FREE RADICAL MEDIATED INJURY IN ISCHEMIC AND REPERFUSED CANINE GRACILIS MUSCLE FLAPS

A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph

by

BRIGITTE A. BMSSON

In partial fulfilment of requirements for the degree of Doctor of Veterinary Science August, 2000

@Brigitte A. Brisson, 2000

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To Simon, for your patience and love

To Mom and Dad, for your support and encouragement

To Franoise, in your memory

ABSTRACT

Free Radical Mediated Injury in Ischemic and Reperfused Canine Gracilis Muscle FIaps

Brigitte Anne-Marie Brisson, DMV University of Guelph, 2000

Advisor: Craig W. Miller DVFVI, MVSc

The first objective was to develop a canine muscle flap model of

ischemia~reperfusion. This model was used to determine whether free radicals were produced in ischemiclreperfused canine skeletal muscle and could be detected fiom effluent blood using electron paramagnetic resonance (EPR) spectroscopy and whcther the effects of ischemialrcperfiision could be detected by light microscopy. In six dogs, one isolated gracilis muscle flap was subjected to 4 hours of ischemia followed by 15 minutes of reperfusion. The contralateral flp served as a control (isolated but perfused).

EPR spectra characteristic of oxygen or carbon centered free radicals were detected in al1
six ischemiclreperfused flaps. Additional signals characteristic of tert-butyl hydronitroxide were detected in 4 flaps. No Free radical adducts were detected in the control flaps. Increased histological abnormalities were detected in the ischemiclreperfused flaps compared to control flaps.

The second objective was to evaluate whether local adenosine treatment prior to ?he ischemir. event could reduce ti-ee radical production and skeletal muscle damage using
the described model. Bilateral gracilis muscle flaps were isolated in 9 dogs. Control

flaps were pre-treated with saline (5mI) and the contralateral flaps were pre-treated with

10 mg of adenosine diluted in saline (5m!).Free radical adducts strong enough to allow

determination of hyperfine splitting constants and to measure signal intensity were detected fi-om the effluent blood in 5 dogs. The EPR spectra from 4 of these 5 dogs demonstrated attenuation of the signals fiom the adenosine treated flaps (mean attenuation from the 5 dogs being 24%). The EPR spectra From the remaining dogs were either absent (n=l), too weak for analysis (n=2), or potential signais were obscured by a copper ion complex (n=l). Histological evaluation revealed no difference between saline and adenosine treated muscle flaps.

These results confirni that frec radicals are produced at the tirnc of reperfusion in ischemic and reperfused muscle flaps and can be detected fiom the effluent blood using

EPR spectroscopy. Histological abnormalities are prcsent aRer 4 hours of ischemia and
15 minutes of reperfusion in this model. Adenosine appeared to reduce Free radical
production in this model but did not diminish histological abnormalities.

1 wish to acknowledge and thank my advisory committee Drs. Craig Miller,

Edward Janzen and Jill McCutcheon for their guidance and support throughout my DVSc program. 1 would specifically like to thank Dr. Craig Miller for helping with many of the research surgeries, and for providing good advice and rnentonng throughout my residency. Dr. Edward Janzen is thanked for the special trips made to Guelph to review papers and provide helpful discussion regarding the interpretation of EPR spectra. Finally, 1 thank Dr. Jill McCutcheon who patiently read the histological slides with me.

1 am grateful to Dr. Simon Pearce, William Sears and Gabrielle Monteith for their

assistance with the statistical analyses and Dr. Lany Haire for answering many questions and for his guidance with the evaluation of the EPR spectra. Finally, a special thank you goes to Amanda Hathaway and Danielle Redhill who assisted with the anesthetic inductions and maintenance, and to the surgical staff specifically Sue Kinsella and Judy Flannigan for their assistance in the operating theater.

1 wish to acknowledge the Ontario Veterinary College Pet Trust Fund for

providing the financial support that made this research possible. 1also extend my gatitude to those who were not directly involved with the project but nevertheless provided encouragement and support.

DECLARATION OF WORK PERFORMED

1 declare that with the exception of the items indicated below, al1 work reported in

this thesis was performed by me.

