated to absolute VO2peak (r = 0.398, p=0.033),

TNF-a (r = 0.454, p= 0.017), and IFN-y (r = 0.487, p = 0.011).














CHAPTER 5
DISCUSSION

Regular physical activity represents and intrinsic means to maintain health and is

recommended to reduce the incidence of many diseases. Exercise is also recognized for

its potential to protect the central nervous system (CNS) from injury as well as promote

restoration of function following insult (142). Thus, regular activity may be an effective

countermeasure to minimize deleterious changes associated with neurodegenerative

diseases such as multiple sclerosis (MS), where immune dysregulation and compromised

neuroprotection are associated with neurodegeneration of the CNS and disease

progression. Early information about the influence of exercise on MS disease activity

comes from research using the animal model of MS. Le Page et al. (81) (1994)

investigated the effect of exercise on the inflammatory phase of experimental

autoimmune encephalomyelitis (EAE). Exercise training reduced the duration and

severity of EAE in rats (81). However, the mechanisms associated with reduced disease

severity remain unknown, but likely involve modulation of the immune system. Regular

aerobic exercise has been shown to modulate immune factors such as pro and anti-

inflammatory cytokines (19, 42, 43), and to increase neuroprotection through elevations

of IGF-1 and BDNF secretion in healthy populations (22, 27, 28, 44). However; to date,

the influence of exercise on factors known to influence disease activity in people with

MS remains unexplored. Therefore, the purpose of this study was to investigate whether

aerobic exercise modulates immune markers, neurotrophins and fatigue in individuals

with MS. We hypothesized that aerobic exercise would modulate cytokines IL-6, TNF-a









and IFN-y, neurotrophins BDNF and IGF-1, and would reduce muscular and perceived

fatigue in MS and matched control subjects.

Resting Cytokine Concentration after 8 Weeks of Exercise Training

Clinical studies investigating the impact of chronic exercise on cytokine

modulation in individuals with MS are limited. Our study is one of the first to provide

evidence that exercise training may influence pro and anti-inflammatory cytokines in

individuals with MS. Resting concentration of plasma IL-6 tended to decrease, which is

consistent with our hypothesis. Schulz et al. (76) (2004) investigated the impact of

chronic aerobic exercise on IL-6, and in contrast to our findings, found that a similar

training regimen did not alter resting concentration of IL-6. Further, Castaneda et al. (16)

(2004) found that interleukin-6 (IL-6) was reduced in patients with kidney inflammatory

disease subjects undergoing 12 weeks of resistance training compared with controls.

Since regulatory changes of systemic IL-6 may be pivotal for the development of

demyelinating lesions in the CNS (143), decreases in this cytokine may have beneficial

outcomes in persons with MS. Previous observations suggest that abnormally high

concentrations of IL-6 in the periphery may result in excess inflammation that may

exacerbate autoimmune disease activity in MS (6). Also, elevated IL-6 may also disrupt

the clearance of microbial pathogens (6) and participate in T cell activation, accelerating

the MS disease process (5, 6). These results provide preliminary evidence that exercise

may modulate immune factors in the periphery of persons with MS.

Resting concentrations of TNF-a and IFN-y increased in MS subjects following

training, which is contrary to our hypothesis. Our results suggest that aerobic training

may also increase concentration of pro-inflammatory cytokines in the periphery of

individuals with MS. However, the consequence of elevated circulatory TNF-a and IFN-y









remain unknown. Previous research suggests that elevated TNF-a concentration in blood

may have beneficial (144) or detrimental effects (143) in people with MS. For example,

while increased TNF-a concentrations in blood and CSF may correlate with the degree of

blood brain barrier dysfunction (143), it may also be associated with favorable decreases

in disease relapses while on interferon-0 treatment (144). In our study, perceived

disability decreased with training suggesting that changes in pro-inflammatory cytokines

may not be linked to negative disease outcomes. In fact, inflammation may be a

prerequisite to activate repair mechanisms such as remyelination as evidenced by studies

showing that inflammation upregulates neurotrophic factors (i.e., BDNF) involved in

neuroprotection (145, 146).

