<%BANNER%>

Pathways of Oxidative Damage to Skeletal Muscle after an Acute Bout of Contractile Claudication


PAGE 1

PATHWAYS OF OXIDATIVE DAMAGE TO SKELETAL MUSCLE AFTER AN ACUTE BOUT OF CONT RACTILE CLAUDICATION By ANDREW JUDGE 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 2003

PAGE 2

Copyright 2003 by Andrew Judge

PAGE 3

ACKNOWLEDGMENTS I would like to thank my mentor and committee chair, Dr. Stephen Dodd, for his continued support throughout my graduate studies at the University of Florida. His encouragement and availability to discuss science have been paramount to my completion of not only this project, but also my doctorate degree. I would also like to thank my doctoral committee (Drs. Scott Powers, Christiaan Leeuwenburgh, and James Jessup) for their encouragement and advice throughout this study; as well as my masters mentor, Dr. Robert Voight, for his belief in my capabilities and encouragement to pursue this goal. Most of all, I am forever grateful to my parents for their love, support, and encouragement through all my many years of school; and to my wife, Sharon (Phaneuf) Judge, for her love, encouragement and patience. iii

PAGE 4

TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE..............................................1 Introduction...................................................................................................................1 Review of Literature.....................................................................................................3 Ischemia-Reperfusion............................................................................................3 Intermittent Claudication.......................................................................................4 Oxidative Stress.....................................................................................................5 Sources of Oxidants after Exercise Claudication..................................................7 Role of Iron............................................................................................................9 Interaction............................................................................................................10 Edema..................................................................................................................11 Muscle Cell Membrane.......................................................................................11 Countermeasures.........................................................................................................12 Allopurinol..........................................................................................................12 Cyclophosphamide..............................................................................................13 Deferoxamine......................................................................................................14 Summary.....................................................................................................................15 Purpose.......................................................................................................................16 Rationale.....................................................................................................................16 Questions and Hypotheses..........................................................................................17 2 METHODS.................................................................................................................19 Animals.......................................................................................................................19 Supplementation Protocol...................................................................................19 Control..........................................................................................................19 Allopurinol...................................................................................................19 Cyclophosphamide.......................................................................................20 Deferoxamine...............................................................................................20 Ligation Procedure......................................................................................................20 iv

PAGE 5

In Vivo Stimulation.....................................................................................................21 Tissue Removal and Storage......................................................................................21 Biochemical Assays....................................................................................................22 Protein Concentrations........................................................................................22 Muscle Water Content.........................................................................................22 Protein Oxidation.................................................................................................22 Lipid Peroxidation...............................................................................................22 4-Hydroxy-2-Nonenal (HNE).............................................................................23 Xanthine Oxidase Activity..................................................................................23 Myeloperoxidase Activity...................................................................................24 Lactate Dehydrogenase Activity.........................................................................24 Statistical Analysis......................................................................................................24 3 RESULTS...................................................................................................................25 Overview of Experimental Findings...........................................................................25 Morphological Measurements....................................................................................25 Contractile Function...................................................................................................25 Lipid Hydroperoxides (LOOH)..................................................................................26 4-Hydroxy-2-Nonenal Levels.....................................................................................26 Protein Carbonyls.......................................................................................................26 Xanthine Oxidase Activity.........................................................................................27 Myeloperoxidase (MPO) Activity..............................................................................27 Wet/Dry Ratio.............................................................................................................27 Lactate Dehydrogenase (LDH) Activity.....................................................................27 4 DISCUSSION.............................................................................................................33 Overview of Experimental Findings...........................................................................33 Contractile Claudication.............................................................................................33 Xanthine Oxidase Inhibition.......................................................................................34 Neutropenia.................................................................................................................36 Iron Chelation.............................................................................................................38 4-Hydroxy-2-Nonenal.................................................................................................40 5 CONCLUSIONS........................................................................................................42 LIST OF REFERENCES...................................................................................................44 BIOGRAPHICAL SKETCH.............................................................................................51 v

PAGE 6

LIST OF TABLES Table page 1 Markers of oxidative stress and edema after 30 min contractile claudication............6 2 Potential sources of oxidants after 30 min contractile claudication...........................8 3 Body weight, wet muscle weight, and protein concentration..................................28 vi

PAGE 7

LIST OF FIGURES Figure page 1 Force production from the triceps surae muscle group during the last minute of the 30-minute stimulation period.........................................................................28 2 Lipid Hydroperoxide levels; values are expressed as mean SEM........................29 3 Total HNE levels......................................................................................................29 4 Protein carbonyls; values are expressed as mean SEM........................................30 5 Xanthine oxidase activity; values are expressed as mean SEM............................30 6 Myeloperoxidase activity; values are expressed as mean SEM............................31 7 Muscle Wet/dry ratio; values are expressed as mean SEM..................................31 8 Lactate dehydrogenase activity; values are expressed as mean SEM...................32 vii

PAGE 8

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 PATHWAYS OF OXIDATIVE DAMAGE TO SKELETAL MUSCLE AFTER AN ACUTE BOUT OF CONTRACTILE CLAUDICATION By Andrew Judge August 2003 Chair: Stephen Dodd Major Department: Exercise and Sports Sciences A limited number of studies have shown an increase in products of lipid peroxidation in the plasma of claudicants after exercise. Previously, we used an animal model to mimic the condition of exercise claudication to show oxidative damage and edema within the muscle. Using this same model, we investigated the sources of this oxidative damage in the gastrocnemius muscle, focusing on xanthine oxidase and neutrophils, in addition to determining the role iron plays in the exercising claudicant. The increase in lipid hydroperoxides, seen in claudicant muscle, was attenuated with independent inhibition of xanthine oxidase activity, depletion of neutrophils, and chelation of iron. An additional marker of lipid peroxidation, 4 hydroxy-2-nonenal (HNE), was also attenuated by depletion of neutrophils. The HNE is formed from oxidant-induced decomposition of lipid hydroperoxides, but may bind to amino acid side chains of proteins, potentially affecting their function. viii

PAGE 9

Protein oxidation, indicated by an increased level of protein carbonyls, was significantly increased in claudicant muscle, but attenuated by depletion of neutrophils. Since oxidants have the potential to modify membrane macromolecules, we also investigated LDH activity, as an indicator of muscle cell membrane permeability; and wet/dry ratio as a marker of edema relective of vascular membrane permeability. The LDH activity was decreased in claudicant muscles, reflecting loss of the cytosolic enzyme due to membrane alterations. This loss was attenuated with independent inhibition of xanthine oxidase activity, depletion of neutrophils, and chelation of iron. Edema, however, was only attenuated by neutrophil depletion. Our findings suggest that neutrophils are the predominant source of oxidants after exercise claudication; and a major cause of edema. However, we also show that xanthine oxidase-derived oxidants contribute to lipid peroxidation and are chemotaxic to neutrophils. Finally, we show that chelation of iron also attenuates lipid peroxidation, despite an increase in xanthine oxidase and neutrophils, demonstrating irons role in propagating oxidant reactions. ix

PAGE 10

CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE Introduction Oxidative stress, due to ischemia followed by reperfusion (I-R) may cause significant muscle damage and dysfunction [1,2]. Most of the research regarding skeletal muscle I-R injury has used prolonged periods of ischemia followed by reperfusion. These studies found convincing evidence of oxidant generation and associated muscle damage. In addition, it has been shown that various countermeasures can attenuate this oxidative damage [2,3]. Recently, exercise claudication has been classified as a form of I-R; and accumulating evidence associates oxidative stress and muscle damage with this condition [4,5]. Using an in vivo rat model to mimic the condition of exercise claudication, we found evidence of protein oxidation, lipid peroxidation, and reduction of total glutathione in both the soleus and gastrocnemius muscles. This further substantiates the claim of oxidative damage after exercise claudication. At present, only two studies have attempted to attenuate this oxidative stress in exercising claudicants, using antioxidant supplementation. Wijen et al. [5] administered Vitamins C and E together orally, over a 4 week period, and attenuated the oxidative stress in exercising claudicants. In a similar study, Silvestro et al. [6] infused Vitamin C intravenously, 5 minutes before the exercise bout; and were likewise successful in attenuating the oxidative stress. 1

PAGE 11

2 While this therapy shows the potential for sequestering oxidants before they can elicit their damaging effects, the antioxidants used were not specific; and therefore may not be the most appropriate countermeasures. Recently, we found evidence of elevated xanthine oxidase activity and activated neutrophils after contractile claudication. Therefore, specifically blocking or inhibiting these pathways of oxidant generation should provide greater protection. In addition, iron mobilization occurs during prolonged I-R [7], and likely occurs after exercise claudication. Since iron is a powerful catalyzing agent in redox reactions, its chelation should provide protection from oxidative damage. Our study attempted to inhibit xanthine oxidase and induce neutropenia, as well as chelate free iron, to establish the significance of each regarding oxidative damage. Xanthine oxidase can be effectively inhibited with allopurinol a purine analog whose metabolite, oxypurinol, forms a tight binding complex with the enzyme, rendering it inactive. Allopurinol-treated animals appear to have attenuation of I-R-induced muscle damage in a variety of tissues [8]. Neutropenia may be induced with cyclophosphamide a compound activated enzymatically in the body to phosphoramide mustard, a powerful and unstable DNA-alkylating agent. Cyclophosphamide has been shown to significantly reduce the number of circulating polymorphonuclear neutrophils [9], thereby diminishing the inflammatory response, and providing protection against I-R-induced injury [10]. This is presumed to occur through a reduction in polymorphonuclear neutrophil superoxide production.

PAGE 12

3 Deferoxamine is a powerful iron chelator, and may be used to prevent iron from participating in redox reactions. Deferoxamine has effectively been used to prevent oxidative stress and muscle damage after prolonged I-R [3]. To date, no studies have used these countermeasures in the exercising claudicant. In fact, until recently no study had measured oxidative stress in muscle after exercise claudication, or considered the potential sources. We administered each of the countermeasures and measured oxidative stress after contractile claudication; and determined muscle edema, as evidenced by an increase in the wet/dry ratio. It is imperative to gain a better understanding of the relative contributions of the various sources of oxidants produced during exercise claudication. At present, the lack of research in this area means that we are relying on somewhat speculative information, based on non specific research, and thus relying on assumptive preventative measures. It seems prudent to gain a specific understanding of the sources of oxidants so that the most appropriate countermeasures can be implemented to attenuate the stress and edema, and reduce further disability from this cardiovascular disease. Review of Literature Ischemia-Reperfusion Ischemia may be defined as a rate of blood flow to an organ that is inadequate to supply sufficient oxygen and maintain aerobic respiration in that organ [11]. Therefore, skeletal muscle becomes ischemic when there is a mismatch between blood supply and blood demand resulting in insufficient oxygen delivery to the muscles. This may result from cardiovascular disease, severe trauma, vascular occlusions, or even strenuous or unaccustomed exercise. Although ischemia may cause muscle damage and, if prolonged enough, necrosis, it appears to be the re-oxygenation, or reperfusion of tissue that causes

PAGE 13

4 the major damage. Reperfusion occurs through restoration of blood flow so that oxygen delivery can once again match the demand. Since this is essential to tissue survival, the potential damage appears to be the lesser of two evils. Intermittent Claudication Skeletal muscle ischemia due to disease is highly dependent on the degree of vessel blockage, and may manifest itself as pain at rest in the most severe cases (or more commonly, as claudication). The term claudication derives from the latin word claudicatio, meaning to limp; and describes a condition whereby pain is felt during exercise or activity (sufficient to require termination of the exercise), with relief upon rest. This exercise-induced pain is a result of the inability to deliver the required substrates to meet the demands of the working muscles a similar condition to that which occurs during strenuous or unaccustomed exercise. Intermittent claudication refers to the continual process of pain upon exercise, and relief with rest. The prevalence of intermittent claudication ranges from 2.9%, in the Speedwell Prospective Heart Disease Study [12] to 4.5% in the Edinburgh Artery Study [13]. The range probably reflects the differences in defining criterion. However, these numbers are likely an underestimate of the true incidence, since only 10 to 50% of people with claudication symptoms consult their general practitioner [14]. Peripheral Arterial Disease (PAD) is probably the most common cause of intermittent claudication. This disease is associated with atherosclerotic lesions, reducing blood flow to the area distal to the lesions. Therefore, the locality of pain can indicate which vessel might be restricted calf pain is most frequently a consequence of femoral artery disease; whereas pain in the thigh/buttock area indicates proximal restriction, most likely due to aortoiliac disease [15].

PAGE 14

5 Except in the most severe cases, the restricted blood flow does not cause ischemia of a resting skeletal muscle, due to its low metabolic demands. However, during physical activity, the metabolic demands of the exercising muscle increase greatly, and blood flow is required to increase many-fold to meet this demand. Although blood flow may be able to increase some, it is often not enough to match the increased demand, rendering the limb ischemic. This is illustrated in a study by Pernow et al. [16], who measured femoral artery blood flow in patients with claudication, both before and during a standard exercise test. During exercise, blood flow in claudicants increased almost threefold; however, this was still only 50% of the exercise blood flow in control subjects. Recently, intermittent claudication has been referred to as a form of low-grade I-R [17]. This is not identical to the traditional I-R, whereby blood flow to a muscle is occluded for a given period (enforcing absolute ischemia) and then restored, allowing re-oxygenation of the tissue. During claudication, the ischemia is relative and the reperfusion does not occur due to restoration of blood flow, but rather due to a reduction in blood demand during the recovery period, allowing the oxygen demands of the muscle to once again be met. However, parallels between the two conditions certainly exist, which has led to the realization that intermittent claudication may generate the same damage that occurs after I-R (contractile dysfunction, oxidative injury [18], cell dysfunction and cell death [19]). Oxidative Stress There is considerable evidence of radical production induced by prolonged ischemia of skeletal muscle, with subsequent reperfusion [1,2,20]. This has applicability to surgical situations where it may be necessary to occlude blood flow for hours, before reperfusion is either allowed, or necessary. These studies have provided evidence of

PAGE 15

6 elevated lipid hydroperoxides, decreased protein thiols, and a loss of glutathione. However, few studies have considered the idea that radicals may be generated after short-term I-R, as would be the case in the exercising claudicant. This form of I-R may be of a lower grade, and skeletal muscle has been shown to be fairly resistant to I-R injury. Indeed, Kadambi et al. [21] found no increase in lipid peroxidation after 30 min of ischemia followed by 1 h of reperfusion. However, when the metabolic demands of a muscle are increased while blood flow to the muscle is reduced, the mismatch between supply and demand becomes greater. This may cause a greater disruption to redox balance. Work from our lab shows convincing evidence of oxidative damage to both lipids and proteins, as well as a reduction in total glutathione; and an increase in wet/dry weight, which is indicative of edema, after a 30-minute bout of contractile claudication (Table 1). Other studies lend support to this, showing increased lipid peroxides [4] and increased ortho-hydroxyantipyrine an indicator of oxidative stress [5]. Table 1. Markers of oxidative stress and edema after 30 min contractile claudication Soleus Gastrocnemius Sham-Stimulated Ligated-Stimulated Sham-Stimulated Ligated-Stimulated Lipid Hydroperoxides (mmol/g wet weight) 8.92 1.43 13.76 1.39 7.39 0.97 10.88 0.86 Protein Carbonyls (nmol/mg protein) 1.80 0.05 2.83 0.07 # 1.87 0.13 2.89 0.13 # Total Glutathione (mM) 1.44 0.03 1.29 0.05 0.90 0.01 0.76 0.04 + Wet/Dry Ratio N/A N/A 4.51 0.08 4.72 0.05 Values are mean SEM; N=8 *Significant at p<0.05; + Significant at p < 0.01; # Significant at p < 0.001 Just as strenuous exercise in healthy individuals causes significant oxidative stress, while moderate intensity exercise does not [22], this too may be the case in claudicants. Silvestro investigated the effects of maximal and submaximal exercise on parameters of oxidative stress in claudicants; and discovered that only maximal exercise caused

PAGE 16

7 significant lipid peroxidation and an increase in intracellular adhesion molecule-1 (ICAM-1). Expressed by endothelial cells, ICAM-1 is a receptor for the neutrophil adhesion molecules CD11/CD18, and is therefore important in determining neutrophil-endothelium cell adhesion. Adherence of these molecules primes the neutrophil for migration into the tissue. These findings are interesting since claudicants are instructed, therapeutically, to exercise until near maximal pain. However, based on Silvestros findings, it might seem logical for claudicants to exercise less intensely. This would presumably cause a milder ischemic insult, reducing oxidative stress and neutrophil infiltration, while potentially still providing the cardiovascular benefits of exercise that claudicants so desperately need. Sources of Oxidants after Exercise Claudication There are a variety of potential biological sources of oxidants. However, most research regarding skeletal muscle I-R has focused on the xanthine oxidase pathway and activated neutrophils. Work from our lab has shown xanthine oxidase activity and activated neutrophils to be elevated after a short bout of contractile-induced skeletal muscle ischemia (Table 2). Thus these potential sources are considered in greater detail. Xanthine oxidase (XO) pathway. During ischemia, or very strenuous exercise, ATP catabolism occurs within the muscle cell, yielding hypoxanthine and xanthine. Normally these metabolites are oxidized via xanthine dehydrogenase (XDH) using NAD as the electron acceptor. However, XDH may be converted to XO by oxidation of thiol groups, or by calcium-dependent proteolytic attack. Since both ischemia and strenuous exercise can disrupt calcium homeostasis, the environment may favor this dehydrogenase to oxidase conversion. XO is still able to oxidize hypoxanthine and xanthine; however,

PAGE 17

8 molecular oxygen acts as the electron acceptor instead of NAD, the consequence being the generation of large amounts of superoxide anion [23]. The necessary components for ROS production are, therefore, provided. The only missing component is molecular oxygen, provided on reperfusion. Table 2. Potential sources of oxidants after 30 min contractile claudication. Soleus Gastrocnemius Sham-Stimulated Ligated-Stimulated Sham-Stimulated Ligated-Stimulated Xanthine Oxidase Activity (mU/g wet weight) 2.85 0.09 3.39 0.19 2.73 0.08 3.29 0.15 Myeloperoxidase Activity (U/g wet weight) 0.80 0.02 1.15 0.03 # 0.66 0.08 1.03 0.06 # Mitochondrial Hydrogen Peroxide Release (nmol/min/mg protein) N/A N/A 0.48 0.04 0.49 0.06 Values are mean SEM; N=6 *Significant at p<0.05; # Significant at p < 0.001 Neutrophils. It has been shown that neutrophil activation is significantly increased after intermittent ischemia of skeletal muscle [23-25]. Plasma neutrophil levels have been shown to peak within 5 min post-exercise in claudicants [25,26]; however infiltration into tissues takes longer. Smith et al. [27] showed what they referred to as a dramatic increase in tissue neutrophils after just 15 min of reperfusion after 4 h ischemia. However, Prem et al. [28] found that although tissue neutrophils increased after 30 min of reperfusion, after 2 h ischemia, the levels peaked after 4 h of reperfusion. This peak in neutrophil infiltration after a longer period of reperfusion is in agreement with others [29]. The mechanisms of neutrophil activation are unquestionably complex, and indeed remain largely unknown. However, it is interesting to note that oxidants themselves have been implicated as chemotactic to neutrophils [30]. This is supported by the observation

PAGE 18

9 that treatment with radical inhibitors before reperfusion prevents tissue neutrophil infiltration [31]. In addition, the lipid peroxidation product 4-hydroxy-trans-2-nonenal has also been shown to be chemotactic to neutrophils [32], weaving a tighter web between oxidant production and propagation of the inflammatory process. Role of Iron Although the generation of superoxide and hydrogen peroxide are potentially damaging, they are poorly reactive and cannot alone explain the toxic effects of superoxide generating systems [33]. Instead, it seems that the highly damaging hydroxyl radical is largely responsible for oxidative injury. The hydroxyl radical may be generated by the reaction of superoxide with hydrogen peroxide (Haber-Weiss reaction), yet this proceeds too slowly to be of biological significance. However, in the presence of a suitable metal catalyst, for example iron or copper, the reaction proceeds much more rapidly. Iron appears to be the best candidate in vivo; and the iron-catalyzed Haber-Weiss reaction proceeds with a rate constant of 76 M-1 s-1, compared to almost zero without a catalyst. In addition to catalyzing hydroxyl radical formation, iron may also reinitiate lipid oxidation by converting lipid hydroperoxides to reactive alkoxyl or peroxyl radicals [7]. Fortunately, under normal physiological conditions, virtually all iron is bound either to proteins, membranes, nucleic acids, or low-molecular-weight chelating agents [33]. Within the muscle cell, iron is stored mainly within ferritin molecules, a protein of 24 polypeptide chains capable of storing up to 4,500 iron atoms [34]. However, iron may be mobilized from ferritin and other storage forms, making the metal redox active. Release of iron from ferritin requires reduction of the ferric iron stored in the molecule, a feat the superoxide anion and other reducing agents are capable of. Biemond et al. [34]

PAGE 19

10 stimulated polymorphonuclear leukocytes to produce superoxide; and observed significant iron mobilization from ferritin. Addition of SOD prevented this, providing convincing evidence that the iron mobilization occurred in a superoxide-dependent manner. Likewise, iron release from ferritin has been shown to be possible with xanthine and xanthine oxidase, a well known source of superoxide [35]. It has also been suggested that NADH and NADPH, which accumulate during ischemia, might interact with ferritin to release ferrous iron [36]. Furthermore, the rate of ferritin iron mobilization by reducing agents is accelerated by acidification. Brain homogenate incubated under aerobic and anaerobic conditions at three different pHs (7, 6 and 5) showed far greater iron delocalization under anaerobic conditions, which was exacerbated with a decrease in pH [36]. Iron has been shown to be an important pro-oxidant in prolonged I-R. Chiao et al. [7] provided evidence of iron delocalization after 2 h ischemia and 30 min reperfusion; and this was associated with membrane dysfunction and lipid peroxidation. Although no study has considered the role of iron in promoting oxidant reactions in the exercising claudicant, the conditions seem appropriate for iron delocalization. Exercising claudicants rely heavily on anaerobic respiration because of reduced oxygen delivery, causing an increase in reducing equivalents and increased lactic acid production. Acidosis, accumulation of reducing equivalents, and superoxide production could release iron from ferritin stores, thereby becoming available to catalyze free radical reactions [37]. Interaction It is doubtful that ROS are derived from a single source in the exercising claudicant. Rather, there is likely some overlap and interaction among the different

PAGE 20

11 sources. This is shown (after prolonged I-R) by Seekamp et al. [29], who measured tissue injury and vascular permeability. Neutrophil depletion, administration of catalase and superoxide dismutase, allopurinol, dimethylthiourea, dimethylsulfoxide, and complement depletion were all individually implemented as countermeasures; and each significantly attenuated muscle damage to some degree. Importantly, pretreatment with antioxidants also significantly reduced neutrophil infiltration, emphasizing the chemotactic potential of ROS. Edema The alteration of cell membranes due to post-ischemic oxidative stress has a strong correlation with the presence of intramuscular edema [38], characterized by fiber swelling, fiber destruction, and an increase in muscle wet weights. Neutrophil depletion or hydroxyl radical scavenging have been shown to significantly reduce both oxidative stress and edema [2], strongly implicating ROS in the muscle damage. We have previously shown significantly increased muscle wet weights in the gastrocnemius and soleus muscles (and a significant increase in the wet/dry ratio in gastrocnemius muscles) of exercise claudication animals, possibly due to oxidative stress-induced edema. Muscle Cell Membrane Lactate Dehydrogenase resides in the cytoplasm of the muscle cell and has a molecular weight of 140,000 kDa, making it highly impermeable to the muscle cell membrane. Therefore, a decrease in LDH may indicate a change in membrane permeability, such that some of the enzyme has escaped into the extracellular compartment. This may be explained by an alteration in cellular ATP levels, which could