Amanda Hatliaway and Danielle Redhill provided assistance with the anesthetic induction and the anesthetic maintenance whilc surgery was being performed. Dr. Craig Miller performed one of the surgical procedures ~ . h e hvo expenments were performed n concurrently and assisted in performing a few of the remaining surgical procedures. Dr. Guoman Chen prepared the blood samples and scanned them by EPR spectroscopy. The determination of the hyperfine splitting constants was performed by myself. The embedding, sectioning and staining of the histological sampIes was performed by the histology department at the Ontario Veterinary College and 1 assisted Dr. Jill McCutcheon for the evaluation of al1 histological samples. Statistical analyses were perfomed by William Sears, Gabrielle Monteith and myself. Artistic assistance for the diagram of the gracilis muscle was provided by Tara Riddell

TABLE OF CONTENTS
PAGE ACKNOWLEDGEMENT DECLARATION OF WORK PERFORMED TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES
1.0 INTRODUCTION

1.1 STATEMENT OF GOALS AND HYPOTHESES 2.0 LITERATURE REVIEW 2.1 Introduction 2.2 Definition of a Free radical 2.3 Mechanism of action 2.4 Xanthine oxidase Free radical production 2.5 Neutrophil mediated cellular injury 2.6 "No reflow" phenornenon 2.7 Detection of Free radicals in biological systernc 2.8 Skeletal muscle damage caused by ischemia/reperf~ision 2.9 Host defense mechanisms 2.9.1 Enzymatic antioxidants 2.9.2 Non enzymatic antioxidants 2.10 Free radical scavengers 2.1 1 Ischemic preconditioning and adenosine 2.12 Models for skeletal ischemia 2.13 Skeletal muscle ischemia
3.0 DETECTION OF FREE RADICALS IN ISCHEMIC AND REPERFUSED CANINE GRACILIS MUSCLE FLAPS BY SPIN TFLAPPING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY 3.1 Abstract 3.2 Introduction 3.3 Materials and Methods 3.3 1 Animal preparation 3.32 Surgical protocol 3.33 EPR spectroscopy 3.34 Histology

3.35 Statistical analysis

PAGE

3.4 Results 3.41 EPR spectroscopy 3.42 Histology 3.5 Discussion 3.6 References
4.0 DETECTION OF FREE RADICALS BY SPIN TRAPPING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY (ST-EPR) IN ISCHEMIC AND REPERFUSED GRACILIS MUSCLE FLAPS AFTER ADENOSINE PRE TREATMENT 4.1 Abstract 4.2 Introduction 4.3 Materials and Methods 4.3 1 Animal preparation 4.32 Surgical protocol 4.33 EPR spectroscopy 4.34 Histology 4.35 Statistical analysis 4.4 Rcsults 4.41 EPR spectroscopy 4.42 Histology 4.5 Discussion 4.6 References
5.0 GENEEWL DISCUSSION AND CONCLUSIONS 5.1 FUTURE STUDIES

6.0 MASTER REFERENCE LIST

7.0 APPENDICES 7.1 APPENDIX - Detemination of hyperfine splitting constants 7.2 APPENDIX - Data From experiment # 1 7.2.1 Histological scores for the control (perfused) muscle

flaps 7.2.2 Histological scores for the ischemic reperfused muscle flaps
7.3 APPENDIX - Data from experiment # 2 7.3.1 Histological scores for the saline treated muscle flaps 7.3.2 Histological scores for the adenosine treated muscle

tlaps

PAGE
7.3.3 Statistical analysis of signal intensity data from experiment #2 7.3.4 Paired EPR spectra (saline and adenosine) 7.4 APPENDIX - Diagram of gracilis muscle flap 7.5 APPENDIX - Diagram of gracilis muscle flap and biopsy sites 7.6 APPENDIX - Gracilis muscle flap during dissection 7.7 APPENDIX - Perfused (control) and ischemic/reperfused gracilis n~uscle flaps during reperfusion 7.8 APPENDIX - Histological abnormalities 7.8.1 Control muscle flap 7.8.2 Ischemic reperfused flap

LIST OF TABLES
TABLE
PAGE

3.1

EPR spectral data for the PBN spin adducts extracted from the ischemic/reperfused muscle flaps
EPR spectral data for the PBN spin adducts extracted fiom the ischemic/reperfused muscle flaps (saline and adenosine treated)

73

4.1

114

LIST OF FIGURES
FIGURE
PAGE

2.1

Reduction of molecular oxygen

2.2

Biochemical pathway for the production of Free radicals in ischemic/reperfused tissue Haber-Weiss reaction or Fenton Chemistry

2.3

3.1

Typical EPR spectrurn from the effluent blood of a control (perfused) gracilis muscle flap