The role of TNF-a in MS is complicated by the observation that TNF-a has dual

roles (4, 143, 145-147) that may be unique to autoimmune diseases such as MS.

Although TNF-a has been linked to inflammatory demyelination in MS (11, 148, 149),

recent reports show strong evidence that TNF-a may also be neuroprotective through

enhancement of oligodedryocyte proliferation and stimulation of remyelination (143,

145, 146). If fact, intravenous anti-TNF-a therapy does not work in MS patients, and may

even worsen MS symptoms (145, 150). It is therefore difficult to resolve the

contradictory roles of TNF-a on disease activity. One explanation may be the existence

of two different signaling pathways mediated by two different TNF-a receptors (p55 and

p75) (143, 145). It is possible that exercise can induce activation of the "good"

inflammatory TNF-a p75 receptor pathway that promotes cell growth and proliferation

(145). Possible mechanisms of action include neuroprotection of the TNF-a p75 receptor

through the induction of superoxide dismutase (151, 152), protecting neurons from









reactive oxygen species, and calbindin stabilization of calcium homeostasis in the CNS

(153). Our study provides preliminary data suggesting that exercise may modulate

cytokines associated with disease activity as well as evidence suggesting that increases in

circulating TNF-a concentration in MS patients may not be associated with negative

outcomes as evidencing by significantly improved disability levels in our MS subjects

following the exercise program.

Similar to TNF-a, plasma concentration of IFN-y also increased following 8 weeks

of aerobic exercise in MS subjects. To date, IFN-y is thought to be present during

relapses and it is considered detrimental to the CNS of individuals with MS (55, 86, 154).

However, the role of IFN-y in the periphery remains unknown. In our study, IFN-y and

TNF-a were highly correlated and seem to follow similar dynamics throughout the study

(r = 0.932, p = 0.001). Work by Moldovan et al. (55) (2003) showed that T cell secreting

IFN-y ex-vivo correlated with functional impairments in MS patients. In contrast, Kraus

et al. (86) (2002) found that circulating pro-inflammatory cytokines did not correlate with

disease activity and severity assessed by lesion load in the brain. As mentioned earlier, it

remains to be elucidated whether exercise plays a positive or negative role in the

pathophysiology of the disease. However, since disability status improved in our subjects

indicating that the observed changes in TNF-a and IFN-y may not be linked to any

changes in perceived disability. Clearly, further investigations are needed to clarify the

roles of exercise-induced pro- inflammatory cytokines in individuals with MS.

Chronic Exercise May Modulate Serum BDNF at rest

Inflammation precedes BDNF production, which is a key factor involved in

neuroprotection (21, 147, 155, 156). In addition, exercise has been shown to increase

neurotrophin production in both the brain and spinal cord (21, 22, 27, 155, 156). In our









study, before the initiation of the exercise training program, we observed lower

concentration of serum BDNF in MS compared to control subjects at rest. These results

are consistent with Sarchielli et al. (23) (2002), where MS subjects also had lower levels

of BDNF at rest. However, in Sarchielli et al. (23) (2002), BDNF was stimulated from

peripheral blood mononuclear cells and the subjects had secondary progressive MS. In

contrast to our results, Gold et al. (77) (2003) found that BDNF concentration at rest was

similar between MS and control subjects. Our results provide further evidence that serum

BDNF may be lower in some individuals with MS compared to matched controls.