PAGE 21

12 increase intracellular calcium levels, activating proteases and lipases, thereby altering normal membrane permeability. Given the degree of ATP catabolism that appears to occur during skeletal muscle ischemia [39] this is a possibility. Alternatively, it could be the result of oxidative modification of membrane lipids and proteins, as we previously showed to occur after contractile claudication. This loss in LDH activity associated with oxidative stress has been reported previously. After downhill running Dawson [40] noted an increase in lipid peroxidation and myeloperoxidase activity; and a decrease in LDH activity in the gastrocnemius muscle, concluding that oxidative modification to the muscle cell membrane made it leaky. In a separate study, Jones [41] stimulated muscles in vitro for 30 min and quantified LDH release into the medium over a 3 h period. Enzyme release peaked 1 h post-stimulation in the soleus muscle under both normoxic and hypoxic conditions, but was significantly more elevated in the hypoxic conditions. In an attempt to distinguish whether ATP depletion or membrane disruption was responsible for this loss of LDH, Jones incubated with iodoacetate and cyanide, to reduce ATP; and then with deoxycholic acid, a detergent, to disrupt the membrane. Both treatments caused even greater release of LDH, confirming both as possibilities. The conclusion drawn was that both metabolic changes and physical damage could play a part. Countermeasures Allopurinol Allopurinol is a purine analog whose metabolite, oxypurinol, forms a tight binding complex with xanthine oxidase, rendering the enzyme inactive. Since xanthine oxidase is a potential source of ROS, inactivating the enzyme could have protective effects against

PAGE 22

13 ROS-induced damage. Animals treated with allopurinol appear to have attenuation of I-R-induced muscle damage in a variety of tissues [8]. Vina et al. [42] showed that blood XO levels were significantly elevated after exhaustive exercise; and that allopurinol administration prevented exercise-induced glutathione oxidation. Although exercising claudicants do not typically perform exercise that would be exhaustive to a healthy individual, even light exercise usually brings about shortness of breath, perspiration, and a general sense of fatigue [26]. Work from our lab, using an animal model of unilateral femoral artery blockage, showed that a mild stimulation protocol elicits much greater metabolic stress to the occluded limb compared to the control, as evidenced by a decline in force production over time. Several studies have shown that oxidative stress is derived from xanthine oxidase during prolonged ischemia, followed by reperfusion, in skeletal muscle. Asami et al. [43] showed that muscular xanthine and malondialdehyde (MDA) levels were elevated during reperfusion after 5 h of ischemia. MDA is an indicator of lipid peroxidation, and its increase was attenuated by allopurinol administration, which strongly implicates XO as the source of the radical-induced lipid damage. Similarly, McCutchan et al. [44] showed that XO activity was significantly elevated with 3 h of ischemia followed by reperfusion; and that this was associated with hydrogen peroxide generation. Administration of either allopurinol or tungsten (which replaced molybdenum in xanthine oxidases active site) reduced XO activity and hydrogen peroxide generation. Cyclophosphamide Cyclophosphamide is a derivative of the nitrogen mustard family, compounds originally developed as chemical weapons. Soldiers exposed to sulfur mustard suffered from low white-blood-cell counts (especially lymphocytes), apparently due to the

PAGE 23

14 mustards cytotoxic effects on dividing tissues. After that observation, nitrogen mustard, a similar but less toxic agent, was developed to treat cancer; and later used as an immunosuppressant. Cyclophosphamide is activated enzymatically in the body to phosphoramide mustard, a powerful and unstable DNA-alkylating agent, interfering with DNA synthesis, therefore proving cytotoxic to dividing lymphocytes [45]. Several studies have used cyclophosphamide to induce neutropenia in rat models. Kuwabara et al. [9] noted the number of polymorphonuclear neutrophils was reduced to 20 per L of blood 4 days after injection, compared to 1224 per L of blood in control animals. Seekamp et al. [29] used cyclophosphamide to induce neutropenia 3 days before I-R exposure and found a protective effect against muscle permeability. This protection from cyclophosphamide-induced neutropenia has also been shown to be effective against an increase in microvascular permeability associated with short-term I-R [10]. Deferoxamine Deferoxamine is a straight-chained molecule with three hydroxamic acid groups. When a ferric ion comes into contact with deferoxamine, the molecule twines itself around the ion, attaching it to its three hydroxamic acid groups. The molecule, therefore, provides a shell, surrounding the iron and becoming a very stable complex ([46]. The powerful iron-chelating properties of deferoxamine mean that it may inhibit lipid peroxidation and the generation of the hydroxyl radical from superoxide and hydrogen peroxide in biological systems where ferrous iron is free. Deferoxamine has also been shown, in high concentrations, to block the conversion of xanthine dehydrogenase to xanthine oxidase in cultured endothelial cells [47]. The mechanism for this is unknown, however the xanthine oxidase protein has four redox-active sites:

PAGE 24

15 molybdenum, flavin adenine dinucleotide (FAD), and two iron sulfur centers of the ferredoxin type [48]. It was speculated that enzyme activity would be impaired by deferoxamine if it bound the iron cofactor. Since myeloperoxidase contains a heme iron essential for its activity, the possibility exists that deferoxamine could inhibit MPO activity if it chelated the heme iron. However, since addiing deferoxamine has no effect on the absorption spectrum of MPO, it doesnt appear to work in this manner [49]. Deferoxamine can, however, be oxidized by MPO and hydrogen peroxide, thereby competing with other electron donors; and deferoxamine has been shown to react with and degrade the highly oxidizing hypochlorous acid a product of the MPO system [49]. Deferoxamine has a relatively low molecular weight (656.79 Da), which facilitates its entry into cells. In fact, deferoxamine has been shown to enter skeletal muscle cells in significant concentrations with a greater intracellular than extracellular distribution [50]. Administration of deferoxamine after prolonged I-R has been shown to prevent lipid peroxidation and to attenuate membrane dysfunction [3]. Additionally, Smith et al. [51] showed that either deferoxamine or apotransferrin (an iron-binding protein) administration prevented an increase in microvascular permeability associated with prolonged I-R. Summary In summary, it has been shown that an acute bout of contractile claudication causes an increase in protein oxidation, lipid peroxidation, and edema; and a loss of total glutathione. This is associated with an increase in xanthine oxidase activity and neutrophil infiltration. In addition, free iron is elevated after prolonged I-R; and is likely elevated after an acute bout of contractile claudication due to favorable conditions for

PAGE 25

16 iron mobilization. Therefore, inhibition of xanthine oxidase activity, induction of neutropenia, and iron chelation have the potential to attenuate the oxidative stress and edema associated with an acute bout of contractile claudication. In addition, reactive oxygen species have themselves been implicated as chemotactic to neutrophils. Therefore, inhibition of xanthine oxidase and iron chelation, have the potential to attenuate neutrophil infiltration into tissue. Purpose Our purpose was to determine if inhibition of xanthine oxidase activity, induction of neutropenia, or iron chelation will protect against oxidative stress and muscle edema induced by an acute bout of contractile claudication. We also determined whether inhibition of xanthine oxidase activity and iron chelation, attenuate the increase in neutrophil infiltration associated with an acute bout of contractile claudication. Rationale We showed that oxidation of proteins and lipids occurs after an acute bout of contractile-induced skeletal muscle ischemia; and that this is associated with an increase in muscle wet weight, likely due to edema. We also showed an increase in xanthine oxidase activity and infiltration of neutrophils after the same conditions. It was shown by others that administering allopurinol inhibits xanthine oxidase activity; and therefore oxidant production [44]. It was also shown that induction of neutropenia by cyclophosphamide attenuates neutrophil infiltration and protects against oxidative stress-induced muscle damage [29]. Thus, it is speculated that allopurinol administration will inhibit xanthine oxidase activity, and that cyclophosphamide administration will attenuate neutrophil infiltration; and that both will independently attenuate protein and lipid oxidation and edema.

PAGE 26

17 Mobilization of iron from its stored sources can catalyze the Haber-Weiss reaction, generating the highly reactive hydroxyl radical; and reinitiating lipid peroxidation, creating the alkoxyl and peroxyl radicals. Delocalization of iron was shown to occur after prolonged I-R; and is associated with lipid peroxidation [7]. In addition, chelation of iron by deferoxamine was shown to prevent lipid peroxidation [3]. Thus, it is expected that deferoxamine administration will attenuate protein and lipid oxidation and edema. Reactive oxygen species have been shown to be chemotactic to neutrophils [30]; and inhibiting their generation has attenuated neutrophil infiltration [31]. Thus, it is anticipated that iron chelation and xanthine oxidase inhibition will reduce the amount of ROS produced; and therefore attenuate neutrophil infiltration. Questions and Hypotheses Question 1. Does an increase in xanthine oxidase activity cause oxidative stress and muscle edema, generated by an acute bout of contractile claudication? Hypothesis 1. Reduction of xanthine oxidase activity will attenuate lipid peroxidation and protein oxidation, as well as muscle edema, induced by an acute bout of contractile claudication. Question 2. Does an increase in xanthine oxidase activity cause neutrophil infiltration into muscle after an acute bout of contractile claudication? Hypothesis 2. Reduction of xanthine oxidase activity will attenuate neutrophil infiltration into muscle after an acute bout of contractile claudication. Question 3. Does neutrophil infiltration cause the oxidative stress and muscle edema induced by an acute bout of contractile claudication?

PAGE 27

18 Hypothesis 3. Reducing neutrophil infiltration into tissue will attenuate lipid peroxidation and protein oxidation, as well as muscle edema, induced by an acute bout of contractile claudication. Question 4. Does reduction of free iron attenuate the oxidative stress and muscle edema induced by an acute bout of contractile claudication? Hypothesis 4. Reduction of free iron will attenuate lipid peroxidation and protein oxidation, as well as muscle edema, induced by an acute bout of contractile claudication. Question 5. Does reduction of free iron attenuate neutrophil infiltration induced by an acute bout of contractile claudication? Hypothesis 5. Reduction of free iron will attenuate neutrophil infiltration into tissue, induced by an acute bout of contractile claudication.

PAGE 28

CHAPTER 2 METHODS Animals All experiments were performed on male Sprague Dawley rats (120 d old) to avoid any antioxidant protection from estrogen. They were fed rat chow, given water ad libitum, and maintained on a 12-h light/dark photoperiod for 7 days before the beginning of these experiments. During this 7 day period, animals were handled daily to prevent a stress hormone-induced reduction in body weight at the beginning of the experiments. Animals were then randomly assigned to one of four experimental groups: Control (CON); Allopurinol supplemented (ALLO); Cyclophosphamide supplemented (CYCLO) and; Deferoxamine supplemented (DFO). The limbs of each rat were then randomly assigned to a ligated/stimulated (LS) or a sham ligated /stimulated (SS) group for the study. Supplementation Protocol Control Animals were injected intraperitoneally with 0.5 mL saline twice daily, beginning 2 days before ischemia. On the day of the experiment, the second injection was given 30 min before the contractile-induced ischemia. Allopurinol Animals were given an intraperitoneal injection of 50 mg of allopurinol per kg body weight twice daily, beginning 2 days before the ischemia. On the day of the experiment, the second injection was given 30 min before the contractile-induced 19

PAGE 29

20 ischemia. This quantity of allopurinol has been shown to yield extracellular fluid concentrations of 10 M, a concentration sufficient to cause an >80% inhibition of xanthine oxidase activity without a scavenging effect [52]. The allopurinol was dissolved in normal saline by adding 1 N sodium hydroxide; and administered slowly. Cyclophosphamide Animals were given intraperitoneal injections of 20 mg of cyclophosphamide per 100 g of body weight, 4 days before contractile-induced ischemia. This dose has been shown to reduce the circulating leukocyte count by 85 to 90%, inhibiting microvascular damage [10]; and, more specifically, to reduce neutrophils to 1.4% of normal levels [53]. Deferoxamine Animals received an IP injection of 100 mg deferoxamine per kg body weight twice a day, beginning 2 days before the ischemia. On the day of the experiment, the second injection was given 30 min before the contractile-induced ischemia. Deferoxamine was dissolved in normal saline and administered slowly. This quantity of deferoxamine has been shown to reduce neutrophil infiltration and muscle damage [54]. Ligation Procedure After isofluorane anesthesia (5% for induction, 1.5 to 2.5% for maintenance), a small incision was made directly above the inguinal fold; and the femoral artery was exposed and isolated by blunt dissection. Two ligatures were placed tightly around the vessel and the vessel was cut between the ties. This procedure produces a 60% to 70% reduction in blood flow during muscle contraction [55,56]. Topical antibiotic powder was placed on the wound before closure with sutures. The sham surgery limbs underwent the identical procedure except the femoral artery was left intact.

PAGE 30

21 In Vivo Stimulation Twenty-four hours post-ligation both hindlimbs were stimulated in vivo for 30 min, and force production was measured. Animals were placed in a prone position in a specially fabricated Plexiglas apparatus that allows the animal to be secured in a reproducible position with limited mobility of the lower leg except at the tibiotarsal joint. Animals were kept warm by an incandescent light, and core temperature maintained from 35 to 38 o C, measured with a rectal thermistor probe. A calibrated force/displacement ergometer was secured to the forefoot between the first and second footpads by a lightweight chain such that the tibiotarsal angle is 90 o The voltage signal from the force transducer was processed via a computerized data acquisition system (LabView, National Instruments, Austin). A stainless steel stimulating electrode was placed transcutaneously near the sciatic nerve midway between the posterior ischeal spine and the greater femoral trochanter. Another stainless steel stimulating electrode (anode) was inserted 3 mm subdermally in the midline of the lower back. The sciatic nerve was then stimulated proximally with 100V, 1.0 pulses per second, and a stimulus time of 0.05 ms (Grass Instruments). Tissue Removal and Storage One hour post-stimulation the gastrocnemius muscle was removed. Each muscle was dissected free, immediately placed in cold antioxidant buffer (100 m EDTA, 50 mM Na 2 HPO 4 1 mM BHT), blotted dry, weighed, and rapidly frozen in liquid nitrogen and stored at -80C until assayed.

PAGE 31

22 Biochemical Assays Protein Concentrations Protein content of muscle homogenates was determined using the biuret technique [57]. Muscle Water Content Total water content of the gastrocnemius muscles was determined by using a freeze drying technique incorporating a vacuum pump with a negative pressure of ~1 mm Hg. A precise frozen wet weight was measured, and then tissues placed in a freeze-dry unit (Virtis Sentry Benchtop 3 L). The dry weight was terminated when the same weight was recorded three times in succession during a six-hour interval. Protein Oxidation Protein carbonyls was measured spectrophotometrically as described by Reznick and Packer [58], with modifications reported by Yan et al. [59]. Briefly, samples were incubated in dinitrophenylhydrazine (DNPH) dissolved in HCl, with blanks incubated in HCl only. Following reaction with DNPH, proteins was precipitated in 20% TCA, washed in ethyl acetate-ethanol (1:1 vol/vol) and dissolved in 6 M guanidine hydrochloride, pH 2.3. Tissue protein carbonyl content was quantified by determining the absorbance at 370 nm and using an extinction coefficient of 22,000 M -1 Protein concentrations was determined using a BSA standard curve in guanidine HCl with absorbance read at 280 nm. Lipid Peroxidation Lipid hydroperoxides were measured using the ferrous oxidation/xylenol orange technique reported by Hermes-Lima et al. [60]. Briefly, samples were homogenized in 100% methanol, centrifuged, and the resulting supernatant mixed in solution with an iron

PAGE 32

23 source (FeSO 4 ), an acid (H 2 SO 4 ) and a reactive dye (xylenol orange). In this mixture, the membrane peroxides oxidize Fe 2+ to Fe 3+ and the peroxides are reduced. The Fe 3+ reacts with xylenol orange to form a Fe 3+ -Xylenol orange complex, yielding a colored product that is accompanied by an absorbance change at 580 nm. 4-Hydroxy-2-Nonenal (HNE) Proteins were separated on a 4-20% precast polyacrylamide gel (BMA, Rockland), using 30 g of protein per well, and then transferred onto a nitrocellulose membrane. The membrane was then blocked overnight using a blocking solution containing 0.05% Tween and 5.0% milk. Membranes were incubated for 1 hour with the primary antibody (Alpha Diagnostics, San Antonio) using a 1:500 dilution, thoroughly washed and then incubated for 1 hour in anti-rabbit IgG horseradish peroxidase (Amersham Life Science, United Kingdom) using a 1:1000 dilution. Blots were developed using ECL (Amersham Pharmacia Biotech, United Kingdom), and imaged using an Image Station (Eastman Kodak Company, model 440cF). Arbitrary OD units were calculated by multiplying the area of each band by its optical density and then normalized to the control group (CON SS), which we made to 100%. Xanthine Oxidase Activity Xanthine oxidase was measured using a modified version of Amplex Red Xanthine/Xanthine Oxidase Assay Kit from Molecular Probes. In this assay xanthine oxidase catalyzes the oxidation of purine bases, hypoxanthine, or xanthine to uric acid and superoxide. Superoxide spontaneously degrades to hydrogen peroxide, which in the presence of horseradish peroxidase, reacts stoichiometrically with Amplex Red reagent to generate the red-fluorescent oxidation product resorufin. The emission wavelength was set at 590 nm and excitation fluorescence measured at 560 nm.

PAGE 33

24 Myeloperoxidase Activity MPO activity was assayed according to methods by Belcastro [61]. Briefly, tissue was homogenized in 0.5% hexadecyltrimethyl ammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. Homogenate was then be sonicated, freeze-thawed three times, sonicated once more, and subsequently centrifuged at 1,300 g for 15 mins. Ten l of supernatant was removed and incubated with 290 l of 50 mM potassium phosphate buffer with 0.6mM hydrogen peroxide and 167 g o-dianisidine dihydrochloride per ml. One unit of MPO was defined as a change in absorbance of 1.0 at an optical density of 480 nm at a temperature of 25C. Lactate Dehydrogenase Activity LDH activity was determined according to methods by Bergmeyer et al [62]. This assay uses pyruvate and muscle homogenate (containing LDH), to oxidize NADH to NAD, which is accompanied by a change in absorbance at 340 nm. Since the NADH being oxidized is equimolar to the pyruvate being reduced, the change in absorbance is directly proportional to the LDH activity in the sample. Statistical Analysis All data was analyzed using a two-way analysis of variance, and the contractile data was analyzed using a two way ANOVA with repeated measures. Significance was established at the P < 0.05 level, and a Bonferroni post-hoc test was used where necessary.

PAGE 34

CHAPTER 3 RESULTS Overview of Experimental Findings This study examined the pathways of oxidative damage after an acute bout of contractile claudication. By inhibiting each of the pathways individually, a cause and effect relationship could be established. The major findings of the study are that neutrophil depletion and iron chelation attenuated the oxidative damage associated with contractile claudication, while edema was only attenuated with neutrophil depletion. Inhibition of xanthine oxidase activity significantly attenuated lipid peroxidation, but not protein oxidation or edema. Morphological Measurements Each of the countermeasures was well tolerated by the animals, with no mortalities or visual side effects noted. The body weights of ALLO and CYCLO animals significantly decreased, by 7% and 13% respectively, from the time of group assignment (pre-injection) to the time of sacrifice, and the muscle weights of CYCLO animals were significantly lower than the muscle weights of CON LS, DFO SS and DFO LS. However, neither the muscle weight/body weight ratio nor total protein concentration was different across any of the groups (Table 1). Contractile Function Force generation from the triceps surae muscle group significantly decreased in each of the ligated-stimulated (LS), or claudicant, limbs during the thirty-minute stimulation period (Figure 1). Compared to their sham-stimulated limbs, both the LS 25

PAGE 35

26 CON and DFO groups were significantly decreased after 3 minutes of stimulation, and the ALLO and CYCLO group after just 2 minutes. However, neither the LS nor SS limbs were different across treatments, providing evidence that each group was made ischemic to the same degree and that administration of the various countermeasures had no effect on force generation during the contractile claudication period itself. Lipid Hydroperoxides (LOOH) LOOHs were significantly elevated (p<0.001) in the LS limbs of control and ALLO supplemented animals, compared to sham. However, the LS limb of ALLO animals was significantly (p<0.05) less than the LS limb of the control group. There were no differences in LOOH levels between the LS and SS limbs of the CYCLO and DFO groups (Figure 2). 4-Hydroxy-2-Nonenal Levels CON SS limbs were used as a control for comparisons of CON LS, ALLO LS, CYCLO LS, and DFO LS. Total HNE binding was significantly attenuated in the CYCLO LS only (Figure 3). Protein Carbonyls Both the control group and ALLO supplemented animals showed a significantly increased (p<0.05) protein carbonyl content in the LS limb compared to SS. There were no differences between the LS limbs of ALLO animals and control. DFO and CYCLO supplementation both attenuated this increase in protein carbonyls in the LS limb (Figure 4).

PAGE 36

27 Xanthine Oxidase Activity In control, DFO supplemented and CYCLO supplemented animals, xanthine oxidase activity significantly increased (p<0.001) in the LS limb, compared to sham. This increase was attenuated in the ALLO supplemented group (Figure 5). Myeloperoxidase (MPO) Activity Control (p<0.001), ALLO supplemented (p<0.01), and DFO supplemented (p<0.05) animals had significantly elevated MPO activity in the LS limb compared to SS. However, MPO activity in LS limbs of both the ALLO group (p<0.01) and the DFO group (p<0.05) was significantly lower than the LS limb of control animals. There was no increase in MPO activity in CYCLO supplemented animals (Figure 6). Wet/Dry Ratio The control (p<0.01), ALLO (p<0.05), and DFO (p<0.05) groups showed a significant increase in muscle wet/dry ratio following contractile claudication, in the LS limb compared to SS (Figure 7). This increase was attenuated in the LS limbs of CYCLO animals. Lactate Dehydrogenase (LDH) Activity The LS limbs of control animals showed a significant decrease (p<0.05) in LDH activity. This reflects damage to the muscle cell membrane since the enzyme may leak from its cytosolic residence into the vasculature, thereby decreasing enzyme activity within the muscle. The decrease in LDH activity was attenuated in the ALLO, CYCLO, and DFO groups (Figure 8).