3.2

EPR spectrum of PBN radical adducts dctccted fiom effluent blood of n gracilis muscle flap containing carbon or oxygcn centered, and unknown free radical adducts
EPR spectra of PBN radical adducts detected from the effluent blood of gracilis muscle flaps containing signals characteristic of tert-butyl hydronitroxide and carbon or oxygen-centered Free radical adducts EPR spectra of PBN radical adducts detected in the effluent blood of gracilis muscle flaps containing large deflections suggesting the presence of manganese ions as well as carbon or oxygen-centered Free radical adducts
Mean 4-SEM summed histological scores according to treatment group for perfused and ischernic/reperfused flaps Mean +/- SEM summed histologicai scores according to hicpsy site fer perfiised and ischemiclreperfused flaps

77

3.3

3.4

3.5

3.6

vii

FIGURE Typical paired (saiine and adenosine treated) EPR spectra of PBN radical adducts containing carbon or oxygen centered free radical adducts. Spectra such as these were strong enough to allow the determination of hyperfine splitting constants and cornparison of signal intensity between saline and adenosine treated flaps Paired EPR spectra of PBN radical adducts showing the attenuation of the signal in the adenosine treated flap compared to the saline treated flap. These spectra also contain signals characteristic of tert-butyl hydronitroxide and di-tert-butyl nitroxide adducts Paired EPR spectra of PBN radical adducts detected in the effluent blood of gracilis muscle flaps containing large deflections suggesting the presence of manganese ions as well as electron centers Mean +/- SEM summed histological scores according to treatmcnt group for saline and adenosine treated flaps Mean +/- SEM summed histological scores according to biopsy site for saline and adenosine treatcd flaps Total histological score for saline and adenosine treated flaps according to each dog Determination of hyperfine splitting constants for oxygen or carbon centered free radical adducts (triplet of doublet) Determination of hyperfine splitting constants for tert-butyl hydronitroxide free radical adducts (doublet of triplet)

PACE

163

viii

CHAPTER 1

1.0 INTRODUCTION

Free radical mediated repertsion injury of skeletal muscle is a significant problem in both human and veterinary medicine. Extensive research efforts have been dedicated to the investigation of free radical mediated reperfusion injury in organs such as the heart and brain. In contrast, little research was initially directed toward skeletal muscle reperfusion injury. With the advent of advanced reconstnictive procedures allowing the re-implantation of severed digits and limbs and the common use of free vascularized muscle flaps, rcsearch investigating reperfusion injury in skeletal muscle has increased.

Several studies have implicated free radicals in the skeletal muscle damage seen following a penod of ischemia and reperfusion. However, the majority of these studies used indirect mcthods to associate fiee radical production with skeletal muscle reperfusion injury. These indirect methods include the measurement of skeletal muscle edema or necrosis, degradation products of lipid peroxidation, antioxidant enzyme levels and neutropliil infiltration. Electron paramagnetic resonance spectroscopy, also narned electron spin resonance (ESR) spectroscopy is a more direct method used tu detect free radicals. Spin trapping (ST) is an associated technique that consists of "binding" Free radicals to spin trapping agents. This "stabilizes" fiee radicals into so-called free radical adducts allowing detection. Spin trapping EPR (ST-EPR) can more directly identify a

number of radicals produced during ischemia/reperfusion. This technique can thereby

increase our understanding of the biology of ischernidreperfusion in skeletal muscle. For example, by measuring the presence, identity and intensity of Free radicals produced it
cm dernonstrate the mechanism and efficacy of dmgs purported to modify free radical

production in ischemic injury. Spin trapping EPR has been used extensively to investigate free radical production in a number of biological and rion-biological systems. To the author's knowlcdge, it has only been applied to the study of skeletal muscle twice.

The present study was divided into ttvo phases. Both phases used a canine mode1

of skeletal muscle ischernidreperfusion injury. The first phase consisted of detcrmining whether free radicals were produced in ischemictreperfused canine skeletal muscle and could be detecled h m effluent blood using EPR spectroscopy and whether light rnicroscopy could detect differences in histological damage between pcrfused (control) and ischemic/reperfused muscle. The second phase of this study evaluated whether preischemic local administration of adenosine was effective in attenuating frec radical production and histological skeletal muscle damage in the mode1 dcveloped in the first part of the study.