Exercise has been shown to increase neurotrophin production in the brain and

spinal cord (21, 22, 27, 155, 156). Our results also show that serum BDNF at rest was

elevated following 4 weeks of exercise training in MS subjects, which supports our

cytokine findings where exercise-induced elevation of pro-inflammatory cytokines may

lead to BDNF upregulation in MS subjects (147). In our study, resting concentration of

BDNF followed a biphasic response to chronic exercise with elevations at 4 weeks while

returning to baseline levels at week 8. This biphasic response of resting BDNF to chronic

exercise has also been observed in healthy animal models (157). During the first weeks of

training, exercise may produce novel effects that can enhance neurogenesis (157).

However, once the individual is accustomed to chronic exercise, "novelty" and molecular

learning effects may diminish and homeostatic mechanisms take over, bringing resting

BDNF back to baseline levels (157). Although accumulating evidence suggests that

exercise provides brain health benefits by increasing neuroprotection (21, 22, 25, 27, 32,

106-108), the mechanisms through which exercise benefits the brain are poorly

understood. The ability of BDNF to cross the blood brain barrier has been demonstrated









(102), suggesting that serum BDNF levels may reflect BDNF levels in the brain (158).

However, the source of origin and the mode of transport of exercise-induced BDNF

actions on the brain remain unknown. Potential sources of BDNF include: 1) Muscle

BDNF anterogradely transported to the CNS, 2) Schwann cell synthesis of BDNF, 3)

injured fiber attracting BDNF to the area, 4) blood borne circulating BDNF (102, 104,

107, 111, 159, 160).

Recent data suggest that BDNF is also elevated in exercised skeletal muscle (109)

and can be transported into the spinal cord (110). BDNF also has effects on skeletal

muscle tissue by inducing the potentiation of spontaneous twitching in myocytes to

enhance muscle contraction (111). In the periphery, exercise can upregulate the

expression of BDNF and maintain skeletal muscle health (25). For example, after

immobilization stress, Adlard and Cotman (25) (2004) found that exercise can override

the negative effects of muscle atrophy in rats through elevations of BDNF secretion after

3 weeks of running. In BDNF deficient rats, 2 months of wheel running increased BDNF

hippocampus concentration (107). Sarchielli et al. (23) (2002) also suggests that BDNF

concentration in the CSF is influenced by its concentration in peripheral blood. BDNF

can be produced in the peripheral circulation and transported by a high capacity, saturable

system that suggests that increased peripheral production of this neurotrophin may

increase its entry into the CNS (23, 102). In addition, a positive correlation between

cortical and serum BDNF concentration has been observed in rats (104). Furthermore, the

role of circulating BDNF on neuroprotection in humans remains to be elucidated.

Circulating BDNF may contribute to increases in neural repair and plasticity mechanisms

in the brain and spinal cord (102, 160). Additionally, BDNF may cross the BBB after a









single bout of exercise in response to a physical stress, which is discussed in the next

section. If circulating BDNF crossed the BBB or was transported to the CNS through

other means (i.e., skeletal muscle), it may positively influence oligodendrocyte survival

and proliferation, and therefore stimulate remyelination (23, 27). However, our data only

captured a snapshot of circulating BDNF at rest, and therefore all these assumptions are

speculative.

BDNF is, among others, regulated by circulating IGF-1, which may promote

neurogenesis, and the ability of exercise to protect the brain from neuronal injury such as

demyelination (28, 44). Aerobic exercise training may elevate IGF-1 concentration

rapidly in a variety of sites (i.e., brain, peripheral circulation, skeletal muscle), and

stimulate remyelination, improve cognitive function, and enhance muscle hypertrophy,

which may ultimately reduce disease progression in persons with MS (21, 116). In our

study, serum IGF-1 concentration at rest was similar between MS and control subjects

before the initiation of the training program and remained unchanged after exercise

training in both groups. Our study corroborates past results where there were no

differences in serum IGF-1 between MS and control subjects at rest (30, 161).

Additionally, our data also corroborates previous findings suggesting that aerobic

exercise training does not alter IGF-1 (162).