PAGE 37

28 Table 3. Body weight, wet muscle weight, and protein concentration from all groups. Values are expressed as mean SEM. a indicates significantly different (p<0.05) from the treatment pre-injection body weight. b indicates significantly different (p<0.05) from CYCLO SS, and c indicates significantly different (p<0.05) from CYCLO LS. (SS = sham-stimulated; LS = ligated-stimulated). Treatment Pre-injection body weight (grams) Body weight at time of sacrifice (grams) Group Muscle weight (grams) Muscle weight/body weight ratio Total protein concentration (mg/gram wet weight) SS 2.05 0.03 5.84 0.12 139.6 3.26 CON 356.3 6.46 350.5 6.840 LS 2.11 0.04 b c 6.04 0.15 140.1 4.98 SS 1.97 0.04 6.07 0.09 136.5 6.04 ALLO 349.2 5.15 324.2 8.432 a LS 2.04 0.07 6.28 0.15 142.0 5.95 SS 1.84 0.05 5.75 0.16 128.1 1.96 CYCLO 365.0 2.88 319.2 6.395 a LS 1.82 0.06 5.71 0.21 132.4 4.05 SS 2.12 0.06 b c 6.09 0.11 131.2 3.93 DFO 370.3 5.91 357.3 7.126 LS 2.26 0.05 b c 6.31 0.14 130.0 2.99 \000\000\000\000\000 )TjETEMC/P <>BDCQ 0 0.502 0 scnBT/T3_2 1 Tf1.92 0 0 -11.52 268.7999 334.08 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8799 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7999 322.56 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8799 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7999 311.04 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8799 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7999 299.52 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8799 288 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7998 288 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8798 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7998 276.48 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8798 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7998 264.96 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8798 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7997 253.44 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8797 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7997 241.92 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8797 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7997 230.4 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8797 218.88 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7997 218.88 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8796 207.36 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 268.7996 207.36 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q267.9 337.5 17.88 -143.4 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 266.8796 195.84 Tm(\000\000\000\000\000 \000\000\000\000\000 )TjETEMC/P <>BDCQ 0 0.502 0 scnBT/T3_2 1 Tf1.92 0 0 -11.52 403.2 241.92 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q402.12 248.58 17.88 -54.48 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 401.2799 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 403.1999 230.4 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q402.12 248.58 17.88 -54.48 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 401.2799 218.88 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 403.1999 218.88 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q402.12 248.58 17.88 -54.48 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 401.2799 207.36 Tm()TjETEMC/P <>BDCQ BT/T3_2 1 Tf1.92 0 0 -11.52 403.1999 207.36 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q402.12 248.58 17.88 -54.48 reW* nBT/T3_2 1 Tf1.92 0 0 -11.52 401.2799 195.84 Tm(\000\000\000\000\000 \000\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_3 1 Tf1.92 0 0 -11.52 241.92 334.08 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 240 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.92 322.56 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 311.04 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 299.52 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 288 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 288 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 276.48 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 264.96 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 253.44 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9199 241.92 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9999 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9198 230.4 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9998 218.88 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9198 218.88 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9998 207.36 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 241.9198 207.36 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q241.02 340.2 17.88 -146.1 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 239.9998 195.84 Tm(\000\000\000\000\000 \000\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_3 1 Tf1.92 0 0 -11.52 376.3199 241.92 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q375.3 246.12 17.88 -52.02 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 374.3999 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 376.3199 230.4 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q375.3 246.12 17.88 -52.02 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 374.3999 218.88 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 376.3199 218.88 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q375.3 246.12 17.88 -52.02 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 374.3999 207.36 Tm()TjETEMC/P <>BDCQ BT/T3_3 1 Tf1.92 0 0 -11.52 376.3199 207.36 Tm(\000\000 \000\000)TjETEMC/P <>BDCQ q375.3 246.12 17.88 -52.02 reW* nBT/T3_3 1 Tf1.92 0 0 -11.52 374.3998 195.84 Tm(\000\000\000\000\000 Sham-StimLig-Stim 0 25 50 75 100CON ALLO \000\000\000\000 CYCLO \000\000\000\000 DFO LimbsForce (% of initial force)************ Figure 1. Force production from the triceps surae muscle group during the last minute of the 30-minute stimulation period. Values are means SEM. indicates significantly different (p<0.001) from sham-stim limbs undergoing the same treatment.

PAGE 38

29 \000\000\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 679.68 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 247.68 679.6799 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.44 679.6799 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 668.1599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.68 668.1599 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.44 668.1599 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 656.6399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 656.6399 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 656.6399 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 645.1199 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 633.5999 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 622.08 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 610.5599 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 599.0399 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6799 587.5199 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6798 575.9999 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 247.6798 564.4799 Tm(\000)TjETEMC/P <>BDCq247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q247.14 686.28 14.16 -137.76 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 552.9599 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ /CS2 CS 0 0 0.753 SCN0.96 w 10 M 1 j 1 J []0 d 247.14 548.52 m247.14 686.28 l247.14 548.52 l247.14 686.28 m261.3 686.28 l247.14 686.28 l261.3 548.52 m261.3 686.28 l261.3 548.52 l247.14 548.52 m261.3 548.52 l247.14 548.52 lS0.48 w 250.68 691.68 m257.82 691.68 l254.22 691.68 m254.22 686.28 lS1 1 1 scn310.74 655.26 14.1 -106.74 refEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 309.12 656.64 Tm(\000\000\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 309.12 645.12 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 645.1199 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7999 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 633.5999 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7999 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 622.08 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7999 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 610.5599 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7999 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 599.0399 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7998 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 587.5199 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7998 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 575.9999 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7998 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 311.0397 564.4799 Tm(\000)TjETEMC/P <>BDCq310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 316.7997 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q310.74 655.26 14.1 -106.74 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.1197 552.9599 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0.96 w 310.74 548.52 m310.74 655.26 l310.74 548.52 l310.74 655.26 m324.84 655.26 l310.74 655.26 l324.84 548.52 m324.84 655.26 l324.84 548.52 l310.74 548.52 m324.84 548.52 l310.74 548.52 lS0.48 w 314.28 662.82 m321.42 662.82 l317.76 662.82 m317.76 655.26 lS1 1 1 scn374.28 617.76 14.16 -69.24 refEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 372.48 622.08 Tm(\000\000\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 372.48 610.56 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 374.3999 610.5599 Tm(\000)TjETEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 380.1599 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 374.3999 599.0399 Tm(\000)TjETEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 380.1599 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 374.3999 587.5199 Tm(\000)TjETEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 380.1599 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 374.3998 575.9999 Tm(\000)TjETEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 380.1599 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 374.3998 564.4799 Tm(\000)TjETEMC/P <>BDCq374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 380.1598 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q374.28 617.76 14.16 -69.24 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 552.9599 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0.96 w 374.28 548.52 m374.28 617.76 l374.28 548.52 l374.28 617.76 m388.44 617.76 l374.28 617.76 l388.44 548.52 m388.44 617.76 l388.44 548.52 l374.28 548.52 m388.44 548.52 l374.28 548.52 lS0.48 w 377.82 624.36 m384.96 624.36 l381.36 624.36 m381.36 617.76 lS1 1 1 scn437.88 627.36 14.16 -78.84 refEMC/P <>BDCq437.88 627.36 14.16 -78.84 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 437.76 633.6 Tm(\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 439.6799 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.76 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 439.6799 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 439.6799 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 439.6799 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 439.6798 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 439.6798 564.4799 Tm(\000 \000\000)TjETEMC/P <>BDCQ q437.88 627.36 14.16 -78.84 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 552.9599 Tm(\000\000\000\000 CONALLOCYCLODFO 0.0 2.5 5.0 7.5 10.0 12.5 15.0**#Lig-Stim \000\000\000\000 \000\000\000\000 Sham-Stim #+#+Treatmenthydroperoxides(mmol per gram wet wt) Figure 2. Lipid Hydroperoxide levels; values are expressed as mean SEM. indicates significantly different (p<0.001) from the sham-stim group undergoing the same treatment. # indicates significantly different (p<0.05) from CON Lig-Stim. + indicates significantly different (p<0.05) from ALLO Lig-Stim. CON LSALLO LSCYCLO LSDFO LS 0 25 50 75 100 125*TreatmentHNE levels (% of CON SS) Figure 3. Total HNE levels in CON LS, ALLO LS, CYCLO LS, and DFO LS limbs. Values are expressed as a percentage of CON SS levels. indicates significantly different from CON LS.

PAGE 39

30 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 679.6799 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 668.1599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 668.1599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 656.6399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4399 656.6399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 645.1199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4398 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4397 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5197 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4397 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5197 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 253.4397 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q252 686.58 13.26 -130.14 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5197 564.4799 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 668.1599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 656.6399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 656.6399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 645.1199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0399 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0398 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9597 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0397 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 312.9597 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q311.64 675.48 13.26 -119.04 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.0397 564.4799 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 645.1199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.28 645.54 13.26 -89.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 564.4799 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 431.9999 645.1199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.08 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9999 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0799 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9999 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0799 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9998 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9998 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9998 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 431.9998 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q430.86 654.24 13.32 -97.8 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 564.4799 Tm(\000\000\000\000 CONALLOCYCLODFO 0.0 0.5 1.0 1.5 2.0 2.5 3.0**#Lig-Stim \000\000\000\000)TjETEMC/P <>BDCQ q392.28 697.38 16.56 -7.92 reW* n0 0 0.753 scnBT/T3_1 1 Tf0 Tc 1.92 0 0 -11.52 391.68 691.2 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0 0 0.753 SCN0 j 0 J 392.52 697.14 16.08 -7.44 reSEMC/P <>BDC0 0 0 scnBT/TT3 1 Tf-0.0198 Tc 10.92 0 0 10.92 410.52 702.4099 Tm(Sham-Stim Treatmentcarbonyls(nmol/mg protein) Figure 4. Protein carbonyls; values are expressed as mean SEM. indicates significantly different (p<0.01) from the sham-stim group undergoing the same treatment. # indicates significantly different (p<0.05) from CON Lig-Stim. \000\000\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 368.64 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 368.64 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.52 368.64 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 357.12 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 357.12 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.52 357.12 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 345.6 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 345.6 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 334.08 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 334.08 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 322.56 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 322.56 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 311.04 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 311.04 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 299.52 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 299.52 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 288 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 288 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 276.48 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 276.48 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 264.96 Tm(\000)TjETEMC/P <>BDCq245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 264.96 Tm(\000\000)TjETEMC/P <>BDCQ q245.34 372 14.04 -127.56 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 253.44 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0 0 0.753 SCN0.48 w 1 j 1 J 245.34 244.44 m245.34 372 l245.34 244.44 l245.34 372 m259.38 372 l245.34 372 l259.38 244.44 m259.38 372 l259.38 244.44 l245.34 244.44 m259.38 244.44 l245.34 244.44 l248.88 374.82 m255.96 374.82 l252.36 374.82 m252.36 372 lS1 1 1 scn308.34 348.42 14.04 -103.98 refEMC/P <>BDCq308.34 348.42 14.04 -103.98 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 307.2 357.12 Tm(\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 345.6 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 334.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 322.56 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 311.04 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 299.52 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 288 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 276.48 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 264.96 Tm(\000 \000\000)TjETEMC/P <>BDCQ q308.34 348.42 14.04 -103.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1997 253.44 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 357.12 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 345.6 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 334.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 322.56 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 311.04 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 299.52 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 288 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4797 276.48 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5597 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4797 264.96 Tm(\000 \000\000)TjETEMC/P <>BDCQ q371.4 367.8 13.98 -123.36 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5597 253.44 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 435.84 357.12 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9199 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 345.6 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9199 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 334.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9199 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 322.56 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9199 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 311.04 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9198 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 299.52 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9198 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 288 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9197 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 276.48 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9197 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 264.96 Tm(\000 \000\000)TjETEMC/P <>BDCQ q434.4 362.46 13.98 -118.02 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 433.9197 253.44 Tm(\000\000\000\000 CONALLOCYCLODFO 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Sham-Stim Lig-Stim \000\000\000\000)TjETEMC/P <>BDCQ q393.48 394.08 14.04 -6.66 reW* n0 0 0.753 scnBT/T3_1 1 Tf0 Tc 1.92 0 0 -11.52 391.68 391.68 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0 0 0.753 SCN393.72 393.84 13.56 -6.18 reSEMC/P <>BDC0 0 0 scnBT/TT3 1 Tf0 Tc 11.94 0 0 11.94 249.84 375.9555 Tm(***#TreatmentXanthine Oxidase Activity(mU/g wet weight) Figure 5. Xanthine oxidase activity; values are expressed as mean SEM. indicates significantly different (p<0.001) from the sham-stim group undergoing the same treatmnt. # indicates significantly different (p<0.001) from CON Lig-Stim.

PAGE 40

31 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 668.1599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 656.6399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 656.6399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 645.1199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 564.4799 Tm(\000 \000\000)TjETEMC/P <>BDCQ q243.9 670.68 14.28 -122.94 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 552.9599 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1199 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1999 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 309.1198 564.4799 Tm(\000 \000\000)TjETEMC/P <>BDCQ q307.98 637.02 14.22 -89.28 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 307.1998 552.9599 Tm(\000\000\000\000 \000\000\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 370.56 610.56 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 610.5599 Tm(\000)TjETEMC/P <>BDCq372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 378.2399 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 599.0399 Tm(\000)TjETEMC/P <>BDCq372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 378.2399 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 587.5199 Tm(\000)TjETEMC/P <>BDCq372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 378.2399 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4799 575.9999 Tm(\000)TjETEMC/P <>BDCq372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 378.2399 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5599 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 372.4798 564.4799 Tm(\000)TjETEMC/P <>BDCq372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 378.2398 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q372.06 621.72 14.22 -73.98 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 370.5598 552.9599 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 372.06 547.74 m372.06 621.72 l372.06 547.74 l372.06 621.72 m386.28 621.72 l372.06 621.72 l386.28 547.74 m386.28 621.72 l386.28 547.74 l372.06 547.74 m386.28 547.74 l372.06 547.74 l375.6 624 m382.8 624 l379.14 624 m379.14 621.72 lS1 1 1 scn436.08 637.92 14.28 -90.18 refEMC/P <>BDCq436.08 637.92 14.28 -90.18 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 435.84 645.12 Tm(\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 633.5999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 622.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 610.5599 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7599 599.0399 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 587.5199 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 575.9999 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 437.7598 564.4799 Tm(\000 \000\000)TjETEMC/P <>BDCQ q436.08 637.92 14.28 -90.18 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 552.9599 Tm(\000\000\000\000 CONALLOCYCLODFO 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2***#Lig-Stim \000\000\000\000 \000\000\000\000 Sham-Stim #TreatmentMyeloperoxidase activity(U/g wet weight) Figure 6. Myeloperoxidase activity; values are expressed as mean SEM. indicates significantly different (p<0.05) from the sham-stim group undergoing the same treatment. # indicates significantly different (p<0.05) from CON Lig-Stim. \000\000\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 236.16 357.12 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 238.08 357.12 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 357.12 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.16 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.08 345.6 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 345.6 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.16 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 334.08 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 334.08 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 322.56 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 322.56 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 311.04 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 311.04 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 299.52 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 299.52 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 288 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 288 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0799 276.48 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 276.48 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1599 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0798 264.96 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 264.96 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1598 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0798 253.44 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 253.44 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1598 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0798 241.92 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 241.92 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1598 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 238.0798 230.4 Tm(\000)TjETEMC/P <>BDCq237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 230.4 Tm(\000\000)TjETEMC/P <>BDCQ q237.6 360.78 14.22 -149.7 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 236.1598 218.88 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0 0 0.753 SCN1 j 1 J 237.6 211.08 m237.6 360.78 l237.6 211.08 l237.6 360.78 m251.82 360.78 l237.6 360.78 l251.82 211.08 m251.82 360.78 l251.82 211.08 l237.6 211.08 m251.82 211.08 l237.6 211.08 l241.14 362.76 m248.34 362.76 l244.74 362.76 m244.74 360.78 lS1 1 1 scn301.62 360 14.28 -148.92 refEMC/P <>BDCq301.62 360 14.28 -148.92 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 301.44 368.64 Tm(\000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 303.36 357.12 Tm(\000 )TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 309.12 357.12 Tm()TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 311.04 357.12 Tm()TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.96 357.12 Tm()TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 314.88 357.12 Tm()TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.44 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3599 345.6 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4399 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3599 334.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4399 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3599 322.56 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4399 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3598 311.04 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4398 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3598 299.52 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4398 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3598 288 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4398 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3597 276.48 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4397 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3597 264.96 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4397 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3597 253.44 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4397 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3596 241.92 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4396 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 303.3596 230.4 Tm(\000 \000\000)TjETEMC/P <>BDCQ q301.62 360 14.28 -148.92 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 301.4396 218.88 Tm(\000\000\000\000 \000\000\000\000 )TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 366.7199 345.6 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7999 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7199 334.08 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7999 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7199 322.56 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7999 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7198 311.04 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7998 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7198 299.52 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7998 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7198 288 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7998 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7198 276.48 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7998 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7197 264.96 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7998 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7197 253.44 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7997 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7197 241.92 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7997 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 366.7197 230.4 Tm(\000 \000\000)TjETEMC/P <>BDCQ q365.7 355.68 14.22 -144.6 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 364.7997 218.88 Tm(\000\000\000\000 \000\000\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 428.16 357.12 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 430.08 357.12 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.84 357.12 Tm()TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 437.76 357.12 Tm()TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 439.68 357.12 Tm()TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 441.6 357.12 Tm()TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 443.52 357.12 Tm()TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.16 345.6 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0799 345.6 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 345.6 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1599 334.08 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0799 334.08 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8399 334.08 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1599 322.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0799 322.56 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 322.56 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1598 311.04 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 311.04 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 311.04 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1598 299.52 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 299.52 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 299.52 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1598 288 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0798 288 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8398 288 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1598 276.48 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0797 276.48 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 276.48 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1597 264.96 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0797 264.96 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 264.96 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1597 253.44 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0797 253.44 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 253.44 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1597 241.92 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0797 241.92 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8397 241.92 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1597 230.4 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 430.0796 230.4 Tm(\000)TjETEMC/P <>BDCq429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 435.8396 230.4 Tm(\000\000)TjETEMC/P <>BDCQ q429.72 359.58 14.28 -148.5 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 428.1596 218.88 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 429.72 211.08 m429.72 359.58 l429.72 211.08 l429.72 359.58 m444 359.58 l429.72 359.58 l444 211.08 m444 359.58 l444 211.08 l429.72 211.08 m444 211.08 l429.72 211.08 l433.32 360.66 m440.52 360.66 l436.86 360.66 m436.86 359.58 lS0.753 0 0 scn216.24 211.08 14.22 142.26 ref0.753 0 0 SCN216.24 211.08 m216.24 353.34 l216.24 211.08 l216.24 353.34 m230.46 353.34 l216.24 353.34 l230.46 211.08 m230.46 353.34 l230.46 211.08 l216.24 211.08 m230.46 211.08 l216.24 211.08 l219.78 354.66 m226.98 354.66 l223.38 354.66 m223.38 353.34 lS280.32 211.08 14.22 142.38 ref280.32 211.08 m280.32 353.46 l280.32 211.08 l280.32 353.46 m294.54 353.46 l280.32 353.46 l294.54 211.08 m294.54 353.46 l294.54 211.08 l280.32 211.08 m294.54 211.08 l280.32 211.08 l283.86 355.68 m291.06 355.68 l287.4 355.68 m287.4 353.46 lS344.34 211.08 14.22 143.16 ref344.34 211.08 m344.34 354.24 l344.34 211.08 l344.34 354.24 m358.56 354.24 l344.34 354.24 l358.56 211.08 m358.56 354.24 l358.56 211.08 l344.34 211.08 m358.56 211.08 l344.34 211.08 l347.88 355.92 m355.08 355.92 l351.48 355.92 m351.48 354.24 lS408.42 211.08 14.22 142.5 ref408.42 211.08 m408.42 353.58 l408.42 211.08 l408.42 353.58 m422.64 353.58 l408.42 353.58 l422.64 211.08 m422.64 353.58 l422.64 211.08 l408.42 211.08 m422.64 211.08 l408.42 211.08 l411.96 355.08 m419.16 355.08 l415.5 355.08 m415.5 353.58 lS0 0 0 SCN0.96 w 209.16 211.08 m451.44 211.08 lSEMC/P <>BDC0 0 0 scnBT/TT3 1 Tf9.36 0 0 9.36 223.68 199.3799 Tm(CONALLOCYCLODFO 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Lig-Stim \000\000\000\000 \000\000\000\000 Sham-Stim ***TreatmentMuscle Wet Weight/Dry Weight Figure 7. Muscle Wet/dry ratio; values are expressed as mean SEM. indicates significantly different (p<0.05) from the sham-stim group undergoing the same treatment.

PAGE 41

32 \000\000\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 645.12 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 245.76 645.1199 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.52 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 633.5999 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.52 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.84 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 622.08 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 610.5599 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 599.0399 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 587.5199 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 575.9999 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 564.4799 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 552.9599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7599 552.9599 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5199 552.9599 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8399 541.4399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 245.7598 541.4399 Tm(\000)TjETEMC/P <>BDCq245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 251.5198 541.4399 Tm(\000\000)TjETEMC/P <>BDCQ q245.46 648.96 15.24 -122.1 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 243.8398 529.9199 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ /CS2 CS 0 0 0.753 SCN0.54 w 10 M 1 j 1 J []0 d 245.46 526.86 m245.46 648.96 l245.46 526.86 l245.46 648.96 m260.7 648.96 l245.46 648.96 l260.7 526.86 m260.7 648.96 l260.7 526.86 l245.46 526.86 m260.7 526.86 l245.46 526.86 l249.24 656.34 m256.98 656.34 l253.08 656.34 m253.08 648.96 lS1 1 1 scn313.92 663.48 15.18 -136.62 refEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 312.96 668.16 Tm(\000\000\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 312.96 656.64 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 314.8799 656.6399 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.64 656.6399 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.96 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8799 645.1199 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6399 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8799 633.5999 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6399 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8799 622.08 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6399 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9599 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8798 610.5599 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6398 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8798 599.0399 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6398 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8798 587.5199 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6398 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9598 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8797 575.9999 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6397 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9597 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8797 564.4799 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6397 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9597 552.9599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8797 552.9599 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6397 552.9599 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9597 541.4399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 314.8796 541.4399 Tm(\000)TjETEMC/P <>BDCq313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 320.6396 541.4399 Tm(\000\000)TjETEMC/P <>BDCQ q313.92 663.48 15.18 -136.62 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 312.9596 529.9199 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 313.92 526.86 m313.92 663.48 l313.92 526.86 l313.92 663.48 m329.1 663.48 l313.92 663.48 l329.1 526.86 m329.1 663.48 l329.1 526.86 l313.92 526.86 m329.1 526.86 l313.92 526.86 l317.7 669.66 m325.38 669.66 l321.48 669.66 m321.48 663.48 lS1 1 1 scn382.32 672.24 15.18 -145.38 refEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 382.08 679.68 Tm(\000\000\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 382.08 668.16 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 383.9999 668.1599 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7599 668.1599 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0799 656.6399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9999 656.6399 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7599 656.6399 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0799 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9999 645.1199 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7599 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0799 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9998 633.5999 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7599 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0799 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9998 622.08 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7598 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0798 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9998 610.5599 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7598 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0798 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9998 599.0399 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7598 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0798 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9998 587.5199 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7597 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0797 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9997 575.9999 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7597 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0797 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9997 564.4799 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7597 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0797 552.9599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9996 552.9599 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7596 552.9599 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0796 541.4399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 383.9996 541.4399 Tm(\000)TjETEMC/P <>BDCq382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 389.7596 541.4399 Tm(\000\000)TjETEMC/P <>BDCQ q382.32 672.24 15.18 -145.38 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 382.0796 529.9199 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 382.32 526.86 m382.32 672.24 l382.32 526.86 l382.32 672.24 m397.5 672.24 l382.32 672.24 l397.5 526.86 m397.5 672.24 l397.5 526.86 l382.32 526.86 m397.5 526.86 l382.32 526.86 l386.1 677.1 m393.78 677.1 l389.94 677.1 m389.94 672.24 lS1 1 1 scn450.72 659.76 15.24 -132.9 refEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 449.28 668.16 Tm(\000\000\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* n0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 449.28 656.64 Tm()TjETEMC/P <>BDCQ 0 0 0.753 scnBT/T3_1 1 Tf1.92 0 0 -11.52 451.2 656.6399 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.96 656.6399 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.28 645.1199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1999 645.1199 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9599 645.1199 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2799 633.5999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1999 633.5999 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9599 633.5999 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2799 622.0799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1999 622.08 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9598 622.08 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2798 610.56 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1998 610.5599 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9598 610.5599 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2798 599.0399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1998 599.0399 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9598 599.0399 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2798 587.5199 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1998 587.5199 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9598 587.5199 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2798 575.9999 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1997 575.9999 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9597 575.9999 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2797 564.4799 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1997 564.4799 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9597 564.4799 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2797 552.9599 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1997 552.9599 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9597 552.9599 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2797 541.4399 Tm()TjETEMC/P <>BDCQ BT/T3_1 1 Tf1.92 0 0 -11.52 451.1996 541.4399 Tm(\000)TjETEMC/P <>BDCq450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 456.9597 541.4399 Tm(\000\000)TjETEMC/P <>BDCQ q450.72 659.76 15.24 -132.9 reW* nBT/T3_1 1 Tf1.92 0 0 -11.52 449.2797 529.9199 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 450.72 526.86 m450.72 659.76 l450.72 526.86 l450.72 659.76 m465.96 659.76 l450.72 659.76 l465.96 526.86 m465.96 659.76 l465.96 526.86 l450.72 526.86 m465.96 526.86 l450.72 526.86 l454.56 666.12 m462.24 666.12 l458.34 666.12 m458.34 659.76 lS0.753 0 0 scn222.66 526.86 15.18 144.54 ref0.753 0 0 SCN222.66 526.86 m222.66 671.4 l222.66 526.86 l222.66 671.4 m237.84 671.4 l222.66 671.4 l237.84 526.86 m237.84 671.4 l237.84 526.86 l222.66 526.86 m237.84 526.86 l222.66 526.86 l226.44 678.96 m234.12 678.96 l230.28 678.96 m230.28 671.4 lS291.06 526.86 15.24 148.14 ref291.06 526.86 m291.06 675 l291.06 526.86 l291.06 675 m306.3 675 l291.06 675 l306.3 526.86 m306.3 675 l306.3 526.86 l291.06 526.86 m306.3 526.86 l291.06 526.86 l294.9 679.62 m302.58 679.62 l298.68 679.62 m298.68 675 lS359.52 526.86 15.18 149.28 ref359.52 526.86 m359.52 676.14 l359.52 526.86 l359.52 676.14 m374.7 676.14 l359.52 676.14 l374.7 526.86 m374.7 676.14 l374.7 526.86 l359.52 526.86 m374.7 526.86 l359.52 526.86 l363.3 681 m370.98 681 l367.14 681 m367.14 676.14 lS427.92 526.86 15.24 148.38 ref427.92 526.86 m427.92 675.24 l427.92 526.86 l427.92 675.24 m443.16 675.24 l427.92 675.24 l443.16 526.86 m443.16 675.24 l443.16 526.86 l427.92 526.86 m443.16 526.86 l427.92 526.86 l431.76 680.7 m439.44 680.7 l435.54 680.7 m435.54 675.24 lS0 0 0 SCN215.04 526.86 m473.88 526.86 lSEMC/P <>BDC0 0 0 scnBT/TT3 1 Tf10.02 0 0 10.02 230.52 514.4406 Tm(CONALLOCYCLODFO 0 100 200 300 400 500 600 700*Lig-Stim \000\000\000\000)TjETEMC/P <>BDCQ q413.94 703.38 15.18 -7.26 reW* n0 0 0.753 scnBT/T3_1 1 Tf0 Tc 1.92 0 0 -11.52 412.8 702.72 Tm(\000\000\000\000)TjETEMC/InlineShape <>BDCQ 0 0 0.753 SCN0 j 0 J 414.18 703.14 14.7 -6.78 reSEMC/P <>BDC0 0 0 scnBT/TT3 1 Tf-0.0213 Tc 10.02 0 0 10.02 430.68 708.6006 Tm(Sham-Stim TreatmentLactate Dehydrogenase Activity(M/g/min) Figure 8. Lactate dehydrogenase activity; values are expressed as mean SEM. indicates significantly different (p<0.05) from the sham-stim group undergoing the same treatment.