1.1 STATEMENT OF GOALS AND HYPOTHESES

The pnmary goal of this study was to develop a model of canine skeletal muscle ischemia/reperfusion injury to determine if fiee radicals are produced in this model and whether or not these cari be detected frorn the effluent blood using spin trapping EPR spectroscopy. It was also intended to determine whether light microscopy could detect differences between control and ischemic/reperfused muscle flaps. The second goal of this study was to detennine whether pre-treating muscle flaps with locaI injection o r adenosine would reduce the quantity of free radicals detected by EPR spcctroscopy and influence light rnicroscopic evidence of skeletal muscle damage.

The hypotheses were as follows:

1)

Free radicals will be generated in the ischemic/reperfused muscle flaps and will be detected from the effluent blood using spin-trapping electron paramagnetic resonance spectroscopy. No free radicals will be generated or detected in the perfused control flaps.

2)

More histological abnormalities will be identified in ischemidreperfused muscle flaps when compared to the perfused control flaps.

3)

Pretreatment of rnuscle flaps with adenosine will result in the attenuation or absence of fiee radical production as detected by spin trapping EPR when

compared to saline treated controls.

4)

More histological abnormalities will be identified in saline treated ischemic/reperfused muscle flaps than in adenosine treated ischemic/reperfused muscle flaps.

CHAPTER 2

LITERATURE REVIEW
2.1 Introduction

Ischemia can be broadly described as a state of inadequate blood supply. Prolonged ischemia results in tissue hypoxia and eventual cell death ifbfood flotv is not re-established. Di fferent tissues and organs Vary in their tolerance of ischemia; however, al1 reach a point of ineversible damage aAer prolonged cessation of blood flow. Reperfusion is necessary to prevent this damage. Ironically the oxygen-rich blood retuming to ischemic tissues may exacerbate the cellular damage of the ischemic event. This phenomenon of further cellular darnage caused by the re-establishment of blood flow to ischemic tissues or organs has been termed reperfiision injury (RI) (Kerrigan & Stotland 1993; McCord 1985). Much of the damage caused by RI is thought to be initiated by the production of free radicals derived from the oxygen thar is carried to the tissues during the resumption of blood flow. These radicals have been shoivn to initiate chemical chaiil reactions that produce vanous cytotoxic products. Toxic metabolites of oxygen are generated during normal cellular aerobic metabolism but under pathological conditions the process is accelerated beyond the tissue's ability to cope with the concentrations of Free radicals produced.

2.2 Definition of a Free Radical

A normal molecular bond consists of a pair ofelectrons with opposite spin

direction sharing the same orbital; this arrangement stabilizes the molecule. An orbital with only one free electron is termed unpaired and has increased reactivity. A free radical is an atom or molecule containing one or more unpaired electrons in its outer orbital. This arrangement makes the molecule very unstable and reactive. Because they are so reactive, free radicals have short half-lives that range from nanoseconds to milliseconds (Flaherty & Weisfeldt 1988).

Some authors prefer the t e m "reactive oxygen metabolite" over "oxygen-denved Free radical" when descnbing these rnoleculcs (McCord 1993). Certain molecules such as hydrogen peroside do not qualify as free radicals because they lack an unpaired electron; however, they are reactive molecules that contribute to reperfusion injury (McCord 1993). This thesis is concerned with the detection of true free radicals.

Each time a radical reacts with a nan radical molecule, another radical is produced

which may lead to a chain reaction. An electron is passed dong in oxidative or reductive reactions that can be thousands of events long. If two radicaIs react, the result is an end to the c h a h reaction and the formation of a stable molecule.

Oxygen is a naturally occurring free radical because it possesses two unpaired electrons in different orbitals and can lose (oxidation) or gain (reduction) electrons. Because the unpaired electrons spin in the same direction, oxygen can only accept one electron at a time from its donors. This creates the potential for free radical formation.

Oxygen is important for energy production through oxidative phosphorylation. In

oxidative phosphorylation, tetravalent electron transfer results in formation of water (Fig

2.1). In contrat, if only one, nvo or three electrons are accepted, Ciee radicals are then

produced.

0,

+ le- -+O2'

O?' + le' + 2H' H,O,- + 1e' + H' -

-+ H202

-+ HO' + H 2 0
+ HzO

HO. + le' + H'

Figure 2.1 The mitochondrial cytochrorne oxidase complex reduces molecular oxygen by the addition of four electrons fonning water. If only one, two or three electrons are added, reactive oxygen species are produced (superoxide, hydrogen peroxide and hydroxyl free radicals).