The potential of IGF-1 to promote remyelination in the CNS makes this growth

factor a therapeutic target. Although it may be attractive to speculate that chronic exercise

may upregulate resting IGF-1 content in the periphery, our study results suggest that

serum IGF-1 concentration at rest did not change after training. In similar fashion, a

limited trial of exogenous subcutaneous IGF-1 treatment in MS was proven unsuccessful









(29), adding to the contradictory results observed by IGF-1 administration on

remyelination in the EAE model (24, 114, 163, 164). Therefore, future research involving

exercise, IGF-1 and MS subjects may be useful to determine whether exercise has an

impact in the production of IGF-1 (acutely and chronically), as well as the impact of

exercise-induced IGF-1 on MS related neuronal repair.

Cytokine and Neurotrophin Response to a Single Bout of Exercise in MS

We investigated the response of immune and neurotrophic factors following a

single bout of aerobic exercise. It has been proposed that the short term release of

cytokines during acute exercise may contribute to the maintenance of an immune

homeostatic environment (79). In addition, many of the acute phase proteins released in

response to elevated cytokine levels are protease inhibitors or free radical scavengers that

attenuate the magnitude of tissue damage associated with release of toxic molecules and

free radicals due to activated neutrophils (79). Therefore, a single bout of exercise may

have an array of effects on immune parameters (76, 80) that could contribute to

neuroprotection (80).

Cytokine Dynamics after a Single Bout of Exercise

Skeletal muscle contractions stimulate IL-6 production and may increase

circulating IL-6 concentration via complex signaling cascades initiated both by Ca2+

dependent and independent stimuli (61). Plasma IL-6 increases in exponential fashion

with exercise and is intensity and duration dependent (42, 60, 75). In our study, MS and

control subjects experienced similar significant increases in plasma IL-6 concentration

following 30 minutes of aerobic exercise at 60% of VO2peak as reported in the literature

by others (See Review by Pedersen (165)). Specifically, IL-6 increased significantly 30

minutes post exercise and tended to stay elevated for 2 hours while returned to baseline 3









hours post exercise in both groups. In contrast to our findings, Schulz et al. (76) (2004)

found that IL-6 remained unchanged immediately after a single bout exercise with no

additional post exercise collections acquired. However, the timing of blood sample

acquisition is important because the dynamics of each cytokine can vary considerably in

response to exercise (53). Therefore, our results provide preliminary data suggesting that

2 to 3 hours are needed to capture the exercise-induced IL-6 response in MS and control

subjects after a moderate exercise bout.

In addition to IL-6, we also assessed the TNF-a and IFN-y response following a

single bout of exercise in both groups. TNF-a and IFN-y plasma concentrations decreased

in similar fashion in MS and control subjects after a single bout of exercise (-42% and -

38% respectively at 3 hours post exercise). The response to exercise of both cytokines did

not change after training in either group. Our results are in contrast to Heesen et al. (19)

(2003), who reported increased TNF-a and IFN-y concentrations 30 minutes post

exercise (30 minutes of cycle ergometry at 60% VO2peak) with no additional post exercise

blood evaluation. In our study, TNF-a and IFN-y concentrations 30 minutes post exercise

were similar to baseline values, with a marked significant decrease 2 and 3 hours post

exercise. The kinetic profile of TNF-a and IFN-y follow the opposite dynamics to IL-6

following a single bout of exercise and provides further information on the impact of

exercise on immune markers.

In our study, MS subjects had a similar cytokine response (IL-6, TNF-a, and IFN-

y) compared to control subjects before the initiation of the exercise training program and

it remained unchanged throughout training. Limited information is available on the

influence of exercise on immune variables that are known to impact disease activity in









MS. These findings suggest that individuals with MS may respond to physical stress

similarly to matched healthy controls. In fact, stabilized levels of interacting Thl/Th2

cytokines are maintained in the benign course of MS and it is hypothesized that benign

MS (EDSS <2) is characterized by a fairly balanced cytokine and neuroendocrine

network (166). Perhaps MS subjects with higher disability (EDSS >5) may exhibit a

different response due to a stronger immune dysregulation. Our subjects reported slightly

higher EDSS score than benign MS (EDSS=3.4), but may still maintain a balanced

cytokine and neuroendocrine network when reacting to a physical stress.