PAGE 42

CHAPTER 4 DISCUSSION Overview of Experimental Findings This is the first study to examine the pathways of oxidative damage and edema after an acute bout of contractile claudication. The major findings of this study are twofold: inhibiting the increase in xanthine oxidase activity and chelation of iron significantly attenuates lipid peroxidation, but not protein oxidation or edema, after contractile claudication. Secondly, induction of neutropenia attenuated lipid peroxidation, protein oxidation, and edema after contractile claudication. Together, these findings indicate that activated neutrophils are the major source of oxidative damage after contractile claudication, and that this damage contributes to edema. In addition, the data suggest that iron plays a pivotal role in contributing to oxidative damage, presumably by being mobilized during contractile claudication from bound sources, thereby becoming available to partake in redox reactions. Contractile Claudication Consistent with our previous findings, an acute bout of contractile claudication causes a significant increase in lipid peroxidation, protein oxidation and edema. This is associated with an increase in xanthine oxidase activity, increased neutrophil infiltration, and a loss of lactate dehydrogenase activity. Since LDH is ordinarily confined to the muscle cell, the loss of LDH activity suggests the muscle cell membrane may be modified by oxidants, making it more permeable and contributing to loss of LDH. 33

PAGE 43

34 Xanthine Oxidase Inhibition Allopurinol is known to be an inhibitor of xanthine oxidase activity. However, we quantified the activity of the enzyme to ensure the dose administered was sufficient to inhibit its activity in this study. As expected, allopurinol supplemented animals showed no increase in xanthine oxidase activity after contractile claudication, whereas the control, cyclophosphamide and deferoxamine groups each showed significant increases. This confirms that the allopurinol served its purpose in inhibiting xanthine oxidase activity. In addition, DFO has been shown to attenuate the increase in xanthine oxidase activity in cultured endothelial cells exposed to radicals. Although the mechanism of this is unknown, speculation suggests that xanthine oxidase activity would be impaired by deferoxamine if it bound the iron cofactor at the enzymes active site. Our findings, however, suggest that DFO has no effect on attenuating xanthine oxidase activity after contractile claudication. Therefore, this potential mechanism of protection may be excluded from the protective effects DFO had in this study. Despite the lack of increase in xanthine oxidase activity in ALLO animals there was still significant lipid peroxidation, protein oxidation, and edema in these animals. However, the lipid peroxidation was significantly less than control animals, showing inhibition of xanthine oxidase activity provided some protection, and suggesting xanthine oxidase as a source of radicals. In addition, inhibition of xanthine oxidase activity attenuated the loss of LDH activity seen in control animals. With leakage of LDH reflective of a disruption to muscle membrane permeability, these data suggest xanthine oxidase-derived oxidants may target primarily membrane lipids. Since oxidants are non-discriminate in their attack, a logical explanation is that oxidants derived from xanthine oxidase are produced within the lipid membrane, where close proximity dictates their

PAGE 44

35 modification. This is an attractive hypothesis since immunolocalization techniques demonstrate xanthine oxidase is concentrated in capillary endothelial cells [63], and histochemical localization studies indicate the enzyme is also localized in the sarcolemma [63]. Both locations are abundant in lipids and could help explain the loss of LDH and the increase in lipid hydroperoxides. Since no other studies have measured xanthine oxidase activity, or the effects of the enzymes inhibition after claudication, parallels can only be drawn from prolonged I-R studies. In one such study [44], the results were very similar to ours in that I-R caused an increase in lipid peroxidation and an associated increase in xanthine oxidase activity. With administration of allopurinol lipid peroxidation was significantly attenuated, confirming that xanthine oxidase-derived radicals can cause peroxidation of cell membranes. If xanthine oxidase-derived radicals cause lipid peroxidation to endothelial cell membranes, membrane permeability might be altered, causing edema. Since CON animals exhibit increased xanthine oxidase, oxidative stress, and edema, this seems to be an attractive possibility. Indeed, this association has been has been found by others following prolonged I-R [51]. However, there was no attenuation of the increase in wet/dry ratio in the ALLO group, suggesting other factors contribute to the edema seen after an acute bout of contractile claudication. In addition, it was hypothesized that inhibiting xanthine oxidase activity would attenuate neutrophil infiltration, and indeed although MPO activity was significantly elevated in the ALLO group, it was 33% lower compared to control animals. Therefore, it can be concluded that xanthine oxidase-derived oxidants are important in the

PAGE 45

36 accumulation of neutrophils after contractile claudication. This chemotaxic potential of oxidants from xanthine oxidase is in agreement with Seekamp [29], who used allopurinol to inhibit xanthine oxidase activity and observed a significant reduction in MPO content following prolonged I-R. The chemotactic potential of xanthine oxidase-derived radicals has several possible explanations. When neutrophils infiltrate tissue from the vasculature, they must first be attracted to, and bind to, the endothelium. Since xanthine oxidase is localized to the endothelium, this places the enzyme at the scene of neutrophil adhesion. Indeed, isolated endothelial cells or isolated vessels exposed to hydrogen peroxide show increased sensitivity for neutrophils [64,65]. There are several potential mechanisms to explain this. One is that oxidants stimulate endothelial cells to synthesize and/or release chemoattractants, such as platelet activating factor and leukotriene B 4 [66] Another potential mechanism is that oxidants may directly induce the expression of endothelial cell adhesion molecules. Indeed endothelial cells exposed to hydrogen peroxide have been shown to induce P-selectin expression [67], and neutrophils incubated in hydrogen peroxide increase their expression of CD11 and CD18 [68]. Neutropenia The use of cyclophosphamide to deplete neutrophils was based on several studies using this agent to cause neutropenia both at baseline and during ischemia-reperfusion injury. Mackie [69] showed the neutrophil count of cyclophosphamide-injected animals was <10% of control animals after 4 days; Lee [70] measured circulating neutrophils at <1% of control, 5 days post-cyclophosphamide injection and; Bertuglia et al [10] showed that cyclophosphamide injected animals had leukocyte counts that were 7% of control animal levels, after 30 minutes of ischemia and 30 minutes reperfusion. This clearly shows the capacity for cyclophosphamide to induce neutropenia.

PAGE 46

37 The CYCLO group in this study showed no significant increase in MPO activity after contractile claudication, confirming attenuation of neutrophil infiltration into the tissue. This lack of neutrophil infiltration attenuated lipid peroxidation, protein oxidation, edema, and muscle membrane damage, clearly demonstrating the ability of neutrophils to cause oxidative damage and edema. Although no previous studies have measured tissue neutrophil levels after claudication, or depleted neutrophils prior to claudication, our findings are in agreement with others showing attenuation of lipid peroxidation and edema [2]; and reduction of muscle permeability [29] with neutrophil depletion prior to prolonged I-R. The attenuation of edema with neutropenia, observed here, has previously been shown after prolonged I-R [10]. It appears that as neutrophils migrate through the vascular endothelium into the muscle they may release lysosomal enzymes and/or oxidizing species. These molecules can damage the endothelium and alter membrane permeability, thereby resulting in edema. A second possibility is that the diapedesis process itself may widen endothelial gap junctions, thereby contributing to edema. Although neither of these potential mechanisms were addressed in this study, it is clear that neutrophils cause significant edema after contractile claudication. Since LDH is confined to the muscle cell and its loss reflects an alteration in membrane permeability, the attenuated loss of this enzymes activity with neutropenia implicates neutrophils. This is the first evidence to show the muscle cell membrane is, at least in part, being oxidized by neutrophil-derived oxidants after contractile claudication, thereby making the membrane more permeable.

PAGE 47

38 Iron Chelation Deferoxamine is a powerful iron chelator that is used broadly in preventing iron-dependent pro-oxidant reactions. It does this by preferentially removing iron from low molecular weight components, including amino acids, organic acids or carbohydrates [71]. Since deferoxamine has been shown to slowly penetrate the plasma membrane [46], thereby entering cells, its protection may be displayed at both the intracellular and extracellular level. In this study, DFO animals showed significant attenuation of lipid peroxidation, neutrophil infiltration, and an attenuation of the loss in LDH activity. However, there was still significant edema in these animals. These findings show irons function in redox reactions, after contractile claudication, but suggest iron has no role in causing edema. Since superoxide and hydrogen peroxide are poorly reactive in an aqueous environment, it is generally thought the more potent, more damaging, hydroxyl radical is responsible for the majority of oxidant-induced cellular damage after skeletal muscle ischemia [72]. It is even suggested that hydroxyl radical formation is critical to cellular injury [73]. With the presence of a suitable transition metal catalyst, such as iron, necessary for the Fenton reaction to proceed at a significant rate, iron is clearly very important to the redox balance. Our findings suggest iron is paramount in causing oxidative damage after contractile claudication. This conclusion is based on the fact that DFO animals have a significant increase in xanthine oxidase activity, and significant neutrophil infiltration, therefore large amounts of oxidants are still being produced. However, despite this oxidant production, significant protection is afforded with chelation of iron. Therefore, the findings of this study are in agreement with others [7], showing that when iron is

PAGE 48

39 chelated, and disruption to the Fenton reaction is presumed, significant attenuation of oxidative damage occurs. Since deferoxamine is thought to enter cells, its protective effects could conceivably occur on either side of the plasma membrane. This issue has been addressed by several investigators with conflicting results. However, those studies conducted in skeletal muscle tissue after I-R appear to lean on the side of extracellular protection. Smith et al [51] administered deferoxamine and apotransferrin independently to address this issue in ischemia-reperfused skeletal muscle. Since apotransferrin cannot cross the cell membrane its protective effects can only be exhibited in the extracellular space. With both compounds exhibiting the same degree of protection, a suggested conclusion was that the iron-catalyzed Haber-Weiss reaction occurs in the extracellular space. In another study of ischemia-reperfused skeletal muscle, Fantini et al [3] administered DFO and DFO conjugated to pentastarch independently to animals. This conjugation alters the physical properties of DFO, so that it cannot cross the cell membrane, while retaining its capacity to chelate. Since the two compounds exerted similar protective effects in inhibiting lipid peroxidation, an extracellular site of action was strongly suggested. Although we are unable to determine from our study which side of the cell membrane DFO is exerting its protective effects, it is certainly a strong possibility that, similar to those studies just discussed, the protection is in the extracellular compartment. This is based on our findings of neutrophils being the predominant source of oxidants after contractile claudication. This would lead to extracellular oxidant production, and potentially extracellular hydroxyl radical formation. Therefore chelation of extracellular

PAGE 49

40 iron would be beneficial in this situation. However, this speculation warrants further investigation. Since we were unable to measure free iron in tissue, the protective effects of DFO could conceivably be explained by alternatives to iron chelation. DFO has been reported to react with O 2 however the rate constant for this reaction at physiological pH is about 10 2 M -1 s -1 [33], which is approximately eight orders of magnitude less than the overall rate of non-enzymatic dismutation of O 2 [74], and therefore this protective possibility is unlikely. A more likely artifact is DFOs scavenging of OH, which proceeds with a rate constant of approximately 10 10 M -1 s -1 [33]. However, using a dose of DFO identical to ours, plasma concentrations appear to stabilize at less than 20 M. This concentration is presumably the same in other extracellular fluids, and is suggested to be too low for significant scavenging of OH or O 2 [75]. In other studies using a similar dose of DFO, and controls with ferrioxamine (which reacts with OH with the same rate constant as DFO), no protection was afforded, reinforcing the conclusion that the protective effects cannot be due to radical scavenging [76]. In any case, as mentioned before, a suitable metal catalyst is necessary for formation of OH, and iron is the best candidate for this role in vivo. Since DFO is accepted to be a very powerful iron chelator, it is likely OH formation is greatly reduced, thereby limiting the very compound for which DFO has the potential to scavenge. 4-Hydroxy-2-Nonenal 4-hydroxy-2-nonenal (HNE) levels were measured as a further marker of oxidative stress, in the LS limbs of all treatment groups, and compared to CON SS levels. Although this aldehyde is formed from the decomposition of lipid hydroperoxides, it may actually

PAGE 50

41 binds to, and modifies, proteins by interacting preferentially with lysine, histidine, serine, and cysteine residues. Since HNE is relatively stable, and can easily diffuse within the cell or escape the cell, it has the potential to interact with many different cellular proteins [77]. This was reflected in our western blot by the appearance of several bands at varying molecular weights. In this study, we summed the net intensity of each band in each lane, in an attempt to quantify total protein-bound HNE levels. Only the CYCLO group showed significant attenuation of total protein-bound HNE levels after contractile claudication. Several studies have demonstrated that increased levels of HNE are potentially very cytotoxic, inhibiting enzymes, protein synthesis, protein degradation, calcium sequestration, and exhibiting chemotaxic potential to neutrophils [78-80]. In addition, HNE can significantly alter the cellular redox balance by rapidly conjugating with the reactive thiol groups of glutathione (GSH). Indeed, GSH is part of the endogenous cellular pathway of HNE metabolism [81]. However, since glutathione is believed to be the primary buffer against reactive oxygen species in skeletal muscle [82], its loss due to HNE conjugation may make the cell more susceptible to oxidative damage. This provides an interesting concept since we have previously shown total GSH levels to be decreased after contractile claudication.

PAGE 51

CHAPTER 5 CONCLUSIONS This is the first study to investigate the pathways of oxidant production after contractile claudication. To establish a cause and effect relationship, we inhibited the major pathways individually. This also provided information on the relative contributions of each pathway. We predicted that both the xanthine oxidase pathway and activated neutrophils are responsible for the oxidative damage and edema seen after contractile claudication. The data supported these expectations, and provided insight into the predominant pathway. Inhibition of xanthine oxidase activity attenuated lipid peroxidation, the loss of LDH activity seen in control animals, and reduced neutrophil infiltration. The conclusion can therefore be made that xanthine oxidase-derived oxidants cause oxidative damage after an acute bout of contractile claudication. In addition, oxidants from this source are chemotactic to neutrophils. Neutropenia reduced neutrophil infiltration by ~82% compared to the ligated-stimulated limb of control animals and had a protective effect on all parameters attenuating lipid peroxidation, protein oxidation, edema and the loss in LDH activity. This clearly shows neutrophils are the predominant source of oxidants, and therefore oxidative damage, after contractile claudication. Although increased neutrophil levels have been documented in the vasculature following exercise claudication in humans, this is the first study to show neutrophil recruitment into skeletal muscle after this condition. This is in agreement with prolonged I-R studies in skeletal muscle, which have firmly 42

PAGE 52

43 established the recruitment of leukocytes into tissue. This causes oxidative damage as the membrane bound NADPH oxidase oxidizes NADPH to NADP + while reducing molecular oxygen to superoxide. Finally, data from the DFO group suggest iron is heavily involved in the oxidative damage seen after contractile claudication. This implies the Fenton reaction causes a large part of the oxidative damage. Indeed, lipid hydroperoxide levels were reduced by ~43% and protein carbonyl content by ~25% in the LS limb of the DFO group compared to the LS limb of the CON group. Since we were unable to actually measure iron levels in this study we cannot definitively conclude that the protection afforded by deferoxamine is solely due to its chelating characteristics. Indeed, deferoxamine has radical scavenging capabilities, which could interfere with the results of this study. However, it is only a weak scavenger of superoxide, and at the dose used in this study the concentration is likely too low for significant scavenging of the hydroxyl radical. Therefore, the probable protective role DFO plays in this study is as an iron chelator. Future research should focus on the infiltration of neutrophils into tissue after exercise claudication, and the signals leading to this infiltration. In addition, it should be determined whether oxidative damage to skeletal muscle after exercise claudication is necessary for, or a hindrance to, the muscular adaptations that occur with this condition.

PAGE 53

LIST OF REFERENCES [1] Pattwell D, McArdle A, Griffiths RD, Jackson MJ. Measurement of free radical production by in vivo microdialysis during ischemia/reperfusion injury to skeletal muscle. Free Radic Biol Med 2001;30:979-85. [2] Hirose J, Yamaga M, Kato T, Ikebe K, Takagi K. Effects of a hydroxyl radical scavenger, EPC-K1, and neutrophil depletion on reperfusion injury in rat skeletal muscle. Acta Orthop Scand 2001;72:404-10. [3] Fantini GA, Yoshioka T. Deferoxamine prevents lipid peroxidation and attenuates reoxygenation injury in postischemic skeletal muscle. Am J Physiol 1993;264:H1953-9. [4] Hickman P, Harrison DK, Hill A, McLaren M, Tamei H, McCollum PT, Belch JJ. Exercise in patients with intermittent claudication results in the generation of oxygen derived free radicals and endothelial damage. Adv Exp Med Biol 1994;361:565-70. [5] Wijnen MH, Coolen SA, Vader HL, Reijenga JC, Huf FA, Roumen RM. Antioxidants reduce oxidative stress in claudicants. J Surg Res 2001;96:183-7. [6] Silvestro A, Scopacasa F, Oliva G, de Cristofaro T, Iuliano L, Brevetti G. Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication. Atherosclerosis 2002;165:277-83. [7] Chiao JJ, Kirschner RE, Fantini GA. Iron delocalization occurs during ischemia and persists on reoxygenation of skeletal muscle. J Lab Clin Med 1994;124:432-8. [8] Sen CK, Packer L, H*anninen O. Handbook of oxidants and antioxidants in exercise. Amsterdam ; Oxford: Elsevier, 2000. [9] Kuwabara Y, Kato T, Sato A, Fujii Y. Prolonged effect of leukocytosis on reperfusion injury of rat intestine: real-time ATP change studied using (31)P MRS. J Surg Res 2000;89:38-42. [10] Bertuglia S, Colantuoni A. Protective effects of leukopenia and tissue plasminogen activator in microvascular ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2000;278:H755-61. [11] Fox SI. Human physiology. Boston: McGraw-Hill, 2002. 44

PAGE 54

45 [12] Bainton D, Sweetnam P, Baker I, Elwood P. Peripheral vascular disease: consequence for survival and association with risk factors in the Speedwell prospective heart disease study. Br Heart J 1994;72:128-32. [13] Fowkes FG, Housley E, Cawood EH, Macintyre CC, Ruckley CV, Prescott RJ. Edinburgh Artery Study: prevalence of asymptomatic and symptomatic peripheral arterial disease in the general population. Int J Epidemiol 1991;20:384-92. [14] Dormandy JA, Murray GD. The fate of the claudicant--a prospective study of 1969 claudicants. Eur J Vasc Surg 1991;5:131-3. [15] Schmieder FA, Comerota AJ. Intermittent claudication: magnitude of the problem, patient evaluation, and therapeutic strategies. Am J Cardiol 2001;87:3D-13D. [16] Pernow B, Saltin B, Wahren J, Cronestrand R, Ekestroom S. Leg blood flow and muscle metabolism in occlusive arterial disease of the leg before and after reconstructive surgery. Clin Sci Mol Med 1975;49:265-75. [17] Tisi PV, Shearman CP. Acute exercise and markers of endothelial injury. Eur J Vasc Endovasc Surg 1998;16:169. [18] Hein S, Scheffold T, Schaper J. Ischemia induces early changes to cytoskeletal and contractile proteins in diseased human myocardium. J Thorac Cardiovasc Surg 1995;110:89-98. [19] Blaisdell FW, Steele M, Allen RE. Management of acute lower extremity arterial ischemia due to embolism and thrombosis. Surgery 1978;84:822-34. [20] Homer-Vanniasinkam S, Gough MJ. Role of lipid mediators in the pathogenesis of skeletal muscle infarction and oedema during reperfusion after ischaemia. Br J Surg 1994;81:1500-3. [21] Kadambi A, Skalak TC. Role of leukocytes and tissue-derived oxidants in short-term skeletal muscle ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2000;278:H435-43. [22] Tozzi-Ciancarelli MG, Penco M, Di Massimo C. Influence of acute exercise on human platelet responsiveness: possible involvement of exercise-induced oxidative stress. Eur J Appl Physiol 2002;86:266-72. [23] Welbourn CR, Goldman G, Paterson IS, Valeri CR, Shepro D, Hechtman HB. Pathophysiology of ischaemia reperfusion injury: central role of the neutrophil. Br J Surg 1991;78:651-5.