2 3 Mechanism of Action .
Several biological sources of reactive oxygen metabolites have been identified. These sources include: the xanthine oxidase system loated within the cytoplasm of endothelial cells and other tissue cells (e.g. muscle cells), the NADPH oxidase reacion in the membrane of activated polymorphonucIear leukocytes, the mitochondrial electron transport chain, the arachidonic acid metabolicm and catecholamine auto-oxidation. Of these, the first two have attracted the most attention in the study of free radical-mediated

RI.

Granger was the firsi to present evidence for the involvement of free radicals in Ri (Granger, Rutili, & McCord 198 1). The administration of superoxide dismutase, an antioxidant enzyme, just before reperfusion prevented injury to capillaries in a feline intestinal mode1 of partial ischemia. Similar evidence supporting the role of oxygenderived free radicals in Ri has been reported to affect many organs including the heart (Arroyo, Kramer, Leiboff, et al. 1987; Bolli, Jeroudi, Patel, et al. 1989; Kuzuya, Hoshida. Kim, et al. 1994), kidney (Baker, C o q , & Autor 1985), liver (Maruba Yshi, Dohi, et al. 1986), brain (DemopouIos, Flamm, Pietronigro, et al. 1980; Flamm, Demopoulos, Seligman, et al. 1978), skin (Angel, Narayanan, Swartz, et al. 1986; Irn, Shen, Pak, et al. 1984; Miller, Chen, & Janzen 1999) and small intestine (Granger 1981; Parks, Bulkley, & Granger 1983; Parks, Granger, Bulkley, et al. 1982). There is also considerable evidence that oxygen-derived fiee radicals are involved in several disease processes besides RI, including shock (Crowell, Jones, & Smith 1969; Itoh & Guth 198S), oncogenesis

(Kozumbo, Trush, & Kensler 1985) and cutaneous bums (Bjork & Arturson 1983; Cetinkale, Belce, Konukoglu et al. 1997).

Proposed mechanisms responsible for reperfiision injury include: 1) Direct cellular toxicity of free radicals
2) Neutrophil rnediated cellular injury (direct or free radical mediated)

3) Microvascular dysfunction with platelet plugging resulting in no reflow

4) The loss of high energy phosphates

It is thought that the xanthine oxidase (XO) system plays an essential role in the production of free radical mediated reperfusion injury in several organs. Initially described in the feline intestine by Granger (Granger 198 l), this system has also been described in the heart, liver, spleen, kidney, small intestine, lung and skin (Al-Khalidi & Chaglassiam 1965; Battelli 1980; Battelli, Della Corte, & Stipe 1972; Engerson, McKelvey, Rhyne, et al. 1987; Ibrahim & Stoward 1978; Picard-Ami, MacKay, & Kemgan 1991; Roy & McCord 1983). It has also been described in skeletal muscle (AIKhalidi 1965; Donon, Zhong, Chiu, et al. 1993; Ibrahim 1978; Jarasch, Bruder, & Heid 1986; Jarasch, Grund, & Bruder 1981; Smith, Carden, Grisham et al. 1989; Smith, Carden, & Korthui; 1989). Controversy exists with regards to the role of XO fiee radical production in skeletal muscle. Roy et al. (Roy 1983) reported that xanthine dehydrogenase (XD) to XO conversion did not occur in rat skeletal muscle subjected to 1 hour of ischemia. Supporting these findings were the results of another study reporting low XO and XJI activities in pig and human skeierai muscie (3oriori 1493j. Xuwwe,

others have dernonstrated significant XD to XO conversion in skeletal muscle subjected to longer periods of ischernia (Smith i989C; Smith l989A). Even if XO concentrations in skeletal muscle are lower than in other organs (Al-KhaIidi 1965; Smith 1989C; Smith 1989A), XO appears to be present in sufficient arnounts to result in skeletal tissue damage (Smith 1989A). In addition, several studies have demonstrated that inhibiting the

XO systern resulted in decreased skeletal tissue injury and irnproved muscle function
(Korthuis, Granger, Townsley, et al. 1985; McCutchan, Schwappach, Enquist, et al. 1990; Smith 1989A).

Experiments using immuriolocalization techniques have found high concentrations of XO in the capillary endothelial cells of human and bovine skeletal muscle (Jarasch 1986; Jarasch 1981). Arroyo et al. (Arroyo, Carnichael, Bouscarel, et al.