Additional studies are needed to provide a more complete and comprehensive

understanding of the dynamic cytokine response to physical stress in MS and its

implications on disease activity. Future research focused on IL-6, IFN-y and TNF-a may

provide further insight because these cytokines are known to directly influence MS

pathophysiology.

Serum BDNF Decreases Following a Single Bout of Exercise

Understanding the impact of exercise on neurotrophic factors and neuroprotection

may yield important information for future therapeutic strategies (26, 167-169).

Previously, Gold et al. (77) (2003) found increases in serum BDNF concentration

immediately after a single bout of aerobic exercise at 60% of VO2peak. However, BDNF

may clear from the circulation very rapidly (within minutes) after subcutaneous injections

of BDNF (102). In our study, BDNF concentration significantly decreased after 2 hours

and 3 hours post exercise in MS and control subjects. Serum BDNF clearance after a

single bout of exercise averaged 73% clearance (of BDNF at baseline) following 2 hours

of post-exercise recovery in both groups. The rapid clearance of BDNF post exercise may

be indicative of 1) of rapid transport of BDNF into the CNS, or 2) BDNF traveling into









the muscle and ultimately transported into the CNS (102, 159). Our findings do not

support the speculation made by Gold that BDNF increases post exercise may be long

lasting (77). However, the fate of circulating BDNF clearance after a single bout of

exercise remains unknown and warrants further study.

Previous studies suggest a positive association between neural repair in the central

nervous and the peripheral concentration of BDNF (77). Kishino and Nakayana (159)

(2003) found that a subcutaneous injection of BDNF enters the blood stream and may be

transferred to the spinal cord axons. In addition, Kishino and Nakayana (159) (2003) also

reported that some circulating BDNF may enter skeletal muscle from the blood stream,

and is transported retrogadely to the motor neuron cell bodies. Kishino and Nakayana

(159) (2003) provide strong evidence that systemic BDNF can enter the CNS and activate

signaling cascades responsible for neural repair. These events are clearly important

because BDNF clearing into the CNS (i.e., exercise bout) could contribute to

remyelination in the CNS and the brain (102).

In a demyelinating disease like MS, blood-derived BDNF may have a beneficial

effect on neuron survival either by transport from a peripheral receptor binding site or by

passage across the BBB (170). Our study provided new evidence that a single bout of

exercise possibly stimulates clearance of circulating BDNF into other areas (i.e., CNS,

skeletal muscle). Whether BDNF crossed the BBB or is transported into the CNS via

skeletal muscle is unclear at this time and further investigations are warranted. Potential

benefits through exercise may be related in part to BDNF availability that increases

neuronal survival (44), facilitated learning (155), and neurogenesis (155, 171) In human

studies, exercise participation predicts better cognitive function (172, 173), lowers risk of









Alzheimer's disease and dementia in general (174, 175). However, whether exercise is a

potent stimulus to provide enhancement of neuroprotective mechanisms in individuals

with MS remains to be elucidated.

Muscle Fatigue

Excessive systemic fatigue and muscle weakness are the most common and

debilitating symptoms of individuals with MS (2). It is known that fatigue can be

counteracted by exercise training in healthy (35, 36) and diseased populations (37, 38).

Although there is evidence indicating that regular exercise may attenuate perceived

systemic fatigue in MS subjects (39-41), less is known about the impact of chronic

exercise training on muscle fatigue in the MS population. Perceived fatigue in MS

subjects is discussed later in this chapter.