PAGE 55

46 [24] Hickman P, McCollum PT, Belch JJ. Neutrophils may contribute to the morbidity and mortality of claudicants. Br J Surg 1994;81:790-8. [25] Edwards AT, Blann AD, Suarez-Mendez VJ, Lardi AM, McCollum CN. Systemic responses in patients with intermittent claudication after treadmill exercise. Br J Surg 1994;81:1738-41. [26] Turton EP, Coughlin PA, Kester RC, Scott DJ. Exercise Training Reduces the Acute Inflammatory ResponseAssociated with Claudication. Eur J Vasc Endovasc Surg 2002;23:309-16. [27] Smith JK, Grisham MB, Granger DN, Korthuis RJ. Free radical defense mechanisms and neutrophil infiltration in postischemic skeletal muscle. Am J Physiol 1989;256:H789-93. [28] Prem JT, Eppinger M, Lemmon G, Miller S, Nolan D, Peoples J. The role of glutamine in skeletal muscle ischemia/reperfusion injury in the rat hind limb model. Am J Surg 1999;178:147-50. [29] Seekamp A, Mulligan MS, Till GO, Ward PA. Requirements for neutrophil products and L-arginine in ischemia-reperfusion injury. Am J Pathol 1993;142:1217-26. [30] Tisi PV, Shearman CP. Biochemical and inflammatory changes in the exercising claudicant. Vasc Med 1998;3:189-98. [31] McCord JM. Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 1987;46:2402-6. [32] Curzio M, Roch-Arveiller M, Negro F, Giroud JP, Esterbaur H, Torrielli MV, Dianzani MU. [Chemotaxis and chemokinesis of rat polymorphonuclear leukocytes in response to 4-hydroxy-2-tetradecenal and 4-hydroxy-2-nonenal.]. Boll Soc Ital Biol Sper 1981;57:2479-85. [33] Halliwell B, Gutteridge JM. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246:501-14. [34] Biemond P, van Eijk HG, Swaak AJ, Koster JF. Iron mobilization from ferritin by superoxide derived from stimulated polymorphonuclear leukocytes. Possible mechanism in inflammation diseases. J Clin Invest 1984;73:1576-9. [35] Mazur A GS, Saha A, and Carleton A. Mechanism of release of ferritin iron in vivo by xanthine oxidase. J. Clin. Invest 1958;37:1809-17.

PAGE 56

47 [36] Bralet J, Schreiber L, Bouvier C. Effect of acidosis and anoxia on iron delocalization from brain homogenates. Biochem Pharmacol 1992;43:979-83. [37] Green CJ, Gower JD, Healing G, Cotterill LA, Fuller BJ, Simpkin S. The importance of iron, calcium and free radicals in reperfusion injury: an overview of studies in ischaemic rabbit kidneys. Free Radic Res Commun 1989;7:255-64. [38] Duran WN, Dillon PK. Effects of ischemia-reperfusion injury on microvascular permeability in skeletal muscle. Microcirc Endothelium Lymphatics 1989;5:223-39. [39] Rubin BB, Romaschin A, Walker PM, Gute DC, Korthuis RJ. Mechanisms of postischemic injury in skeletal muscle: intervention strategies. J Appl Physiol 1996;80:369-87. [40] Dawson R, Jr., Biasetti M, Messina S, Dominy J. The cytoprotective role of taurine in exercise-induced muscle injury. Amino Acids 2002;22:309-24. [41] Jones DA, Jackson MJ, Edwards RH. Release of intracellular enzymes from an isolated mammalian skeletal muscle preparation. Clin Sci (Lond) 1983;65:193-201. [42] Vina J, Gimeno A, Sastre J, Desco C, Asensi M, Pallardo FV, Cuesta A, Ferrero JA, Terada LS, Repine JE. Mechanism of free radical production in exhaustive exercise in humans and rats; role of xanthine oxidase and protection by allopurinol. IUBMB Life 2000;49:539-44. [43] Asami A, Orii M, Shirasugi N, Yamazaki M, Akiyama Y, Kitajima M. The effect of allopurinol on interstitial purine metabolism and tissue damage in skeletal muscle I-R injury. J Cardiovasc Surg (Torino) 1996;37:209-16. [44] McCutchan HJ, Schwappach JR, Enquist EG, Walden DL, Terada LS, Reiss OK, Leff JA, Repine JE. Xanthine oxidase-derived H2O2 contributes to reperfusion injury of ischemic skeletal muscle. Am J Physiol 1990;258:H1415-9. [45] Janeway C. Immunobiology 5 : the immune system in health and disease. New York: Garland Pub., 2001. [46] Keberie H. The biochemistry of desferrioxamine and its relation to iron metabolism. Ann. N Y Acad. Sci. 1964;119:758-68. [47] Rinaldo JE, Gorry M. Protection by deferoxamine from endothelial injury: a possible link with inhibition of intracellular xanthine oxidase. Am J Respir Cell Mol Biol 1990;3:525-33.

PAGE 57

48 [48] Parks DA, Granger DN. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol Scand Suppl 1986;548:87-99. [49] Klebanoff SJ, Waltersdorph AM. Inhibition of peroxidase-catalyzed reactions by deferoxamine. Arch Biochem Biophys 1988;264:600-6. [50] Bobeck-Rutsaert MM, Wiltink WF, Op den Kelder AM, van Euk HG, Leijnse B. The distribution pattern of radioactive ferrioxamine administered intravenously in rats. I. Acta Haematol 1972;48:125-8. [51] Smith JK, Carden DL, Grisham MB, Granger DN, Korthuis RJ. Role of iron in postischemic microvascular injury. Am J Physiol 1989;256:H1472-7. [52] Zimmerman BJ, Parks DA, Grisham MB, Granger DN. Allopurinol does not enhance antioxidant properties of extracellular fluid. Am J Physiol 1988;255:H202-6. [53] Leong JC, Knight KR, Hickey MJ, Morrison WA, Stewart AG. Neutrophil-independent protective effect of r-metHuG-CSF in ischaemia-reperfusion injury in rat skeletal muscle. Int J Exp Pathol 2000;81:41-9. [54] Sundin BM, Hussein MA, Glasofer S, El-Falaky MH, Abdel-Aleem SM, Sachse RE, Klitzman B. The role of allopurinol and deferoxamine in preventing pressure ulcers in pigs. Plast Reconstr Surg 2000;105:1408-21. [55] Challiss RA, Hayes DJ, Petty RF, Radda GK. An investigation of arterial insufficiency in rat hindlimb. A combined 31P-n.m.r. and bloodflow study. Biochem J 1986;236:461-7. [56] Mathien GM, Terjung RL. Muscle blood flow in trained rats with peripheral arterial insufficiency. Am J Physiol 1990;258:H759-65. [57] Watters C. A one-step biuret assay for protein in the presence of detergent. Anal Biochem 1978;88:695-8. [58] Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994;233:357-63. [59] Yan LJ, Traber MG, Kobuchi H, Matsugo S, Tritschler HJ, Packer L. Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch Biochem Biophys 1996;327:330-4. [60] Hermes-Lima M, Willmore WG, Storey KB. Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation. Free Radic Biol Med 1995;19:271-80.

PAGE 58

49 [61] Belcastro AN, Arthur GD, Albisser TA, Raj DA. Heart, liver, and skeletal muscle myeloperoxidase activity during exercise. J Appl Physiol 1996;80:1331-5. [62] Bergmeyer H, Bernt E, Hess B. Methods in Enzymatic Analysis. New York: Academic Press, 1965. [63] Ibrahim B, Stoward PJ. The histochemical localization of xanthine oxidase. Histochem J 1978;10:615-7. [64] Lewis MS, Whatley RE, Cain P, McIntyre TM, Prescott SM, Zimmerman GA. Hydrogen peroxide stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion. J Clin Invest 1988;82:2045-55. [65] Gasic AC, McGuire G, Krater S, Farhood AI, Goldstein MA, Smith CW, Entman ML, Taylor AA. Hydrogen peroxide pretreatment of perfused canine vessels induces ICAM-1 and CD18-dependent neutrophil adherence. Circulation 1991;84:2154-66. [66] Suzuki M, Asako H, Kubes P, Jennings S, Grisham MB, Granger DN. Neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules. Microvasc Res 1991;42:125-38. [67] McEver RP. Selectins: novel receptors that mediate leukocyte adhesion during inflammation. Thromb Haemost 1991;65:223-8. [68] Patel KD, Zimmerman GA, Prescott SM, McEver RP, McIntyre TM. Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils. J Cell Biol 1991;112:749-59. [69] Mackie EJ. Immunosuppressive effects of cyclophosphamide in pigs. Am J Vet Res 1981;42:189-94. [70] Lee C, Kerrigan CL, Picard-Ami LA, Jr. Cyclophosphamide-induced neutropenia: effect on postischemic skin-flap survival. Plast Reconstr Surg 1992;89:1092-7. [71] Ninfali P, Perini MP, Bresolin N, Aluigi G, Cambiaggi C, Ferrali M, Pompella A. Iron release and oxidant damage in human myoblasts by divicine. Life Sci 2000;66:PL85-91. [72] Weiss SJ. Oxygen, ischemia and inflammation. Acta Physiol Scand Suppl 1986;548:9-37. [73] Korthuis RJ, Granger DN, Townsley MI, Taylor AE. The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability. Circ Res 1985;57:599-609.

PAGE 59

50 [74] Halliwell B. Use of desferrioxamine as a 'probe' for iron-dependent formation of hydroxyl radicals. Evidence for a direct reaction between desferal and the superoxide radical. Biochem Pharmacol 1985;34:229-33. [75] Symons MCR, Gutteridge JMC. Free radicals and iron : chemistry, biology, and medicine. Oxford ; New York: Oxford University Press, 1998. [76] Summers MR, Jacobs A, Tudway D, Perera P, Ricketts C. Studies in desferrioxamine and ferrioxamine metabolism in normal and iron-loaded subjects. Br J Haematol 1979;42:547-55. [77] Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 2003;42:318-43. [78] Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991;11:81-128. [79] Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, Uchida K. 4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules. J Biol Chem 1999;274:23787-93. [80] Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem 1997;68:255-64. [81] Spitz DR, Malcolm RR, Roberts RJ. Cytotoxicity and metabolism of 4-hydroxy-2-nonenal and 2-nonenal in H2O2-resistant cell lines. Do aldehydic by-products of lipid peroxidation contribute to oxidative stress? Biochem J 1990;267:453-9. [82] Sirsjo A, Kagedal B, Arstrand K, Lewis DH, Nylander G, Gidlof A. Altered glutathione levels in ischemic and postischemic skeletal muscle: difference between severe and moderate ischemic insult. J Trauma 1996;41:123-8.

PAGE 60

BIOGRAPHICAL SKETCH Andrew R. Judge was born in Northampton, England and raised in the village of Harpole, just outside Northampton. He graduated from Loughborough University, Leicestershire, England in 1996 with a bachelors degree. In 1997 he moved to Lake Charles, Louisiana where he attended McNeese State University. Here he received a masters degree with a specialization in Exercise physiology. Andrew began his Doctor of Philosophy degree in exercise physiology in 1999, at the University of Florida. 51


Permanent Link: http://ufdc.ufl.edu/UFE0000954/00001

Material Information

Title: Pathways of Oxidative Damage to Skeletal Muscle after an Acute Bout of Contractile Claudication
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: UFE0000954:00001

Permanent Link: http://ufdc.ufl.edu/UFE0000954/00001

Material Information

Title: Pathways of Oxidative Damage to Skeletal Muscle after an Acute Bout of Contractile Claudication
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: UFE0000954:00001


This item has the following downloads:


Full Text












PATHWAYS OF OXIDATIVE DAMAGE TO SKELETAL MUSCLE AFTER AN
ACUTE BOUT OF CONTRACTILE CLAUDICATION

















By

ANDREW JUDGE


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


2003

































Copyright 2003

by

Andrew Judge














ACKNOWLEDGMENTS

I would like to thank my mentor and committee chair, Dr. Stephen Dodd, for his

continued support throughout my graduate studies at the University of Florida. His

encouragement and availability to discuss science have been paramount to my

completion of not only this project, but also my doctorate degree. I would also like to

thank my doctoral committee (Drs. Scott Powers, Christiaan Leeuwenburgh, and James

Jessup) for their encouragement and advice throughout this study; as well as my master's

mentor, Dr. Robert Voight, for his belief in my capabilities and encouragement to pursue

this goal.

Most of all, I am forever grateful to my parents for their love, support, and

encouragement through all my many years of school; and to my wife, Sharon (Phaneuf)

Judge, for her love, encouragement and patience.















TABLE OF CONTENTS
page

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

LIST OF TABLES .............. .......... ........................... ...................... .. vi

L IST O F FIG U R E S .... ....... ................................................ .... ..... .. ............. vii

ABSTRAC T ... ................... .................................. ........... .... .. ........ viii

CHAPTER

1 INTRODUCTION AND REVIEW OF LITERATURE ..............................................1

In tro d u ctio n ...................................... .................................. .................. .
R review of L literature .................................................................. .. ................ .
Isch em ia-R ep erfu sion .................................................................. ................ ...... 3
Interm ittent C laudication .............................................................. ............... 4
O xidative Stress .................. ................. ................ ....... .... .......... 5
Sources of Oxidants after Exercise Claudication ...............................................7
R ole of Iron................................................................ ...... .........9
In tera ctio n ...................................... .............................. ................ 10
E dem a .................................... ........................... ................... ........ 11
M uscle Cell M em brane ..................................................... ..... ............... 11
C ou n term easu res ................................................................................. .. ..... 12
A llo p u rin o l ................................................................12
C y clophospham ide ............................................................ .. .. .. .... .. ..... 13
D eferox am in e ...............................................................14
Summary ............... ....................................................15
P u rp o se ...............................................................................1 6
R rationale ............. .. ........................ ..................... ......... 16
Questions and Hypotheses ............... ......... ...... .........17

2 METHODS ....... ........... ................................. 19

A n im als ..............................................................19
Supplementation Protocol ................. .................................19
Control ............... ......... .................. 19
A llo p u rin o l .............................................................................................. 1 9
Cyclophosphamide ................ .............................. 20
Deferoxamine ...... ......... ......... .......... ........20
Ligation Procedure.............................................. 20









In Vivo Stim ulation ......... .... .. .......... .. ........................... .... 21
T issue R em oval and Storage ........................................................... .....................2 1
B iochem ical A ssay s............ ............................................................ .......... ....... 22
Protein C concentrations ............................................... ............................. 22
M uscle W ater C ontent........... ................. ............................ ............... 22
P rotein O xidation ............ ............................................................ .... .... ..... .. 22
L ipid P eroxidation ............................................................. ...... .... .... .. 22
4-Hydroxy-2-Nonenal (HNE) ........................................................ ...............23
X anthine Oxidase A activity ............................................................................ 23
M yeloperoxidase A activity ............................................................................24
Lactate D ehydrogenase A activity ........................................ ...... ............... 24
Statistical Analysis................ .. ... .................. ......... 24

3 R E S U L T S .............................................................................2 5

Overview of Experim ental Findings................................... ..................................... 25
Morphological Measurements .............................................................................25
C ontractile Function ......................... ............................................ .. ........... 25
Lipid Hydroperoxides (LOOH) ............................................................................26
4-Hydroxy-2-N onenal Levels....................................... ..................................... 26
Protein C arbonyls ............................................ .. .. ........... ......... 26
X anthine O xidase A activity .............................................................. .....................27
M yeloperoxidase (M PO) A activity ........................................ ......................... 27
Wet/Dry Ratio........................................................ .... ........... 27
Lactate Dehydrogenase (LDH) Activity................................... ...................... 27

4 D ISCU SSION ...................................................................... .......... 33

Overview of Experim ental Findings................................... ..................................... 33
C ontractile Claudication ............................................................................ .. 33
X anthine O xidase Inhibition ............................................................ .....................34
N eu trop en ia ................................36............................
Iro n C h e latio n ............................................................................................................. 3 8
4-H ydroxy-2-N onenal.......................................................................... ..............40

5 CON CLU SION S .................................. .. .......... .. .............42

LIST OF REFEREN CES ............................................................................. 44

B IO G R A PH IC A L SK E TCH .................................................................. .....................51










v















LIST OF TABLES


Table page

1 Markers of oxidative stress and edema after 30 min contractile claudication............6

2 Potential sources of oxidants after 30 min contractile claudication......................

3 Body weight, wet muscle weight, and protein concentration. .................................28















LIST OF FIGURES

Figure pge

1 Force production from the triceps surae muscle group during the last minute
of the 30-m inute stim ulation period...................................................................... 28

2 Lipid Hydroperoxide levels; values are expressed as mean SEM ......................29

3 T otal H N E lev els.......... .................................................................. .......... ....... 29

4 Protein carbonyls; values are expressed as mean SEM ........................................30

5 Xanthine oxidase activity; values are expressed as mean SEM.........................30

6 Myeloperoxidase activity; values are expressed as mean + SEM............................31

7 Muscle Wet/dry ratio; values are expressed as mean SEM ..............................31

8 Lactate dehydrogenase activity; values are expressed as mean SEM...................32















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

PATHWAYS OF OXIDATIVE DAMAGE TO SKELETAL MUSCLE AFTER AN
ACUTE BOUT OF CONTRACTILE CLAUDICATION

By

Andrew Judge

August 2003

Chair: Stephen Dodd
Major Department: Exercise and Sports Sciences

A limited number of studies have shown an increase in products of lipid

peroxidation in the plasma of claudicants after exercise. Previously, we used an animal

model to mimic the condition of exercise claudication to show oxidative damage and

edema within the muscle. Using this same model, we investigated the sources of this

oxidative damage in the gastrocnemius muscle, focusing on xanthine oxidase and

neutrophils, in addition to determining the role iron plays in the exercising claudicant.

The increase in lipid hydroperoxides, seen in claudicant muscle, was attenuated

with independent inhibition of xanthine oxidase activity, depletion of neutrophils, and

chelation of iron. An additional marker of lipid peroxidation, 4 hydroxy-2-nonenal

(HNE), was also attenuated by depletion of neutrophils. The HNE is formed from

oxidant-induced decomposition of lipid hydroperoxides, but may bind to amino acid side

chains of proteins, potentially affecting their function.









Protein oxidation, indicated by an increased level of protein carbonyls, was

significantly increased in claudicant muscle, but attenuated by depletion of neutrophils.

Since oxidants have the potential to modify membrane macromolecules, we also

investigated LDH activity, as an indicator of muscle cell membrane permeability; and

wet/dry ratio as a marker of edema relective of vascular membrane permeability. The

LDH activity was decreased in claudicant muscles, reflecting loss of the cytosolic

enzyme due to membrane alterations. This loss was attenuated with independent

inhibition of xanthine oxidase activity, depletion of neutrophils, and chelation of iron.

Edema, however, was only attenuated by neutrophil depletion.

Our findings suggest that neutrophils are the predominant source of oxidants after

exercise claudication; and a major cause of edema. However, we also show that xanthine

oxidase-derived oxidants contribute to lipid peroxidation and are chemotaxic to

neutrophils. Finally, we show that chelation of iron also attenuates lipid peroxidation,

despite an increase in xanthine oxidase and neutrophils, demonstrating iron's role in

propagating oxidant reactions.














CHAPTER 1
INTRODUCTION AND REVIEW OF LITERATURE

Introduction

Oxidative stress, due to ischemia followed by reperfusion (I-R) may cause

significant muscle damage and dysfunction [1,2]. Most of the research regarding skeletal

muscle I-R injury has used prolonged periods of ischemia followed by reperfusion. These

studies found convincing evidence of oxidant generation and associated muscle damage.

In addition, it has been shown that various countermeasures can attenuate this oxidative

damage [2,3].

Recently, exercise claudication has been classified as a form of I-R; and

accumulating evidence associates oxidative stress and muscle damage with this condition

[4,5]. Using an in vivo rat model to mimic the condition of exercise claudication, we

found evidence of protein oxidation, lipid peroxidation, and reduction of total glutathione

in both the soleus and gastrocnemius muscles. This further substantiates the claim of

oxidative damage after exercise claudication.

At present, only two studies have attempted to attenuate this oxidative stress in

exercising claudicants, using antioxidant supplementation. Wijen et al. [5] administered

Vitamins C and E together orally, over a 4 week period, and attenuated the oxidative

stress in exercising claudicants. In a similar study, Silvestro et al. [6] infused Vitamin C

intravenously, 5 minutes before the exercise bout; and were likewise successful in

attenuating the oxidative stress.









While this therapy shows the potential for sequestering oxidants before they can

elicit their damaging effects, the antioxidants used were not specific; and therefore may

not be the most appropriate countermeasures. Recently, we found evidence of elevated

xanthine oxidase activity and activated neutrophils after contractile claudication.

Therefore, specifically blocking or inhibiting these pathways of oxidant generation

should provide greater protection. In addition, iron mobilization occurs during prolonged

I-R [7], and likely occurs after exercise claudication. Since iron is a powerful catalyzing

agent in redox reactions, its chelation should provide protection from oxidative damage.

Our study attempted to inhibit xanthine oxidase and induce neutropenia, as well as

chelate free iron, to establish the significance of each regarding oxidative damage.

Xanthine oxidase can be effectively inhibited with allopurinol a purine analog

whose metabolite, oxypurinol, forms a tight binding complex with the enzyme, rendering

it inactive. Allopurinol-treated animals appear to have attenuation of I-R-induced muscle

damage in a variety of tissues [8].

Neutropenia may be induced with cyclophosphamide a compound activated

enzymatically in the body to phosphoramide mustard, a powerful and unstable

DNA-alkylating agent. Cyclophosphamide has been shown to significantly reduce the

number of circulating polymorphonuclear neutrophils [9], thereby diminishing the

inflammatory response, and providing protection against I-R-induced injury [10]. This is

presumed to occur through a reduction in polymorphonuclear neutrophil superoxide

production.









Deferoxamine is a powerful iron chelator, and may be used to prevent iron from

participating in redox reactions. Deferoxamine has effectively been used to prevent

oxidative stress and muscle damage after prolonged I-R [3].

To date, no studies have used these countermeasures in the exercising claudicant. In

fact, until recently no study had measured oxidative stress in muscle after exercise

claudication, or considered the potential sources. We administered each of the

countermeasures and measured oxidative stress after contractile claudication; and

determined muscle edema, as evidenced by an increase in the wet/dry ratio.

It is imperative to gain a better understanding of the relative contributions of the

various sources of oxidants produced during exercise claudication. At present, the lack of

research in this area means that we are relying on somewhat speculative information,

based on non specific research, and thus relying on assumptive preventative measures. It

seems prudent to gain a specific understanding of the sources of oxidants so that the most

appropriate countermeasures can be implemented to attenuate the stress and edema, and

reduce further disability from this cardiovascular disease.

Review of Literature

Ischemia-Reperfusion

Ischemia may be defined as "a rate of blood flow to an organ that is inadequate to

supply sufficient oxygen and maintain aerobic respiration in that organ" [11]. Therefore,

skeletal muscle becomes ischemic when there is a mismatch between blood supply and

blood demand resulting in insufficient oxygen delivery to the muscles. This may result

from cardiovascular disease, severe trauma, vascular occlusions, or even strenuous or

unaccustomed exercise. Although ischemia may cause muscle damage and, if prolonged

enough, necrosis, it appears to be the re-oxygenation, or reperfusion of tissue that causes









the major damage. Reperfusion occurs through restoration of blood flow so that oxygen

delivery can once again match the demand. Since this is essential to tissue survival, the

potential damage appears to be the lesser of two evils.

Intermittent Claudication

Skeletal muscle ischemia due to disease is highly dependent on the degree of vessel

blockage, and may manifest itself as pain at rest in the most severe cases (or more

commonly, as claudication). The term claudication derives from the latin word

claudicatio, meaning to limp; and describes a condition whereby pain is felt during

exercise or activity (sufficient to require termination of the exercise), with relief upon

rest. This exercise-induced pain is a result of the inability to deliver the required

substrates to meet the demands of the working muscles a similar condition to that

which occurs during strenuous or unaccustomed exercise. Intermittent claudication refers

to the continual process of pain upon exercise, and relief with rest.

The prevalence of intermittent claudication ranges from 2.9%, in the Speedwell

Prospective Heart Disease Study [12] to 4.5% in the Edinburgh Artery Study [13]. The

range probably reflects the differences in defining criterion. However, these numbers are

likely an underestimate of the true incidence, since only 10 to 50% of people with

claudication symptoms consult their general practitioner [14].