1990) documented free radical production by endothelial cells; this was associated with
endothelial ceIl injury or death. brahim et al. (Ibrahim 1978) further localized XO to the sarcolemma and mitochondria of aerobic muscle fibers. Species differences in XO levels have also been reported in the skin of pigs, rats and humans (Picard-.Mi 1991).

2.4 Xanthine Oxidase Free Radical Production

During hypoxia, depletion of high energy phosphates occurs (Haljamae & Enger
1975) and intraceilular adenosiiie triphosphate (ATP) is degradcd to hypoxanthine (Fig

2.2). Larsson et al. (Larsson, Gidioff, Lewis, et al. 1982) documented a marked rise in
the venous hypoxantnine ieveis upon reperfusion of iscivmiic: iiiuscft: i k c iii iii~ii.

Hypoxanthine is normally oxidized by XD to form xanthine using nicotinamide adenine dinucleotide (NAD) as an electron acceptor. With this process, NAD is converted to NADH. Free radicals are not produced through this normal pathway. Shortly after the onset of ischemia, the ce11 membrane electroiyte gradient is compromised and the sodium-calcium pumps are affected. Increased intra-cellular calcium concentration is thought to increase the activity of proteases. Such enzymes permit the transfom~ation (reversible or irreversible) of XD (type D, present in large quantities) to XO (type O) (Battelli 1980; Battelli 1972; Engerson 1987; Roy 1983). Xanthine oxidase, unlike XD, is unable to catalyze the transformation of xanthine using NAD; instead it must use oxygen therefore it also generates free radicals.

When the ischemic tissues are reperfused, accumulated XO catalyzes the transfornlation of hypoxanthine to santhine, producing within seconds, large amounts of superoxide anion (o~'). This is the begiming of a free radical chain reaction. The superoxide anion is very unstable and reacts rapidly to form hydrogen peroxide (H202). Hydrogen peroxide by itself is not a potent oxidizing agent; however, hydroxyl radicals (HO') are very potent and form in the presence of iron, superoxide radical and hydrogen peroxide through the iron derived Haber-Weiss reaction also known as the modified Fenton reduction (Haber & Weiss 1934). Iron is normally bound in the femc state (Fe ' to transfemn or stored in the 7
)+'

ferric state (Fe

bound to femtin. These complexes are especially difficult to reduce.

However, the superoxide radical is capable of reducing fefic iron (Fe j ' ) released from

femtin during ischemia to the ferrous state (Fe ") pennitting the Haber-Weiss reaction to occur to form hydroxyl radicals (Fig 2.3). The hydroxyl radical is considered to be the most destructive biological fiee radical. It c m initiate lipid peroxidation in the phospholipid bilayer of organelle membranes by removing hydrogen From the polyunsaturated fatty acids and forming lipid fiee radicals. These free radicals also react to form new reactive metabolites. These may propagate ceIl injury by causing loss of membrane structure and function, initiating free radical chain reactions, attacking structural molecules and dismptin DNA strands. Free radicals can also increase capillary penneability and enhance pIatelet and neutrophil adherence to vesse1 walls.

The importance of the XO mechanism is confirmed by the attenuation of tissue injury when oxygen deficient blood is used to reperfuse the ischemic tissue (Korthuis, Smith, & Carden 1989; Walker, Lindsay, Labbe, et al. 1987).

ATP
.I. v

ADP

4
AMP

.1
Adenosine Xanthine Dehydrogenase

4
Inosine Ca *

& Calpain

4
Hypoxanthine

Xanthine oxidase
)

O,' + H,Oz + Urate

l '
0 2

Reperfusion

Figure 2.2 Biochemical pathway for the production of free radicals in ischernic and
reperfused tissue. Adapted fiom McCord et al., 1987

O;

+ ~ e "+ O, +

~ e "

~ e "+ H202 + ~ e ' ) + HO' + OH-

0; + H202 --+O2 + HO. + OH-

Figure 2.3 Combination of reactions known as the iron-catalyzed Haber-Weiss reaction

or as superoxide-dnven Fenton reaction. Superoxide radicals reduce femc iron (Fe '3) to the ferrous state (Fe ") permitting the Haber-Weiss reaction to occur.