Few studies have reported the impact of chronic aerobic exercise on muscular

fatigue in individuals with MS. Consistent with previous findings, MS subjects have

lower strength and more muscular fatigue compared to controls (176, 177). However,

contrary to our hypothesis, 8 weeks of aerobic exercise did not alter muscular fatigue in

MS subjects as measured in this study. Our results corroborate previous investigations

reporting no improvements in muscular fatigue after participating in an exercise program

(178). Others have observed mild improvements in fatigue after regular exercise training

(179, 180). For example, Surakka et al. (180) (2004) found that after an unsupervised

combined aerobic and resistance exercise program for 6 months, women but not men

with MS reduced extensor fatigue. However, in Surakka et al. (180) (2004), fatigue was

measured as a 30 second maximal static contraction. Patti et al. (179) (2003) also showed

mild improvements in muscular fatigue after a 6 week outpatient rehabilitation program

including mild exercise.









The lack of observed change in muscular endurance may be explained in several

ways. First, it has been hypothesized that individuals with MS are unable to fully activate

motor units and consequently hinder possible skeletal adaptations to overload stress (39,

177). Second, our muscle fatigue testing protocol may not have been specific to detect

actual changes in muscle endurance. Based on the observed improvements in 6-minute

walking distance and increased performance during the assessment of maximal oxygen

consumption, it can be argued that muscle endurance improved with training. The

observed changes in aerobic capacity of our subjects is consistent with previous reports

showing a 10-22% gain in aerobic capacity after engaging in an aerobic exercise training

program (40, 41, 181, 182).

Functional Mobility

As expected, MS subjects walked a shorter distance during the 6 minute walk, and

were slower during the timed up and go tests, and the 25 and 100 ft walking tests

compared to control subjects. Multiple sclerosis and control subjects experienced

improvements in functional mobility assessments following training, but did not reach

statistical significance. In fact, our study found similar outcomes compared to Kileff and

Ashburn (183)(2004), where a similar aerobic training program did not translate into 25ft

and 6 minute walk improvements in MS subjects with moderate disability (EDSS = 4-6).

Additionally, our results also found similar observations to White et al. (39) (2004),

where after 8 weeks of resistance training, the 25 ft walking speed remained unchanged

as well. Debolt and McCubbin (184) (2004) also observed no changes in functional

mobility following 8 weeks of unsupervised resistance training. However, the fact that

MS subjects in our study were able to walk 25 meters further during the 6 minute walk

test and performed the timed up and go test one second faster (15% improvement) after









the training program is of clinical importance. These results may also help explain

reductions in perceived disability and may increase their ability to engage in more

activities of daily living. In individuals with MS, enhancing the ability to improve

mobility could have a large impact in their quality of life. In individuals with MS,

reducing disability may enhance daily activity and help offset the cycle of declining

fitness with inactivity.

Quality of Life in Health and Disease

Exercise has been shown to improve psychological and cognitive functioning in

humans (172, 185, 186). Specifically, regular exercise has antidepressant properties

(186), has been shown to decrease anxiety (187), and elevates mood and coping skills in

response to stress (188). In our study, MS subjects significantly decreased perceived

disability, and had marginal improvements in perceived fatigue and quality of life

measures after 8 weeks of exercise training.

Perceived Disability

The observed significant reduction in perceived disability (self-assessed EDSS

score) following training is an important clinical finding because despite the lack of

statistical changes in functional mobility, MS subjects perceived their disability to be

reduced. The fact that MS subjects were able to improve their aerobic capacity and

walking distance likely reflects the change in perceived disability. These findings support

the role of exercise as a positive therapeutic strategy to attenuate functional declines often

observed in this population.