Peripheral Arterial Disease (PAD) is probably the most common cause of

intermittent claudication. This disease is associated with atherosclerotic lesions, reducing

blood flow to the area distal to the lesions. Therefore, the locality of pain can indicate

which vessel might be restricted calf pain is most frequently a consequence of femoral

artery disease; whereas pain in the thigh/buttock area indicates proximal restriction, most

likely due to aortoiliac disease [15].









Except in the most severe cases, the restricted blood flow does not cause ischemia

of a resting skeletal muscle, due to its low metabolic demands. However, during physical

activity, the metabolic demands of the exercising muscle increase greatly, and blood flow

is required to increase many-fold to meet this demand. Although blood flow may be able

to increase some, it is often not enough to match the increased demand, rendering the

limb ischemic. This is illustrated in a study by Pernow et al. [16], who measured femoral

artery blood flow in patients with claudication, both before and during a standard exercise

test. During exercise, blood flow in claudicants increased almost threefold; however, this

was still only 50% of the exercise blood flow in control subjects.

Recently, intermittent claudication has been referred to as a form of low-grade I-R

[17]. This is not identical to the traditional I-R, whereby blood flow to a muscle is

occluded for a given period (enforcing absolute ischemia) and then restored, allowing

re-oxygenation of the tissue. During claudication, the ischemia is relative and the

reperfusion does not occur due to restoration of blood flow, but rather due to a reduction

in blood demand during the recovery period, allowing the oxygen demands of the muscle

to once again be met. However, parallels between the two conditions certainly exist,

which has led to the realization that intermittent claudication may generate the same

damage that occurs after I-R contractilee dysfunction, oxidative injury [18], cell

dysfunction and cell death [19]).

Oxidative Stress

There is considerable evidence of radical production induced by prolonged

ischemia of skeletal muscle, with subsequent reperfusion [1,2,20]. This has applicability

to surgical situations where it may be necessary to occlude blood flow for hours, before

reperfusion is either allowed, or necessary. These studies have provided evidence of









elevated lipid hydroperoxides, decreased protein thiols, and a loss of glutathione.

However, few studies have considered the idea that radicals may be generated after

short-term I-R, as would be the case in the exercising claudicant. This form of I-R may be

of a lower grade, and skeletal muscle has been shown to be fairly resistant to I-R injury.

Indeed, Kadambi et al. [21] found no increase in lipid peroxidation after 30 min of

ischemia followed by 1 h of reperfusion. However, when the metabolic demands of a

muscle are increased while blood flow to the muscle is reduced, the mismatch between

supply and demand becomes greater. This may cause a greater disruption to redox

balance. Work from our lab shows convincing evidence of oxidative damage to both

lipids and proteins, as well as a reduction in total glutathione; and an increase in wet/dry

weight, which is indicative of edema, after a 30-minute bout of contractile claudication

(Table 1). Other studies lend support to this, showing increased lipid peroxides [4] and

increased ortho-hydroxyantipyrine an indicator of oxidative stress [5].

Table 1. Markers of oxidative stress and edema after 30 min contractile claudication
Soleus Gastrocnemius
Sham- Ligated- Sham- Ligated-
Stimulated Stimulated Stimulated Stimulated
Lipid Hydroperoxides
(mmol/g wet weight) 8.92 + 1.43 13.76 + 1.39 7.39 0.97 10.88 0.86 *
Protein Carbonyls
(nmol/mg protein) 1.80 + 0.05 2.83 0.07# 1.87 0.13 2.89 0.13 #
Total Glutathione (mM) 1.44 + 0.03 1.29 + 0.05 0.90 + 0.01 0.76 0.04
Wet/Dry Ratio N/A N/A 4.51 0.08 4.72 0.05*
Values are mean SEM; N=8
*Significant at p<0.05; Significant at p < 0.01; #Significant at p < 0.001

Just as strenuous exercise in healthy individuals causes significant oxidative stress,

while moderate intensity exercise does not [22], this too may be the case in claudicants.

Silvestro investigated the effects of maximal and submaximal exercise on parameters of

oxidative stress in claudicants; and discovered that only maximal exercise caused









significant lipid peroxidation and an increase in intracellular adhesion molecule-1

(ICAM-1). Expressed by endothelial cells, ICAM-1 is a receptor for the neutrophil

adhesion molecules CD11/CD18, and is therefore important in determining

neutrophil-endothelium cell adhesion. Adherence of these molecules primes the

neutrophil for migration into the tissue.

These findings are interesting since claudicants are instructed, therapeutically, to

exercise until near maximal pain. However, based on Silvestro's findings, it might seem

logical for claudicants to exercise less intensely. This would presumably cause a milder

ischemic insult, reducing oxidative stress and neutrophil infiltration, while potentially

still providing the cardiovascular benefits of exercise that claudicants so desperately

need.

Sources of Oxidants after Exercise Claudication

There are a variety of potential biological sources of oxidants. However, most

research regarding skeletal muscle I-R has focused on the xanthine oxidase pathway and

activated neutrophils. Work from our lab has shown xanthine oxidase activity and

activated neutrophils to be elevated after a short bout of contractile-induced skeletal

muscle ischemia (Table 2). Thus these potential sources are considered in greater detail.

Xanthine oxidase (XO) pathway. During ischemia, or very strenuous exercise,

ATP catabolism occurs within the muscle cell, yielding hypoxanthine and xanthine.

Normally these metabolites are oxidized via xanthine dehydrogenase (XDH) using NAD

as the electron acceptor. However, XDH may be converted to XO by oxidation of thiol

groups, or by calcium-dependent proteolytic attack. Since both ischemia and strenuous

exercise can disrupt calcium homeostasis, the environment may favor this dehydrogenase

to oxidase conversion. XO is still able to oxidize hypoxanthine and xanthine; however,









molecular oxygen acts as the electron acceptor instead of NAD, the consequence being

the generation of large amounts of superoxide anion [23]. The necessary components for

ROS production are, therefore, provided. The only missing component is molecular

oxygen, provided on reperfusion.

Table 2. Potential sources of oxidants after 30 min contractile claudication.
Soleus Gastrocnemius
Sham- Ligated- Sham- Ligated-
Stimulated Stimulated Stimulated Stimulated
Xanthine Oxidase Activity
(mU/g wet weight) 2.85 0.09 3.39 0.19 2.73 0.08 3.29 0.15 *
Myeloperoxidase Activity
(U/g wet weight) 0.80 + 0.02 1.15 + 0.03# 0.66 + 0.08 1.03 + 0.06#
Mitochondrial Hydrogen
Peroxide Release N/A N/A 0.48 + 0.04 0.49 + 0.06
(nmol/min/mg protein)
Values are mean SEM; N=6
*Significant at p<0.05; #Significant at p < 0.001

Neutrophils. It has been shown that neutrophil activation is significantly increased

after intermittent ischemia of skeletal muscle [23-25]. Plasma neutrophil levels have been

shown to peak within 5 min post-exercise in claudicants [25,26]; however infiltration into

tissues takes longer. Smith et al. [27] showed what they referred to as a "dramatic"

increase in tissue neutrophils after just 15 min of reperfusion after 4 h ischemia.

However, Prem et al. [28] found that although tissue neutrophils increased after 30 min of

reperfusion, after 2 h ischemia, the levels peaked after 4 h of reperfusion. This peak in

neutrophil infiltration after a longer period of reperfusion is in agreement with others

[29].

The mechanisms of neutrophil activation are unquestionably complex, and indeed

remain largely unknown. However, it is interesting to note that oxidants themselves have

been implicated as chemotactic to neutrophils [30]. This is supported by the observation









that treatment with radical inhibitors before reperfusion prevents tissue neutrophil

infiltration [31]. In addition, the lipid peroxidation product 4-hydroxy-trans-2-nonenal

has also been shown to be chemotactic to neutrophils [32], weaving a tighter web

between oxidant production and propagation of the inflammatory process.

Role of Iron

Although the generation of superoxide and hydrogen peroxide are potentially

damaging, they are poorly reactive and cannot alone explain the toxic effects of

superoxide generating systems [33]. Instead, it seems that the highly damaging hydroxyl

radical is largely responsible for oxidative injury. The hydroxyl radical may be generated

by the reaction of superoxide with hydrogen peroxide (Haber-Weiss reaction), yet this

proceeds too slowly to be of biological significance. However, in the presence of a

suitable metal catalyst, for example iron or copper, the reaction proceeds much more

rapidly. Iron appears to be the best candidate in vivo; and the iron-catalyzed Haber-Weiss

reaction proceeds with a rate constant of 76 M-1 s-1, compared to almost zero without a

catalyst. In addition to catalyzing hydroxyl radical formation, iron may also reinitiate

lipid oxidation by converting lipid hydroperoxides to reactive alkoxyl or peroxyl radicals

[7].

Fortunately, under normal physiological conditions, virtually all iron is bound

either to proteins, membranes, nucleic acids, or low-molecular-weight chelating agents

[33]. Within the muscle cell, iron is stored mainly within ferritin molecules, a protein of

24 polypeptide chains capable of storing up to 4,500 iron atoms [34]. However, iron may

be mobilized from ferritin and other storage forms, making the metal redox active.

Release of iron from ferritin requires reduction of the ferric iron stored in the molecule, a

feat the superoxide anion and other reducing agents are capable of. Biemond et al. [34]









stimulated polymorphonuclear leukocytes to produce superoxide; and observed

significant iron mobilization from ferritin. Addition of SOD prevented this, providing

convincing evidence that the iron mobilization occurred in a superoxide-dependent

manner. Likewise, iron release from ferritin has been shown to be possible with xanthine

and xanthine oxidase, a well known source of superoxide [35]. It has also been suggested

that NADH and NADPH, which accumulate during ischemia, might interact with ferritin

to release ferrous iron [36].

Furthermore, the rate of ferritin iron mobilization by reducing agents is

accelerated by acidification. Brain homogenate incubated under aerobic and anaerobic

conditions at three different pH's (7, 6 and 5) showed far greater iron delocalization

under anaerobic conditions, which was exacerbated with a decrease in pH [36].

Iron has been shown to be an important pro-oxidant in prolonged I-R. Chiao et al.

[7] provided evidence of iron delocalization after 2 h ischemia and 30 min reperfusion;

and this was associated with membrane dysfunction and lipid peroxidation. Although no

study has considered the role of iron in promoting oxidant reactions in the exercising

claudicant, the conditions seem appropriate for iron delocalization. Exercising

claudicants rely heavily on anaerobic respiration because of reduced oxygen delivery,

causing an increase in reducing equivalents and increased lactic acid production.

Acidosis, accumulation of reducing equivalents, and superoxide production could release

iron from ferritin stores, thereby becoming available to catalyze free radical reactions

[37].

Interaction

It is doubtful that ROS are derived from a single source in the exercising

claudicant. Rather, there is likely some overlap and interaction among the different









sources. This is shown (after prolonged I-R) by Seekamp et al. [29], who measured tissue

injury and vascular permeability. Neutrophil depletion, administration of catalase and

superoxide dismutase, allopurinol, dimethylthiourea, dimethylsulfoxide, and complement

depletion were all individually implemented as countermeasures; and each significantly

attenuated muscle damage to some degree. Importantly, pretreatment with antioxidants

also significantly reduced neutrophil infiltration, emphasizing the chemotactic potential

of ROS.

Edema

The alteration of cell membranes due to post-ischemic oxidative stress has a

strong correlation with the presence of intramuscular edema [38], characterized by fiber

swelling, fiber destruction, and an increase in muscle wet weights. Neutrophil depletion

or hydroxyl radical scavenging have been shown to significantly reduce both oxidative

stress and edema [2], strongly implicating ROS in the muscle damage.

We have previously shown significantly increased muscle wet weights in the

gastrocnemius and soleus muscles (and a significant increase in the wet/dry ratio in

gastrocnemius muscles) of exercise claudication animals, possibly due to oxidative

stress-induced edema.

Muscle Cell Membrane

Lactate Dehydrogenase resides in the cytoplasm of the muscle cell and has a

molecular weight of 140,000 kDa, making it highly impermeable to the muscle cell

membrane. Therefore, a decrease in LDH may indicate a change in membrane

permeability, such that some of the enzyme has escaped into the extracellular

compartment. This may be explained by an alteration in cellular ATP levels, which could









increase intracellular calcium levels, activating proteases and lipases, thereby altering

normal membrane permeability. Given the degree of ATP catabolism that appears to

occur during skeletal muscle ischemia [39] this is a possibility. Alternatively, it could be

the result of oxidative modification of membrane lipids and proteins, as we previously

showed to occur after contractile claudication.

This loss in LDH activity associated with oxidative stress has been reported

previously. After downhill running Dawson [40] noted an increase in lipid peroxidation

and myeloperoxidase activity; and a decrease in LDH activity in the gastrocnemius

muscle, concluding that oxidative modification to the muscle cell membrane made it

leaky. In a separate study, Jones [41] stimulated muscles in vitro for 30 min and

quantified LDH release into the medium over a 3 h period. Enzyme release peaked 1 h

post-stimulation in the soleus muscle under both normoxic and hypoxic conditions, but

was significantly more elevated in the hypoxic conditions. In an attempt to distinguish

whether ATP depletion or membrane disruption was responsible for this loss of LDH,

Jones incubated with iodoacetate and cyanide, to reduce ATP; and then with deoxycholic

acid, a detergent, to disrupt the membrane. Both treatments caused even greater release of

LDH, confirming both as possibilities. The conclusion drawn was that both metabolic

changes and physical damage could play a part.

Countermeasures

Allopurinol

Allopurinol is a purine analog whose metabolite, oxypurinol, forms a tight binding

complex with xanthine oxidase, rendering the enzyme inactive. Since xanthine oxidase is

a potential source of ROS, inactivating the enzyme could have protective effects against









ROS-induced damage. Animals treated with allopurinol appear to have attenuation of

I-R-induced muscle damage in a variety of tissues [8].

Vina et al. [42] showed that blood XO levels were significantly elevated after

exhaustive exercise; and that allopurinol administration prevented exercise-induced

glutathione oxidation. Although exercising claudicants do not typically perform exercise

that would be exhaustive to a healthy individual, even light exercise usually brings about

shortness of breath, perspiration, and a general sense of fatigue [26]. Work from our lab,

using an animal model of unilateral femoral artery blockage, showed that a mild

stimulation protocol elicits much greater metabolic stress to the occluded limb compared

to the control, as evidenced by a decline in force production over time.

Several studies have shown that oxidative stress is derived from xanthine oxidase

during prolonged ischemia, followed by reperfusion, in skeletal muscle. Asami et al. [43]

showed that muscular xanthine and malondialdehyde (MDA) levels were elevated during

reperfusion after 5 h of ischemia. MDA is an indicator of lipid peroxidation, and its

increase was attenuated by allopurinol administration, which strongly implicates XO as

the source of the radical-induced lipid damage. Similarly, McCutchan et al. [44] showed

that XO activity was significantly elevated with 3 h of ischemia followed by reperfusion;

and that this was associated with hydrogen peroxide generation. Administration of either

allopurinol or tungsten (which replaced molybdenum in xanthine oxidase's active site)

reduced XO activity and hydrogen peroxide generation.

Cyclophosphamide

Cyclophosphamide is a derivative of the nitrogen mustard family, compounds

originally developed as chemical weapons. Soldiers exposed to sulfur mustard suffered

from low white-blood-cell counts (especially lymphocytes), apparently due to the









mustard's cytotoxic effects on dividing tissues. After that observation, nitrogen mustard,

a similar but less toxic agent, was developed to treat cancer; and later used as an

immunosuppressant. Cyclophosphamide is activated enzymatically in the body to

phosphoramide mustard, a powerful and unstable DNA-alkylating agent, interfering with

DNA synthesis, therefore proving cytotoxic to dividing lymphocytes [45].

Several studies have used cyclophosphamide to induce neutropenia in rat models.

Kuwabara et al. [9] noted the number of polymorphonuclear neutrophils was reduced to

20 per ptL of blood 4 days after injection, compared to 1224 per p.L of blood in control

animals. Seekamp et al. [29] used cyclophosphamide to induce neutropenia 3 days before

I-R exposure and found a protective effect against muscle permeability. This protection

from cyclophosphamide-induced neutropenia has also been shown to be effective against

an increase in microvascular permeability associated with short-term I-R [10].

Deferoxamine

Deferoxamine is a straight-chained molecule with three hydroxamic acid groups.

When a ferric ion comes into contact with deferoxamine, the molecule twines itself

around the ion, attaching it to its three hydroxamic acid groups. The molecule, therefore,

provides a shell, surrounding the iron and becoming a very stable complex ([46].

The powerful iron-chelating properties of deferoxamine mean that it may inhibit

lipid peroxidation and the generation of the hydroxyl radical from superoxide and

hydrogen peroxide in biological systems where ferrous iron is free. Deferoxamine has

also been shown, in high concentrations, to block the conversion of xanthine

dehydrogenase to xanthine oxidase in cultured endothelial cells [47]. The mechanism for

this is unknown, however the xanthine oxidase protein has four redox-active sites:









molybdenum, flavin adenine dinucleotide (FAD), and two iron sulfur centers of the

ferredoxin type [48]. It was speculated that enzyme activity would be impaired by

deferoxamine if it bound the iron cofactor.

Since myeloperoxidase contains a heme iron essential for its activity, the possibility

exists that deferoxamine could inhibit MPO activity if it chelated the heme iron.

However, since adding deferoxamine has no effect on the absorption spectrum of MPO,

it doesn't appear to work in this manner [49]. Deferoxamine can, however, be oxidized

by MPO and hydrogen peroxide, thereby competing with other electron donors; and

deferoxamine has been shown to react with and degrade the highly oxidizing

hypochlorous acid a product of the MPO system [49].

Deferoxamine has a relatively low molecular weight (656.79 Da), which facilitates

its entry into cells. In fact, deferoxamine has been shown to enter skeletal muscle cells in

significant concentrations with a greater intracellular than extracellular distribution [50].

Administration of deferoxamine after prolonged I-R has been shown to prevent

lipid peroxidation and to attenuate membrane dysfunction [3]. Additionally, Smith et al.

[51] showed that either deferoxamine or apotransferrin (an iron-binding protein)

administration prevented an increase in microvascular permeability associated with

prolonged I-R.

Summary

In summary, it has been shown that an acute bout of contractile claudication causes

an increase in protein oxidation, lipid peroxidation, and edema; and a loss of total

glutathione. This is associated with an increase in xanthine oxidase activity and

neutrophil infiltration. In addition, free iron is elevated after prolonged I-R; and is likely

elevated after an acute bout of contractile claudication due to favorable conditions for









iron mobilization. Therefore, inhibition of xanthine oxidase activity, induction of

neutropenia, and iron chelation have the potential to attenuate the oxidative stress and

edema associated with an acute bout of contractile claudication. In addition, reactive

oxygen species have themselves been implicated as chemotactic to neutrophils.

Therefore, inhibition of xanthine oxidase and iron chelation, have the potential to

attenuate neutrophil infiltration into tissue.

Purpose

Our purpose was to determine if inhibition of xanthine oxidase activity, induction

of neutropenia, or iron chelation will protect against oxidative stress and muscle edema

induced by an acute bout of contractile claudication. We also determined whether

inhibition of xanthine oxidase activity and iron chelation, attenuate the increase in

neutrophil infiltration associated with an acute bout of contractile claudication.

Rationale

We showed that oxidation of proteins and lipids occurs after an acute bout of

contractile-induced skeletal muscle ischemia; and that this is associated with an increase

in muscle wet weight, likely due to edema. We also showed an increase in xanthine

oxidase activity and infiltration of neutrophils after the same conditions. It was shown by

others that administering allopurinol inhibits xanthine oxidase activity; and therefore

oxidant production [44]. It was also shown that induction of neutropenia by

cyclophosphamide attenuates neutrophil infiltration and protects against oxidative stress-

induced muscle damage [29]. Thus, it is speculated that allopurinol administration will

inhibit xanthine oxidase activity, and that cyclophosphamide administration will attenuate

neutrophil infiltration; and that both will independently attenuate protein and lipid

oxidation and edema.









Mobilization of iron from its stored sources can catalyze the Haber-Weiss reaction,

generating the highly reactive hydroxyl radical; and reinitiating lipid peroxidation,

creating the alkoxyl and peroxyl radicals. Delocalization of iron was shown to occur after

prolonged I-R; and is associated with lipid peroxidation [7]. In addition, chelation of iron

by deferoxamine was shown to prevent lipid peroxidation [3]. Thus, it is expected that

deferoxamine administration will attenuate protein and lipid oxidation and edema.

Reactive oxygen species have been shown to be chemotactic to neutrophils [30];

and inhibiting their generation has attenuated neutrophil infiltration [31]. Thus, it is

anticipated that iron chelation and xanthine oxidase inhibition will reduce the amount of

ROS produced; and therefore attenuate neutrophil infiltration.

Questions and Hypotheses

Question 1. Does an increase in xanthine oxidase activity cause oxidative stress

and muscle edema, generated by an acute bout of contractile claudication?

Hypothesis 1. Reduction of xanthine oxidase activity will attenuate lipid

peroxidation and protein oxidation, as well as muscle edema, induced by an acute bout of

contractile claudication.

Question 2. Does an increase in xanthine oxidase activity cause neutrophil

infiltration into muscle after an acute bout of contractile claudication?

Hypothesis 2. Reduction of xanthine oxidase activity will attenuate neutrophil

infiltration into muscle after an acute bout of contractile claudication.

Question 3. Does neutrophil infiltration cause the oxidative stress and muscle

edema induced by an acute bout of contractile claudication?









Hypothesis 3. Reducing neutrophil infiltration into tissue will attenuate lipid

peroxidation and protein oxidation, as well as muscle edema, induced by an acute bout of

contractile claudication.

Question 4. Does reduction of free iron attenuate the oxidative stress and muscle

edema induced by an acute bout of contractile claudication?

Hypothesis 4. Reduction of free iron will attenuate lipid peroxidation and protein

oxidation, as well as muscle edema, induced by an acute bout of contractile claudication.

Question 5. Does reduction of free iron attenuate neutrophil infiltration induced by

an acute bout of contractile claudication?

Hypothesis 5. Reduction of free iron will attenuate neutrophil infiltration into

tissue, induced by an acute bout of contractile claudication.














CHAPTER 2
METHODS

Animals

All experiments were performed on male Sprague Dawley rats (120 d old) to avoid

any antioxidant protection from estrogen. They were fed rat chow, given water ad

libitum, and maintained on a 12-h light/dark photoperiod for 7 days before the beginning

of these experiments. During this 7 day period, animals were handled daily to prevent a

stress hormone-induced reduction in body weight at the beginning of the experiments.

Animals were then randomly assigned to one of four experimental groups: Control

(CON); Allopurinol supplemented (ALLO); Cyclophosphamide supplemented (CYCLO)

and; Deferoxamine supplemented (DFO). The limbs of each rat were then randomly

assigned to a ligated/stimulated (LS) or a sham ligated /stimulated (SS) group for the

study.

Supplementation Protocol

Control

Animals were injected intraperitoneally with 0.5 mL saline twice daily, beginning 2

days before ischemia. On the day of the experiment, the second injection was given 30

min before the contractile-induced ischemia.

Allopurinol

Animals were given an intraperitoneal injection of 50 mg of allopurinol per kg

body weight twice daily, beginning 2 days before the ischemia. On the day of the

experiment, the second injection was given 30 min before the contractile-induced









ischemia. This quantity of allopurinol has been shown to yield extracellular fluid

concentrations of 10 [LM, a concentration sufficient to cause an >80% inhibition of

xanthine oxidase activity without a scavenging effect [52]. The allopurinol was dissolved

in normal saline by adding 1 N sodium hydroxide; and administered slowly.

Cyclophosphamide

Animals were given intraperitoneal injections of 20 mg of cyclophosphamide per

100 g of body weight, 4 days before contractile-induced ischemia. This dose has been

shown to reduce the circulating leukocyte count by 85 to 90%, inhibiting microvascular

damage [10]; and, more specifically, to reduce neutrophils to 1.4% of normal levels [53].