2.5 Neutrophil Mediated Cellular Injury

Under conrrolled conditions superoxide has a physiologically protective role. Activated polyrnorphonuclear leukocytes possess a nicotin-amide adenine dinucleotide oxidase on their plasma membrane that reduces molecular oxygen phosphate (NADPH) to superoxide (Goldstein, Cerqueira, Lind, et al. 1977; lyer & Quastel 1963). The enzyme (NADPH oxidase) that catalyzes the transformation of O, to O!' is dormant in resting phagocytes but becomes functional when the leukocyte is activated by opsonized bacteria, the complement fragment C5a, or other stimulating factors (Sacks, Moldow, Craddock, et al. 1978). Because the onset of oxidant production in activated neutrophils is associated with an abrupt increase in phagocyte oxygen consumption, the metabolic event that gives nse to these oxidants is known as "the respiratoty oxidative burst". In

HO' addition to O!', several oxidizing agents such as H202, and hypochloms acid (HOC])
are produced enabling leukocytes to destroy invading microorganisms (Thomas, Gnsham, & Jefferson 1983). Although oxidants are manufstured by the phagocyte strictly for intemal use they have the potential to leak into surrounding tissues and result in tissue darnage. The genetic condition resulting in ar. inability of neutrophils to produce
O?' causes a life threatening condition known as chronic granulomatous disease. The

neutrophils of affected individuals are impaired in their ability to kill ingested microorganisrns resulting in rccurrent Iocal infections, septicernia and possibly death at a

young age. Unfortunately, excessive activation of neutrophils c m result in significant

leakage of oxidants and severe injury to the surrounding tissues.

Free radical mediated reperfusion injury appears to occur in hvo phases. Initially, reperfusion of ischemic tissues produces superoxide radicaIs through the XO system mainly at the level of the endothelial cells, as explained previously. Superoxide radicals have been shown to activate a superoxide-dependent neutrophil chemotactic factor present in plasma (Petrone, English, Wong, et al. 1980). Secondary injury occurs when neutrophils attracted to the site of inflammation adhere to the endothelium and are activated (Cambria, Anderson. Dikdan, et al. 1991; Carden, Smith, & Korthuis 1990; Menger, Pelikan, Steiner, et al. 1992). Accumulation of leukocytes within skeletal muscle following ischemia has been documented as early as 15 to 30 minutes after the beginning of reperfusion (Menger 1992; Smith, Grisham, Granger, et al. 1989). Cambria et al. (Cambria 1991) demonstrated that ischemiafreperfusion injury in skeletal muscle directly activated polyrnorphonuclear (PMN) Leukocytes resulting in increased superoxide radical production. That study also revealed that the degree of PMN activation was directly related to the extent of ischemic injury (Cambria 1991).

Leukocyte mediated reperfusion injury was first described by Romson et al. (Romson, Hook, Kunkel, et al. 1983) in a canine myocardial infarction model, The results of hvo studies perfomed by this group of investigators demonstrated that leukocyte depletion or the administration oFSOD and CAT before reperfusion effectively decreased myocardial infarct size (Romson 1983; Jolly, Cane, Bailie, et al. 1984). Based on these findings the authors concluded that leukocytes exacerbated ischemia reperfusion injury in myocardial tissue and that reperfusion injury was at least in part mediated by
r 1 toxic metaboiites of oxygen producea by neurropiiiis (lbrrisuri *nn.i , ~-u 1 .i. y* n o m \ L YO J i I Y O-+).

Similar studjes perfoimed in ischemic and reperfused skeletal muscle have also supported these findings and documented decreased vascular permeability afler white blood ce11 depletion (Iwahon, Ishiguro, Shimizu, et al. 1998; Korthuis, Grisham, & Granger 1988). Inhibiting leukocyte adherence to the endothelial wall has also been shown to prevent increases in vascular permeability supporting once more the role of leukocytes in reperfusion injury (Carden 1990). Excessive adherence of neutrophils to the endotheliurn is thought to be in part responsible for the "no reflow" phenomenon causing perpetuation of ischeniia and eventually necrosis of the tissues (Carden 1990).