Perceived Fatigue

As expected, perceived fatigue was significantly higher in MS compared to control

subjects in total MFIS and all MFIS subscales prior to the beginning of the exercise









program. Total MFIS scores decreased 19% in MS subjects while only decreased 1% in

controls. Although it was not statistically significant, exercise training improved

perceived fatigue in the MS group. Since fatigue is one the most debilitating symptoms in

MS, with up to two-thirds of patients describing fatigue as their main complaint (189),

fatigue reduction represents a clinically significant outcome. Using the same perceived

fatigue assessment, White et al. (39) (2004) found a comparable 24% decrease in

perceived fatigue after 8 weeks of resistance training in MS subjects. Using the fatigue

severity scale (FSS) for their fatigue assessment, Mostert and Kesselring (41) reported

reductions in fatigue (-14%) of MS subjects after only 4 weeks of aerobic exercise

training. MS subjects also increased their activity level by 17% after only 4 weeks of

regular aerobic exercise. After 15 weeks of aerobic exercise at moderate intensity, MS

subjects experienced a significant reduction in fatigue (measured with the profile of mood

states questionnaire) and a negative association between improvement in aerobic fitness

and fatigue perception (40). As a result of increases in aerobic capacity, MS subjects

were able to perform activities of daily living at lower relative intensity, preventing

excessive fatigue (40). Consequently, therapeutic interventions involving exercise have

the potential to provide a means to control fatigue in people with MS and perhaps

improve daily activity.

Short Form-36 Quality of Life Questionnaire

The SF-36 total mental and physical components were significantly different

between MS and control subjects and remained unchanged following 8 weeks of aerobic

exercise training in both groups. MS subjects improved the total mental and physical

score in the SF-36 questionnaire by 9% and 6% respectively, but these changes were not

significant. Our findings are consistent with previous investigations reporting no effect of









aerobic exercise training on total physical and mental SF-36 outcomes. Mostert and

Kesselring (41) (2002) did not find significant changes in total mental and physical SF-36

scores after 4 weeks of aerobic exercise either. In addition, Heesen et al. (19) (2003) did

not find any effects of 8 weeks of aerobic exercise training on SF-36 outcomes. However,

other quality of life measurements such as the profile of mood states (POMS) have been

shown to be affected by aerobic exercise training (40). In Petajan et al. (40) (1996), MS

subjects experienced decreases in fatigue, anger and depression measured by POMS after

15 weeks of aerobic exercise training. Longer exercise interventions may have an impact

on the quality of life of individuals with MS and may provide additional benefits related

to their psychological state in addition to fitness improvements.

Future Directions

Due to the neuroprotective potential of exercise training in neurodegenerative and

autoimmune diseases, further research is warranted in this area. Additionally, studies

focusing on the impact of exercise on immune markers are important because exercise

may be used as a model for stress after a single bout or as a long term therapeutic

intervention. Further, investigating the immune response to chronic and acute exercise of

other immune markers such as IL-4 or IL-10 may add important information regarding

the potential of exercise to modulate the Thl/Th2 balance. Since regular exercise has

been shown to be immunomodulatory in healthy populations, further investigations may

provide further information regarding the impact of exercise on autoimmune diseases and

their progression.

Determining the role of exercise and BDNF regulation may provide further insight

involving neuroprotective therapeutic strategies in degenerative diseases such as MS.

Additionally, studies focusing on the fate of cleared immune markers and neurotrophins









after exercise are important to understand exercise-induced mechanisms affecting neural

health. The study of IGF-1 may provide additional information regarding the observed

neuroprotective effects of exercise in mammals. Clearly, a combination of both animal

and human studies are needed to clarify the role of exercise on pathways associated with

disease progression in MS.

Since exercise provides an intrinsic means to modify functional outcomes in the

MS population, further study in this area may compliment other therapeutic strategies

designed to attenuate MS disease progression. In addition, increasing activity levels in

MS patients is crucial for long term health. Enhancing muscle strength and endurance

through exercise training may increase daily activity, reduce fatigue and depression and

increase quality of life in the MS population. Therefore, therapeutic interventions such as

regular exercise training are pivotal because they may stimulate protective mechanisms

that not only protect against secondary diseases, but have the potential to impact disease

progression in individuals with MS.















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