Deferoxamine

Animals received an IP injection of 100 mg deferoxamine per kg body weight

twice a day, beginning 2 days before the ischemia. On the day of the experiment, the

second injection was given 30 min before the contractile-induced ischemia.

Deferoxamine was dissolved in normal saline and administered slowly. This quantity of

deferoxamine has been shown to reduce neutrophil infiltration and muscle damage [54].

Ligation Procedure

After isofluorane anesthesia (5% for induction, 1.5 to 2.5% for maintenance), a

small incision was made directly above the inguinal fold; and the femoral artery was

exposed and isolated by blunt dissection. Two ligatures were placed tightly around the

vessel and the vessel was cut between the ties. This procedure produces a 60% to 70%

reduction in blood flow during muscle contraction [55,56]. Topical antibiotic powder was

placed on the wound before closure with sutures. The sham surgery limbs underwent the

identical procedure except the femoral artery was left intact.









In Vivo Stimulation

Twenty-four hours post-ligation both hindlimbs were stimulated in vivo for 30 min,

and force production was measured. Animals were placed in a prone position in a

specially fabricated Plexiglas apparatus that allows the animal to be secured in a

reproducible position with limited mobility of the lower leg except at the tibiotarsal joint.

Animals were kept warm by an incandescent light, and core temperature maintained from

35 to 38 C, measured with a rectal thermistor probe. A calibrated force/displacement

ergometer was secured to the forefoot between the first and second footpads by a

lightweight chain such that the tibiotarsal angle is 900. The voltage signal from the force

transducer was processed via a computerized data acquisition system (LabView, National

Instruments, Austin).

A stainless steel stimulating electrode was placed transcutaneously near the sciatic

nerve midway between the posterior ischeal spine and the greater femoral trochanter.

Another stainless steel stimulating electrode (anode) was inserted 3 mm subdermally in

the midline of the lower back. The sciatic nerve was then stimulated proximally with

100V, 1.0 pulses per second, and a stimulus time of 0.05 ms (Grass Instruments).

Tissue Removal and Storage

One hour post-stimulation the gastrocnemius muscle was removed. Each muscle

was dissected free, immediately placed in cold antioxidant buffer (100 jtm EDTA, 50

mM Na2HPO4, 1 mM BHT), blotted dry, weighed, and rapidly frozen in liquid nitrogen

and stored at -800C until assayed.









Biochemical Assays

Protein Concentrations

Protein content of muscle homogenates was determined using the biuret technique

[57].

Muscle Water Content

Total water content of the gastrocnemius muscles was determined by using a freeze

drying technique incorporating a vacuum pump with a negative pressure of -1 mm Hg. A

precise frozen wet weight was measured, and then tissues placed in a freeze-dry unit

(Virtis Sentry Benchtop 3 L). The dry weight was terminated when the same weight was

recorded three times in succession during a six-hour interval.

Protein Oxidation

Protein carbonyls was measured spectrophotometrically as described by Reznick

and Packer [58], with modifications reported by Yan et al. [59]. Briefly, samples were

incubated in dinitrophenylhydrazine (DNPH) dissolved in HC1, with blanks incubated in

HC1 only. Following reaction with DNPH, proteins was precipitated in 20% TCA,

washed in ethyl acetate-ethanol (1:1 vol/vol) and dissolved in 6 M guanidine

hydrochloride, pH 2.3. Tissue protein carbonyl content was quantified by determining

the absorbance at 370 nm and using an extinction coefficient of 22,000 M-1. Protein

concentrations was determined using a BSA standard curve in guanidine HC1 with

absorbance read at 280 nm.

Lipid Peroxidation

Lipid hydroperoxides were measured using the ferrous oxidation/xylenol orange

technique reported by Hermes-Lima et al. [60]. Briefly, samples were homogenized in

100% methanol, centrifuged, and the resulting supernatant mixed in solution with an iron









source (FeSO4), an acid (H2S04) and a reactive dye (xylenol orange). In this mixture, the

membrane peroxides oxidize Fe2+ to Fe3+ and the peroxides are reduced. The Fe3+ reacts

with xylenol orange to form a Fe3+-Xylenol orange complex, yielding a colored product

that is accompanied by an absorbance change at 580 nm.

4-Hydroxy-2-Nonenal (HNE)

Proteins were separated on a 4-20% precast polyacrylamide gel (BMA, Rockland),

using 30 .g of protein per well, and then transferred onto a nitrocellulose membrane. The

membrane was then blocked overnight using a blocking solution containing 0.05%

Tween and 5.0% milk. Membranes were incubated for 1 hour with the primary antibody

(Alpha Diagnostics, San Antonio) using a 1:500 dilution, thoroughly washed and then

incubated for 1 hour in anti-rabbit IgG horseradish peroxidase (Amersham Life Science,

United Kingdom) using a 1:1000 dilution. Blots were developed using ECL (Amersham

Pharmacia Biotech, United Kingdom), and imaged using an Image Station (Eastman

Kodak Company, model 440cF). Arbitrary OD units were calculated by multiplying the

area of each band by its optical density and then normalized to the control group (CON

SS), which we made to 100%.

Xanthine Oxidase Activity

Xanthine oxidase was measured using a modified version of Amplex Red

Xanthine/Xanthine Oxidase Assay Kit from Molecular Probes. In this assay xanthine

oxidase catalyzes the oxidation of purine bases, hypoxanthine, or xanthine to uric acid

and superoxide. Superoxide spontaneously degrades to hydrogen peroxide, which in the

presence of horseradish peroxidase, reacts stoichiometrically with Amplex Red reagent to

generate the red-fluorescent oxidation product resorufin. The emission wavelength was

set at 590 nm and excitation fluorescence measured at 560 nm.









Myeloperoxidase Activity

MPO activity was assayed according to methods by Belcastro [61]. Briefly, tissue

was homogenized in 0.5% hexadecyltrimethyl ammonium bromide in 50 mM potassium

phosphate buffer, pH 6.0. Homogenate was then be sonicated, freeze-thawed three times,

sonicated once more, and subsequently centrifuged at 1,300 g for 15 mins. Ten [l of

supernatant was removed and incubated with 290 ul of 50 mM potassium phosphate

buffer with 0.6mM hydrogen peroxide and 167 tg o-dianisidine dihydrochloride per ml.

One unit of MPO was defined as a change in absorbance of 1.0 at an optical density of

480 nm at a temperature of 250C.

Lactate Dehydrogenase Activity

LDH activity was determined according to methods by Bergmeyer et al [62]. This

assay uses pyruvate and muscle homogenate (containing LDH), to oxidize NADH to

NAD, which is accompanied by a change in absorbance at 340 nm. Since the NADH

being oxidized is equimolar to the pyruvate being reduced, the change in absorbance is

directly proportional to the LDH activity in the sample.

Statistical Analysis

All data was analyzed using a two-way analysis of variance, and the contractile

data was analyzed using a two way ANOVA with repeated measures. Significance was

established at the P < 0.05 level, and a Bonferroni post-hoc test was used where

necessary.














CHAPTER 3
RESULTS

Overview of Experimental Findings

This study examined the pathways of oxidative damage after an acute bout of

contractile claudication. By inhibiting each of the pathways individually, a cause and

effect relationship could be established. The major findings of the study are that

neutrophil depletion and iron chelation attenuated the oxidative damage associated with

contractile claudication, while edema was only attenuated with neutrophil depletion.

Inhibition of xanthine oxidase activity significantly attenuated lipid peroxidation, but not

protein oxidation or edema.

Morphological Measurements

Each of the countermeasures was well tolerated by the animals, with no mortalities

or visual side effects noted. The body weights of ALLO and CYCLO animals

significantly decreased, by 7% and 13% respectively, from the time of group assignment

(pre-injection) to the time of sacrifice, and the muscle weights of CYCLO animals were

significantly lower than the muscle weights of CON LS, DFO SS and DFO LS. However,

neither the muscle weight/body weight ratio nor total protein concentration was different

across any of the groups (Table 1).

Contractile Function

Force generation from the triceps surae muscle group significantly decreased in

each of the ligated-stimulated (LS), or claudicant, limbs during the thirty-minute

stimulation period (Figure 1). Compared to their sham-stimulated limbs, both the LS









CON and DFO groups were significantly decreased after 3 minutes of stimulation, and

the ALLO and CYCLO group after just 2 minutes. However, neither the LS nor SS limbs

were different across treatments, providing evidence that each group was made ischemic

to the same degree and that administration of the various countermeasures had no effect

on force generation during the contractile claudication period itself.

Lipid Hydroperoxides (LOOH)

LOOH's were significantly elevated (p<0.001) in the LS limbs of control and

ALLO supplemented animals, compared to sham. However, the LS limb of ALLO

animals was significantly (p<0.05) less than the LS limb of the control group. There were

no differences in LOOH levels between the LS and SS limbs of the CYCLO and DFO

groups (Figure 2).

4-Hydroxy-2-Nonenal Levels

CON SS limbs were used as a control for comparisons of CON LS, ALLO LS,

CYCLO LS, and DFO LS. Total HNE binding was significantly attenuated in the

CYCLO LS only (Figure 3).

Protein Carbonyls

Both the control group and ALLO supplemented animals showed a significantly

increased (p<0.05) protein carbonyl content in the LS limb compared to SS. There were

no differences between the LS limbs of ALLO animals and control. DFO and CYCLO

supplementation both attenuated this increase in protein carbonyls in the LS limb (Figure

4).









Xanthine Oxidase Activity

In control, DFO supplemented and CYCLO supplemented animals, xanthine

oxidase activity significantly increased (p<0.001) in the LS limb, compared to sham. This

increase was attenuated in the ALLO supplemented group (Figure 5).

Myeloperoxidase (MPO) Activity

Control (p<0.001), ALLO supplemented (p<0.01), and DFO supplemented

(p<0.05) animals had significantly elevated MPO activity in the LS limb compared to SS.

However, MPO activity in LS limbs of both the ALLO group (p<0.01) and the DFO

group (p<0.05) was significantly lower than the LS limb of control animals. There was

no increase in MPO activity in CYCLO supplemented animals (Figure 6).

Wet/Dry Ratio

The control (p<0.01), ALLO (p<0.05), and DFO (p<0.05) groups showed a

significant increase in muscle wet/dry ratio following contractile claudication, in the LS

limb compared to SS (Figure 7). This increase was attenuated in the LS limbs of CYCLO

animals.

Lactate Dehydrogenase (LDH) Activity

The LS limbs of control animals showed a significant decrease (p<0.05) in LDH

activity. This reflects "damage" to the muscle cell membrane since the enzyme may

"leak" from its cytosolic residence into the vasculature, thereby decreasing enzyme

activity within the muscle. The decrease in LDH activity was attenuated in the ALLO,

CYCLO, and DFO groups (Figure 8).










Table 3. Body weight, wet muscle weight, and protein concentration from all groups.
Values are expressed as mean SEM. a indicates significantly different
(p<0.05) from the treatment pre-injection body weight. b indicates
significantly different (p<0.05) from CYCLO SS, and c indicates significantly
different (p<0.05) from CYCLO LS. (SS = sham-stimulated; LS = ligated-
stimulated).


Pre-injection
body weight
Treatment (grams)


Body weight at
time of sacrifice
(grams)


Muscle
Muscle weight weight/body


Group (grams)


weight ratio


Total protein
concentration
(mg/gram wet
weight)


356.3 6.46 350.5 6.840


SS 2.05 0.03 5.84 0.12 139.6 3.26


LS 2.11 0.04 bc 6.04 0.15


140.1 4.98


349.2 + 5.15 324.2 + 8.432 a


CYCLO 365.0 2.88 319.2 6.395 a


370.3 5.91 357.3 7.126


SS 1.97 0.04 6.07 0.09 136.5 6.04
LS 2.04 0.07 6.28 0.15 142.0 5.95

SS 1.84 0.05 5.75 0.16 128.1 + 1.96

LS 1.82 0.06 5.71 0.21 132.4 4.05


SS 2.12 0.06 bc 6.09 0.11
LS 2.26 0.05 bc 6.31 0.14


131.2 3.93
130.0 2.99


ICON
M ALLO
= CYCLO
I DFO


*** ***


Ik


Sham-Stim Lig-Stim
Limbs


Figure 1. Force production from the triceps surae muscle group during the last minute
of the 30-minute stimulation period. Values are means SEM. indicates
significantly different (p<0.001) from sham-stim limbs undergoing the same
treatment.


CON


ALLO


DFO












-Sham-Stim
Mi Lig-Stim


CON


ALLO CYCLO
Treatment


DFO


Figure 2. Lipid Hydroperoxide levels; values are expressed as mean SEM. indicates
significantly different (p<0.001) from the sham-stim group undergoing the
same treatment. # indicates significantly different (p<0.05) from CON Lig-
Stim. + indicates significantly different (p<0.05) from ALLO Lig-Stim.


125-


2 100-
0

o 75-


w 50-

LU
Z 25-
M-


0-


ALLO LS CYCLO LS
Treatment


DFO LS


Figure 3. Total HNE levels in CON LS, ALLO LS, CYCLO LS, and DFO LS limbs.
Values are expressed as a percentage of CON SS levels. indicates
significantly different from CON LS.























CON ALLO CYCLO DFO
Treatment


Figure 4. Protein carbonyls; values are expressed as mean SEM. indicates
significantly different (p<0.01) from the sham-stim group undergoing the
same treatment. # indicates significantly different (p<0.05) from CON Lig-
Stim.


CON ALLO CYCLO DFO
Treatment


Figure 5. Xanthine oxidase activity; values are expressed as mean SEM. indicates
significantly different (p<0.001) from the sham-stim group undergoing the
same treatment. # indicates significantly different (p<0.001) from CON Lig-
Stim.












> Sham-Stim
1.0 i Lig-Stim

0.8 *
Cu c 0.7



0.3-
0.2-
0.1

0.0
CON ALLO CYCLO DFO
Treatment


Figure 6. Myeloperoxidase activity; values are expressed as mean + SEM. indicates
significantly different (p<0.05) from the sham-stim group undergoing the
same treatment. # indicates significantly different (p<0.05) from CON Lig-
Stim.




Sham-Stim
EM Lig-Stim

S5.0- *
4.5.
S4.0
-0 3.5
0 3.0

a, 2.5
2.0-
1.5-

o 1.0-
g 0.5-
0.0
CON ALLO CYCLO DFO
Treatment

Figure 7. Muscle Wet/dry ratio; values are expressed as mean SEM. indicates
significantly different (p<0.05) from the sham-stim group undergoing the
same treatment.










Sham-Stim
> 700- ME Lig-Stim
._
U 600-

500-

E 400

M I-300
4
200-

i 100

0
CON ALLO CYCLO DFO
Treatment

Figure 8. Lactate dehydrogenase activity; values are expressed as mean + SEM.
indicates significantly different (p<0.05) from the sham-stim group
undergoing the same treatment.














CHAPTER 4
DISCUSSION

Overview of Experimental Findings

This is the first study to examine the pathways of oxidative damage and edema

after an acute bout of contractile claudication. The major findings of this study are

twofold: inhibiting the increase in xanthine oxidase activity and chelation of iron

significantly attenuates lipid peroxidation, but not protein oxidation or edema, after

contractile claudication. Secondly, induction of neutropenia attenuated lipid peroxidation,

protein oxidation, and edema after contractile claudication. Together, these findings

indicate that activated neutrophils are the major source of oxidative damage after

contractile claudication, and that this damage contributes to edema. In addition, the data

suggest that iron plays a pivotal role in contributing to oxidative damage, presumably by

being mobilized during contractile claudication from bound sources, thereby becoming

available to partake in redox reactions.

Contractile Claudication

Consistent with our previous findings, an acute bout of contractile claudication

causes a significant increase in lipid peroxidation, protein oxidation and edema. This is

associated with an increase in xanthine oxidase activity, increased neutrophil infiltration,

and a loss of lactate dehydrogenase activity. Since LDH is ordinarily confined to the

muscle cell, the loss of LDH activity suggests the muscle cell membrane may be

modified by oxidants, making it more permeable and contributing to loss of LDH.









Xanthine Oxidase Inhibition

Allopurinol is known to be an inhibitor of xanthine oxidase activity. However, we

quantified the activity of the enzyme to ensure the dose administered was sufficient to

inhibit its activity in this study. As expected, allopurinol supplemented animals showed

no increase in xanthine oxidase activity after contractile claudication, whereas the

control, cyclophosphamide and deferoxamine groups each showed significant increases.

This confirms that the allopurinol served its purpose in inhibiting xanthine oxidase

activity. In addition, DFO has been shown to attenuate the increase in xanthine oxidase

activity in cultured endothelial cells exposed to radicals. Although the mechanism of this

is unknown, speculation suggests that xanthine oxidase activity would be impaired by

deferoxamine if it bound the iron cofactor at the enzyme's active site. Our findings,

however, suggest that DFO has no effect on attenuating xanthine oxidase activity after

contractile claudication. Therefore, this potential mechanism of protection may be

excluded from the protective effects DFO had in this study.

Despite the lack of increase in xanthine oxidase activity in ALLO animals there

was still significant lipid peroxidation, protein oxidation, and edema in these animals.

However, the lipid peroxidation was significantly less than control animals, showing

inhibition of xanthine oxidase activity provided some protection, and suggesting xanthine

oxidase as a source of radicals. In addition, inhibition of xanthine oxidase activity

attenuated the loss of LDH activity seen in control animals. With leakage of LDH

reflective of a disruption to muscle membrane permeability, these data suggest xanthine

oxidase-derived oxidants may target primarily membrane lipids. Since oxidants are non-

discriminate in their attack, a logical explanation is that oxidants derived from xanthine

oxidase are produced within the lipid membrane, where close proximity dictates their









modification. This is an attractive hypothesis since immunolocalization techniques

demonstrate xanthine oxidase is concentrated in capillary endothelial cells [63], and

histochemical localization studies indicate the enzyme is also localized in the sarcolemma

[63]. Both locations are abundant in lipids and could help explain the loss of LDH and

the increase in lipid hydroperoxides.

Since no other studies have measured xanthine oxidase activity, or the effects of the

enzyme's inhibition after claudication, parallels can only be drawn from prolonged I-R

studies. In one such study [44], the results were very similar to ours in that I-R caused an

increase in lipid peroxidation and an associated increase in xanthine oxidase activity.

With administration of allopurinol lipid peroxidation was significantly attenuated,

confirming that xanthine oxidase-derived radicals can cause peroxidation of cell

membranes.

If xanthine oxidase-derived radicals cause lipid peroxidation to endothelial cell

membranes, membrane permeability might be altered, causing edema. Since CON

animals exhibit increased xanthine oxidase, oxidative stress, and edema, this seems to be

an attractive possibility. Indeed, this association has been has been found by others

following prolonged I-R [51]. However, there was no attenuation of the increase in

wet/dry ratio in the ALLO group, suggesting other factors contribute to the edema seen

after an acute bout of contractile claudication.

In addition, it was hypothesized that inhibiting xanthine oxidase activity would

attenuate neutrophil infiltration, and indeed although MPO activity was significantly

elevated in the ALLO group, it was 33% lower compared to control animals. Therefore, it

can be concluded that xanthine oxidase-derived oxidants are important in the









accumulation of neutrophils after contractile claudication. This chemotaxic potential of

oxidants from xanthine oxidase is in agreement with Seekamp [29], who used allopurinol

to inhibit xanthine oxidase activity and observed a significant reduction in MPO content

following prolonged I-R. The chemotactic potential of xanthine oxidase-derived radicals

has several possible explanations. When neutrophils infiltrate tissue from the vasculature,

they must first be attracted to, and bind to, the endothelium. Since xanthine oxidase is

localized to the endothelium, this places the enzyme at the scene of neutrophil adhesion.

Indeed, isolated endothelial cells or isolated vessels exposed to hydrogen peroxide show

increased sensitivity for neutrophils [64,65]. There are several potential mechanisms to

explain this. One is that oxidants stimulate endothelial cells to synthesize and/or release

chemoattractants, such as platelet activating factor and leukotriene B4 [66]. Another

potential mechanism is that oxidants may directly induce the expression of endothelial

cell adhesion molecules. Indeed endothelial cells exposed to hydrogen peroxide have

been shown to induce P-selectin expression [67], and neutrophils incubated in hydrogen

peroxide increase their expression of CD 11 and CD18 [68].

Neutropenia

The use of cyclophosphamide to deplete neutrophils was based on several studies

using this agent to cause neutropenia both at baseline and during ischemia-reperfusion

injury. Mackie [69] showed the neutrophil count of cyclophosphamide-injected animals

was <10% of control animals after 4 days; Lee [70] measured circulating neutrophils at

<1% of control, 5 days post-cyclophosphamide injection and; Bertuglia et al [10] showed

that cyclophosphamide injected animals had leukocyte counts that were 7% of control

animal levels, after 30 minutes of ischemia and 30 minutes reperfusion. This clearly

shows the capacity for cyclophosphamide to induce neutropenia.









The CYCLO group in this study showed no significant increase in MPO activity

after contractile claudication, confirming attenuation of neutrophil infiltration into the

tissue. This lack of neutrophil infiltration attenuated lipid peroxidation, protein oxidation,

edema, and muscle membrane damage, clearly demonstrating the ability of neutrophils to

cause oxidative damage and edema. Although no previous studies have measured tissue

neutrophil levels after claudication, or depleted neutrophils prior to claudication, our

findings are in agreement with others showing attenuation of lipid peroxidation and

edema [2]; and reduction of muscle permeability [29] with neutrophil depletion prior to

prolonged I-R.

The attenuation of edema with neutropenia, observed here, has previously been

shown after prolonged I-R [10]. It appears that as neutrophils migrate through the

vascular endothelium into the muscle they may release lysosomal enzymes and/or

oxidizing species. These molecules can damage the endothelium and alter membrane

permeability, thereby resulting in edema. A second possibility is that the diapedesis

process itself may widen endothelial gap junctions, thereby contributing to edema.

Although neither of these potential mechanisms were addressed in this study, it is clear

that neutrophils cause significant edema after contractile claudication.

Since LDH is confined to the muscle cell and its loss reflects an alteration in

membrane permeability, the attenuated loss of this enzyme's activity with neutropenia

implicates neutrophils. This is the first evidence to show the muscle cell membrane is, at

least in part, being oxidized by neutrophil-derived oxidants after contractile claudication,

thereby making the membrane more permeable.









Iron Chelation

Deferoxamine is a powerful iron chelator that is used broadly in preventing iron-

dependent pro-oxidant reactions. It does this by preferentially removing iron from low

molecular weight components, including amino acids, organic acids or carbohydrates

[71]. Since deferoxamine has been shown to slowly penetrate the plasma membrane [46],

thereby entering cells, its protection may be displayed at both the intracellular and

extracellular level.

In this study, DFO animals showed significant attenuation of lipid peroxidation,

neutrophil infiltration, and an attenuation of the loss in LDH activity. However, there was

still significant edema in these animals. These findings show iron's function in redox

reactions, after contractile claudication, but suggest iron has no role in causing edema.

Since superoxide and hydrogen peroxide are poorly reactive in an aqueous

environment, it is generally thought the more potent, more damaging, hydroxyl radical is

responsible for the majority of oxidant-induced cellular damage after skeletal muscle

ischemia [72]. It is even suggested that hydroxyl radical formation is critical to cellular

injury [73]. With the presence of a suitable transition metal catalyst, such as iron,

necessary for the Fenton reaction to proceed at a significant rate, iron is clearly very

important to the redox balance.

Our findings suggest iron is paramount in causing oxidative damage after

contractile claudication. This conclusion is based on the fact that DFO animals have a

significant increase in xanthine oxidase activity, and significant neutrophil infiltration,

therefore large amounts of oxidants are still being produced. However, despite this

oxidant production, significant protection is afforded with chelation of iron. Therefore,

the findings of this study are in agreement with others [7], showing that when iron is









chelated, and disruption to the Fenton reaction is presumed, significant attenuation of

oxidative damage occurs.

Since deferoxamine is thought to enter cells, its protective effects could

conceivably occur on either side of the plasma membrane. This issue has been addressed

by several investigators with conflicting results. However, those studies conducted in

skeletal muscle tissue after I-R appear to lean on the side of extracellular protection.

Smith et al [51] administered deferoxamine and apotransferrin independently to address

this issue in ischemia-reperfused skeletal muscle. Since apotransferrin cannot cross the

cell membrane its protective effects can only be exhibited in the extracellular space. With

both compounds exhibiting the same degree of protection, a suggested conclusion was

that the iron-catalyzed Haber-Weiss reaction occurs in the extracellular space. In another

study of ischemia-reperfused skeletal muscle, Fantini et al [3] administered DFO and

DFO conjugated to pentastarch independently to animals. This conjugation alters the

physical properties of DFO, so that it cannot cross the cell membrane, while retaining its

capacity to chelate. Since the two compounds exerted similar protective effects in

inhibiting lipid peroxidation, an extracellular site of action was strongly suggested.

Although we are unable to determine from our study which side of the cell

membrane DFO is exerting its protective effects, it is certainly a strong possibility that,

similar to those studies just discussed, the protection is in the extracellular compartment.

This is based on our findings of neutrophils being the predominant source of oxidants

after contractile claudication. This would lead to extracellular oxidant production, and

potentially extracellular hydroxyl radical formation. Therefore chelation of extracellular









iron would be beneficial in this situation. However, this speculation warrants further

investigation.

Since we were unable to measure free iron in tissue, the protective effects of DFO

could conceivably be explained by alternatives to iron chelation. DFO has been reported

to react with 0'2-, however the rate constant for this reaction at physiological pH is about

102 M-1 s-1 [33], which is approximately eight orders of magnitude less than the overall

rate of non-enzymatic dismutation of 0'2 [74], and therefore this protective possibility is

unlikely. A more likely artifact is DFO's scavenging of'OH, which proceeds with a rate

constant of approximately 1010 M-1 s-1 [33]. However, using a dose of DFO identical to

ours, plasma concentrations appear to stabilize at less than 20 iM. This concentration is

presumably the same in other extracellular fluids, and is suggested to be too low for

significant scavenging of'OH or O2- [75]. In other studies using a similar dose of DFO,

and controls with ferrioxamine (which reacts with 'OH with the same rate constant as

DFO), no protection was afforded, reinforcing the conclusion that the protective effects

cannot be due to radical scavenging [76].

In any case, as mentioned before, a suitable metal catalyst is necessary for

formation of 'OH, and iron is the best candidate for this role in vivo. Since DFO is

accepted to be a very powerful iron chelator, it is likely 'OH formation is greatly reduced,

thereby limiting the very compound for which DFO has the potential to scavenge.

4-Hydroxy-2-Nonenal

4-hydroxy-2-nonenal (HNE) levels were measured as a further marker of oxidative

stress, in the LS limbs of all treatment groups, and compared to CON SS levels. Although

this aldehyde is formed from the decomposition of lipid hydroperoxides, it may actually









binds to, and modifies, proteins by interacting preferentially with lysine, histidine, serine,

and cysteine residues.

Since HNE is relatively stable, and can easily diffuse within the cell or escape the

cell, it has the potential to interact with many different cellular proteins [77]. This was

reflected in our western blot by the appearance of several bands at varying molecular

weights. In this study, we summed the net intensity of each band in each lane, in an

attempt to quantify total protein-bound HNE levels. Only the CYCLO group showed

significant attenuation of total protein-bound HNE levels after contractile claudication.

Several studies have demonstrated that increased levels of HNE are potentially very

cytotoxic, inhibiting enzymes, protein synthesis, protein degradation, calcium

sequestration, and exhibiting chemotaxic potential to neutrophils [78-80]. In addition,

HNE can significantly alter the cellular redox balance by rapidly conjugating with the

reactive thiol groups of glutathione (GSH). Indeed, GSH is part of the endogenous

cellular pathway of HNE metabolism [81]. However, since glutathione is believed to be

the primary buffer against reactive oxygen species in skeletal muscle [82], its loss due to

HNE conjugation may make the cell more susceptible to oxidative damage. This provides

an interesting concept since we have previously shown total GSH levels to be decreased

after contractile claudication.














CHAPTER 5
CONCLUSIONS

This is the first study to investigate the pathways of oxidant production after

contractile claudication. To establish a cause and effect relationship, we inhibited the

major pathways individually. This also provided information on the relative contributions

of each pathway. We predicted that both the xanthine oxidase pathway and activated

neutrophils are responsible for the oxidative damage and edema seen after contractile

claudication. The data supported these expectations, and provided insight into the

predominant pathway.

Inhibition of xanthine oxidase activity attenuated lipid peroxidation, the loss of

LDH activity seen in control animals, and reduced neutrophil infiltration. The conclusion

can therefore be made that xanthine oxidase-derived oxidants cause oxidative damage

after an acute bout of contractile claudication. In addition, oxidants from this source are

chemotactic to neutrophils.

Neutropenia reduced neutrophil infiltration by -82% compared to the ligated-

stimulated limb of control animals and had a protective effect on all parameters -

attenuating lipid peroxidation, protein oxidation, edema and the loss in LDH activity.

This clearly shows neutrophils are the predominant source of oxidants, and therefore

oxidative damage, after contractile claudication. Although increased neutrophil levels

have been documented in the vasculature following exercise claudication in humans, this

is the first study to show neutrophil recruitment into skeletal muscle after this condition.

This is in agreement with prolonged I-R studies in skeletal muscle, which have firmly









established the recruitment of leukocytes into tissue. This causes oxidative damage as the

membrane bound NADPH oxidase oxidizes NADPH to NADP+ while reducing

molecular oxygen to superoxide.

Finally, data from the DFO group suggest iron is heavily involved in the oxidative

damage seen after contractile claudication. This implies the Fenton reaction causes a

large part of the oxidative damage. Indeed, lipid hydroperoxide levels were reduced by

-43% and protein carbonyl content by -25% in the LS limb of the DFO group compared

to the LS limb of the CON group.

Since we were unable to actually measure iron levels in this study we cannot

definitively conclude that the protection afforded by deferoxamine is solely due to its

chelating characteristics. Indeed, deferoxamine has radical scavenging capabilities, which

could interfere with the results of this study. However, it is only a weak scavenger of

superoxide, and at the dose used in this study the concentration is likely too low for

significant scavenging of the hydroxyl radical. Therefore, the probable protective role

DFO plays in this study is as an iron chelator.

Future research should focus on the infiltration of neutrophils into tissue after

exercise claudication, and the "signals" leading to this infiltration. In addition, it should

be determined whether oxidative damage to skeletal muscle after exercise claudication is

necessary for, or a hindrance to, the muscular adaptations that occur with this condition.














LIST OF REFERENCES


[1] Pattwell D, McArdle A, Griffiths RD, Jackson MJ. Measurement of free radical
production by in vivo microdialysis during ischemia/reperfusion injury to skeletal
muscle. Free Radic Biol Med 2001;30:979-85.

[2] Hirose J, Yamaga M, Kato T, Ikebe K, Takagi K. Effects of a hydroxyl radical
scavenger, EPC-K1, and neutrophil depletion on reperfusion injury in rat skeletal
muscle. Acta Orthop Scand 2001;72:404-10.

[3] Fantini GA, Yoshioka T. Deferoxamine prevents lipid peroxidation and attenuates
reoxygenation injury in postischemic skeletal muscle. Am J Physiol
1993;264:H1953-9.

[4] Hickman P, Harrison DK, Hill A, McLaren M, Tamei H, McCollum PT, Belch JJ.
Exercise in patients with intermittent claudication results in the generation of
oxygen derived free radicals and endothelial damage. Adv Exp Med Biol
1994;361:565-70.

[5] Wijnen MH, Coolen SA, Vader HL, Reijenga JC, HufFA, Roumen RM.
Antioxidants reduce oxidative stress in claudicants. J Surg Res 2001;96:183-7.

[6] Silvestro A, Scopacasa F, Oliva G, de Cristofaro T, luliano L, Brevetti G. Vitamin
C prevents endothelial dysfunction induced by acute exercise in patients with
intermittent claudication. Atherosclerosis 2002; 165:277-83.

[7] Chiao JJ, Kirschner RE, Fantini GA. Iron delocalization occurs during ischemia
and persists on reoxygenation of skeletal muscle. J Lab Clin Med 1994; 124:432-
8.

[8] Sen CK, Packer L, H*anninen O. Handbook of oxidants and antioxidants in
exercise. Amsterdam ; Oxford: Elsevier, 2000.

[9] Kuwabara Y, Kato T, Sato A, Fujii Y. Prolonged effect ofleukocytosis on
reperfusion injury of rat intestine: real-time ATP change studied using (31)P
MRS. J Surg Res 2000;89:38-42.

[10] Bertuglia S, Colantuoni A. Protective effects of leukopenia and tissue
plasminogen activator in microvascular ischemia-reperfusion injury. Am J
Physiol Heart Circ Physiol 2000;278:H755-61.

[11] Fox SI. Human physiology. Boston: McGraw-Hill, 2002.









[12] Bainton D, Sweetnam P, Baker I, Elwood P. Peripheral vascular disease:
consequence for survival and association with risk factors in the Speedwell
prospective heart disease study. Br Heart J 1994;72:128-32.

[13] Fowkes FG, Housley E, Cawood EH, Macintyre CC, Ruckley CV, Prescott RJ.
Edinburgh Artery Study: prevalence of asymptomatic and symptomatic peripheral
arterial disease in the general population. Int J Epidemiol 1991;20:384-92.

[14] Dormandy JA, Murray GD. The fate of the claudicant--a prospective study of
1969 claudicants. Eur J Vasc Surg 1991;5:131-3.

[15] Schmieder FA, Comerota AJ. Intermittent claudication: magnitude of the
problem, patient evaluation, and therapeutic strategies. Am J Cardiol 2001;87:3D-
13D.

[16] Pernow B, Saltin B, Wahren J, Cronestrand R, Ekestroom S. Leg blood flow and
muscle metabolism in occlusive arterial disease of the leg before and after
reconstructive surgery. Clin Sci Mol Med 1975;49:265-75.

[17] Tisi PV, Shearman CP. Acute exercise and markers of endothelial injury. Eur J
Vasc Endovasc Surg 1998;16:169.

[18] Hein S, Scheffold T, Schaper J. Ischemia induces early changes to cytoskeletal
and contractile proteins in diseased human myocardium. J Thorac Cardiovasc
Surg 1995;110:89-98.

[19] Blaisdell FW, Steele M, Allen RE. Management of acute lower extremity arterial
ischemia due to embolism and thrombosis. Surgery 1978;84:822-34.

[20] Homer-Vanniasinkam S, Gough MJ. Role of lipid mediators in the pathogenesis
of skeletal muscle infarction and oedema during reperfusion after ischaemia. Br J
Surg 1994;81:1500-3.

[21] Kadambi A, Skalak TC. Role ofleukocytes and tissue-derived oxidants in short-
term skeletal muscle ischemia-reperfusion injury. Am J Physiol Heart Circ
Physiol 2000;278:H435-43.

[22] Tozzi-Ciancarelli MG, Penco M, Di Massimo C. Influence of acute exercise on
human platelet responsiveness: possible involvement of exercise-induced
oxidative stress. Eur J Appl Physiol 2002;86:266-72.

[23] Welboum CR, Goldman G, Paterson IS, Valeri CR, Shepro D, Hechtman HB.
Pathophysiology of ischaemia reperfusion injury: central role of the neutrophil. Br
J Surg 1991;78:651-5.









[24] Hickman P, McCollum PT, Belch JJ. Neutrophils may contribute to the morbidity
and mortality of claudicants. Br J Surg 1994;81:790-8.

[25] Edwards AT, Blann AD, Suarez-Mendez VJ, Lardi AM, McCollum CN. Systemic
responses in patients with intermittent claudication after treadmill exercise. Br J
Surg 1994;81:1738-41.

[26] Turton EP, Coughlin PA, Kester RC, Scott DJ. Exercise Training Reduces the
Acute Inflammatory ResponseAssociated with Claudication. Eur J Vasc Endovasc
Surg 2002;23:309-16.

[27] Smith JK, Grisham MB, Granger DN, Korthuis RJ. Free radical defense
mechanisms and neutrophil infiltration in postischemic skeletal muscle. Am J
Physiol 1989;256:H789-93.

[28] Prem JT, Eppinger M, Lemmon G, Miller S, Nolan D, Peoples J. The role of
glutamine in skeletal muscle ischemia/reperfusion injury in the rat hind limb
model. Am J Surg 1999;178:147-50.

[29] Seekamp A, Mulligan MS, Till GO, Ward PA. Requirements for neutrophil
products and L-arginine in ischemia-reperfusion injury. Am J Pathol
1993;142:1217-26.

[30] Tisi PV, Shearman CP. Biochemical and inflammatory changes in the exercising
claudicant. Vasc Med 1998;3:189-98.

[31] McCord JM. Oxygen-derived radicals: a link between reperfusion injury and
inflammation. Fed Proc 1987;46:2402-6.

[32] Curzio M, Roch-Arveiller M, Negro F, Giroud JP, Esterbaur H, Torrielli MV,
Dianzani MU. [Chemotaxis and chemokinesis of rat polymorphonuclear
leukocytes in response to 4-hydroxy-2-tetradecenal and 4-hydroxy-2-nonenal.].
Boll Soc Ital Biol Sper 1981;57:2479-85.

[33] Halliwell B, Gutteridge JM. Oxygen free radicals and iron in relation to biology
and medicine: some problems and concepts. Arch Biochem Biophys
1986;246:501-14.

[34] Biemond P, van Eijk HG, Swaak AJ, Koster JF. Iron mobilization from ferritin by
superoxide derived from stimulated polymorphonuclear leukocytes. Possible
mechanism in inflammation diseases. J Clin Invest 1984;73:1576-9.

[35] Mazur A GS, Saha A, and Carleton A. Mechanism of release of ferritin iron in
vivo by xanthine oxidase. J. Clin. Invest 1958;37:1809-17.









[36] Bralet J, Schreiber L, Bouvier C. Effect of acidosis and anoxia on iron
delocalization from brain homogenates. Biochem Pharmacol 1992;43:979-83.

[37] Green CJ, Gower JD, Healing G, Cotterill LA, Fuller BJ, Simpkin S. The
importance of iron, calcium and free radicals in reperfusion injury: an overview of
studies in ischaemic rabbit kidneys. Free Radic Res Commun 1989;7:255-64.

[38] Duran WN, Dillon PK. Effects of ischemia-reperfusion injury on microvascular
permeability in skeletal muscle. Microcirc Endothelium Lymphatics 1989;5:223-
39.

[39] Rubin BB, Romaschin A, Walker PM, Gute DC, Korthuis RJ. Mechanisms of
postischemic injury in skeletal muscle: intervention strategies. J Appl Physiol
1996;80:369-87.

[40] Dawson R, Jr., Biasetti M, Messina S, Dominy J. The cytoprotective role of
taurine in exercise-induced muscle injury. Amino Acids 2002;22:309-24.

[41] Jones DA, Jackson MJ, Edwards RH. Release of intracellular enzymes from an
isolated mammalian skeletal muscle preparation. Clin Sci (Lond) 1983;65:193-
201.

[42] Vina J, Gimeno A, Sastre J, Desco C, Asensi M, Pallardo FV, Cuesta A, Ferrero
JA, Terada LS, Repine JE. Mechanism of free radical production in exhaustive
exercise in humans and rats; role of xanthine oxidase and protection by
allopurinol. IUBMB Life 2000;49:539-44.

[43] Asami A, Orii M, Shirasugi N, Yamazaki M, Akiyama Y, Kitajima M. The effect
of allopurinol on interstitial purine metabolism and tissue damage in skeletal
muscle I-R injury. J Cardiovasc Surg (Torino) 1996;37:209-16.

[44] McCutchan HJ, Schwappach JR, Enquist EG, Walden DL, Terada LS, Reiss OK,
Leff JA, Repine JE. Xanthine oxidase-derived H202 contributes to reperfusion
injury of ischemic skeletal muscle. Am J Physiol 1990;258:H1415-9.

[45] Janeway C. Immunobiology 5 : the immune system in health and disease. New
York: Garland Pub., 2001.

[46] Keberie H. The biochemistry of desferrioxamine and its relation to iron
metabolism. Ann. NY Acad. Sci. 1964; 119:758-68.

[47] Rinaldo JE, Gorry M. Protection by deferoxamine from endothelial injury: a
possible link with inhibition of intracellular xanthine oxidase. Am J Respir Cell
Mol Biol 1990;3:525-33.









[48] Parks DA, Granger DN. Xanthine oxidase: biochemistry, distribution and
physiology. Acta Physiol Scand Suppl 1986;548:87-99.

[49] Klebanoff SJ, Waltersdorph AM. Inhibition of peroxidase-catalyzed reactions by
deferoxamine. Arch Biochem Biophys 1988;264:600-6.

[50] Bobeck-Rutsaert MM, Wiltink WF, Op den Kelder AM, van Euk HG, Leijnse B.
The distribution pattern of radioactive ferrioxamine administered intravenously in
rats. I. Acta Haematol 1972;48:125-8.

[51] Smith JK, Carden DL, Grisham MB, Granger DN, Korthuis RJ. Role of iron in
postischemic microvascular injury. Am J Physiol 1989;256:H1472-7.

[52] Zimmerman BJ, Parks DA, Grisham MB, Granger DN. Allopurinol does not
enhance antioxidant properties of extracellular fluid. Am J Physiol
1988;255:H202-6.

[53] Leong JC, Knight KR, Hickey MJ, Morrison WA, Stewart AG. Neutrophil-
independent protective effect of r-metHuG-CSF in ischaemia-reperfusion injury
in rat skeletal muscle. Int J Exp Pathol 2000;81:41-9.

[54] Sundin BM, Hussein MA, Glasofer S, El-Falaky MH, Abdel-Aleem SM, Sachse
RE, Klitzman B. The role of allopurinol and deferoxamine in preventing pressure
ulcers in pigs. Plast Reconstr Surg 2000;105:1408-21.

[55] Challiss RA, Hayes DJ, Petty RF, Radda GK. An investigation of arterial
insufficiency in rat hindlimb. A combined 31P-n.m.r. and bloodflow study.
Biochem J 1986;236:461-7.

[56] Mathien GM, Terjung RL. Muscle blood flow in trained rats with peripheral
arterial insufficiency. Am J Physiol 1990;258:H759-65.

[57] Watters C. A one-step biuret assay for protein in the presence of detergent. Anal
Biochem 1978;88:695-8.

[58] Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method
for carbonyl assay. Methods Enzymol 1994;233:357-63.

[59] Yan LJ, Traber MG, Kobuchi H, Matsugo S, Tritschler HJ, Packer L. Efficacy of
hypochlorous acid scavengers in the prevention of protein carbonyl formation.
Arch Biochem Biophys 1996;327:330-4.

[60] Hermes-Lima M, Willmore WG, Storey KB. Quantification of lipid peroxidation
in tissue extracts based on Fe(III)xylenol orange complex formation. Free Radic
Biol Med 1995;19:271-80.









[61] Belcastro AN, Arthur GD, Albisser TA, Raj DA. Heart, liver, and skeletal muscle
myeloperoxidase activity during exercise. J Appl Physiol 1996;80:1331-5.

[62] Bergmeyer H, Bernt E, Hess B. Methods in Enzymatic Analysis. New York:
Academic Press, 1965.

[63] Ibrahim B, Stoward PJ. The histochemical localization of xanthine oxidase.
Histochem J 1978;10:615-7.

[64] Lewis MS, Whatley RE, Cain P, McIntyre TM, Prescott SM, Zimmerman GA.
Hydrogen peroxide stimulates the synthesis of platelet-activating factor by
endothelium and induces endothelial cell-dependent neutrophil adhesion. J Clin
Invest 1988;82:2045-55.

[65] Gasic AC, McGuire G, Krater S, Farhood AI, Goldstein MA, Smith CW, Entman
ML, Taylor AA. Hydrogen peroxide pretreatment of perfused canine vessels
induces ICAM-1 and CD18-dependent neutrophil adherence. Circulation
1991;84:2154-66.

[66] Suzuki M, Asako H, Kubes P, Jennings S, Grisham MB, Granger DN. Neutrophil-
derived oxidants promote leukocyte adherence in postcapillary venules.
Microvasc Res 1991;42:125-38.

[67] McEver RP. Selectins: novel receptors that mediate leukocyte adhesion during
inflammation. Thromb Haemost 1991;65:223-8.

[68] Patel KD, Zimmerman GA, Prescott SM, McEver RP, McIntyre TM. Oxygen
radicals induce human endothelial cells to express GMP-140 and bind
neutrophils. J Cell Biol 1991;112:749-59.

[69] Mackie EJ. Immunosuppressive effects of cyclophosphamide in pigs. Am J Vet
Res 1981;42:189-94.

[70] Lee C, Kerrigan CL, Picard-Ami LA, Jr. Cyclophosphamide-induced neutropenia:
effect on postischemic skin-flap survival. Plast Reconstr Surg 1992;89:1092-7.

[71] Ninfali P, Perini MP, Bresolin N, Aluigi G, Cambiaggi C, Ferrali M, Pompella A.
Iron release and oxidant damage in human myoblasts by divicine. Life Sci
2000;66:PL85-91.

[72] Weiss SJ. Oxygen, ischemia and inflammation. Acta Physiol Scand Suppl
1986;548:9-37.

[73] Korthuis RJ, Granger DN, Townsley MI, Taylor AE. The role of oxygen-derived
free radicals in ischemia-induced increases in canine skeletal muscle vascular
permeability. Circ Res 1985;57:599-609.









[74] Halliwell B. Use of desferrioxamine as a 'probe' for iron-dependent formation of
hydroxyl radicals. Evidence for a direct reaction between desferal and the
superoxide radical. Biochem Pharmacol 1985;34:229-33.

[75] Symons MCR, Gutteridge JMC. Free radicals and iron : chemistry, biology, and
medicine. Oxford ; New York: Oxford University Press, 1998.

[76] Summers MR, Jacobs A, Tudway D, Perera P, Ricketts C. Studies in
desferrioxamine and ferrioxamine metabolism in normal and iron-loaded subjects.
Br J Haematol 1979;42:547-55.

[77] Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog
Lipid Res 2003;42:318-43.

[78] Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-
hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med
1991;11:81-128.

[79] Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, Uchida K. 4-
Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during
oxidative stress. Identification of proteasomes as target molecules. J Biol Chem
1999;274:23787-93.

[80] Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-
hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion
homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem
1997;68:255-64.

[81] Spitz DR, Malcolm RR, Roberts RJ. Cytotoxicity and metabolism of 4-hydroxy-
2-nonenal and 2-nonenal in H202-resistant cell lines. Do aldehydic by-products
of lipid peroxidation contribute to oxidative stress? Biochem J 1990;267:453-9.

[82] Sirsjo A, Kagedal B, Arstrand K, Lewis DH, Nylander G, Gidlof A. Altered
glutathione levels in ischemic and postischemic skeletal muscle: difference
between severe and moderate ischemic insult. J Trauma 1996;41:123-8.














BIOGRAPHICAL SKETCH

Andrew R. Judge was born in Northampton, England and raised in the village of

Harpole, just outside Northampton. He graduated from Loughborough University,

Leicestershire, England in 1996 with a bachelor's degree. In 1997 he moved to Lake

Charles, Louisiana where he attended McNeese State University. Here he received a

master's degree with a specialization in Exercise physiology. Andrew began his Doctor

of Philosophy degree in exercise physiology in 1999, at the University of Florida.