2.6 "No Reflow" Phenornenon

The "no reflow" phenomenon is described as a failure to adequately reperfuse ischernic tissues aAer blood supply is re-established (Guilford 1994). Strock et al. (Strock & Majno 1969) confirmed 30 years ago that some capillaries fail to perfuse on reinstitution of blood flow in skeletal muscle. Ultimately, this leads to further ischemia (partial or cornpiete) and potentially irreversible tissue damage. Conflicting reports have been published regarding the cause and mechanism of the "no reflow" phenomenon. The rnicrocircuIation of a reperfused organ may be compromised by several factors: persistent vasoconstriction (Pemberton, Anderson, & Barker 1996), vascular obstruction caused by thrombosis (Hartsock, Seaber, & Urbaniak 1989), extnnsic compression from interstitial ederna (Jerome, Akimitsu, & Korthuis 1994) ancilor neutrophil migration resulting in capillary blocking (Engler, Schrnid-Schonbein, & Pavalee 1983), post-ischemic

hypotension, and increased blood viscosity due to leakage of plasma through damaged capillaries (Menger, Sack, Barker, et al. 1988).

2.7 Detection of Free Radicals in Biological Systems

As previously discussed free radicals are extremely reactive rnolecules and their half-lives are short. This makes their detection difficult even in controlled experimental conditions. These difficulties are compounded by the variables inherent in biological systems. Because of these limitations, most of the methods used to detect free radicals are indirect. These indirect techniques do not detect free radicals themselves but detect secondary metabolites. Included in this category are the measurement of lipid peroxidation metabolites and conjugated dienes. A number of lipid derivatives can be assayed when investigating oxidative stress. Malondialdehyde (MDA) is a controversial but widely used indicator of lipid peroxidation. Other indirect methods consist of evaluating parameters that are affected by ischemia and reperfusion. Such parameters include vascular penneability and tissue infarction. In these cases, if moleciiles known to inhibit or scavenge free radicals successfully reduce the changes in the measured parameter after ischemia and reperfusion it is assumed that Free radical production was responsible for the abnormalities. More direct methods for detection of free radicals include chemiluminescence and spin trapping electron paramagnetic resonance (ST-EPR) spectroscopy. Chemiluminescence allows evaluation of oxidative stress in vivo. Products of oxidative stress such as peroxides and singlet oxygen spontaneously emit light. Uniortunateiy, the ernitted iignt is ofien very weak. hpiificaiiuii uC iiic ~igiiai

c m be achieved by using probes such a luminol. This molecule andfor its products emit s light when they react with oxygen radicals.

Electron paramagnetic resonance spectroscopy depends on the use of hornogenous static magnetic fields and electromagnetic radiation. The unpaired electrons behave as small magnets when placed in a magnetic field (Halliwell & Gutteridge 1989). Electron paramagnetic resonance spectroscopy can directly detect the amount of energy absorbed by an electron when its spin flips frorn "down" to "up" (Poole 1983). The detection of free radicals using this technique gives rise to a spectrum. Therefore, if a spectrum is obtained, free radicals are definitely present (Halliwell 1989). Unfortunately, because of the very short half-life of hee radicals if a spectrum is not obtained it does not preclude the presence of free radicals.

Spin trapping is a technique in which free radicals that are not detectable by EPR because of their very short half-lives react with a scavenger or "spin trap" producing a new, inore stable free radical, with a longer half-life, and with a spectrum that can be detected by EPR (Halliweil 1989), The hydroxyl and superoxide radicals produced in rnany biological systems after ischernia and reperhsion are undetectable by EPR and need to be converted to more stable species. A number of compounds are used to stabilize oxygen radicals. The most commonly used spin trap compounds are nitrones such as a-phenyl N-tert-butylnitrone (PBN) and 5,5-dimethylpyrroline-N-oxide (DMPO) (Halliwell 1989).

2.8 Skeletal Damage Caused by Ischemia/Reperfusion

Skeletal muscle, although more resilient to ischemia and reperfusion injury than other organs such as the brain or heart, may suffer irreversible damage resulting in skeletal muscle dysfunction and infarction afier prolonged ischemia (Kenigan 1993). The higher tolerance of skeletal muscle to ischemia and reperfusion rnay be explained by the reported lower rate of D- to O- conversion of XO (Dorion 1993; Roy 1983), higher phosphocreatine store and slower ATP depletion of skeletal muscle during ischemia (Hams, Wafker, Mickle, et al. 1986; Pang, Yang, Zhong, et al. 1995) when compared to other tissues.

Situations in which skeletal muscle may be subjected to ischemia and reperfusion include limb revascularization afier use of a tourniquet, at the time of re-implantation of a sevcred digit or limb or with free n~uscle transfer used for reconstnictivs surgery. flap However, any situation involving cessation or diminution of blood flow to skeletal muscle for a period of time followed by reperfusion may result in tissue injury. The skeletal muscle injury created by ischemidreperfusion can result in serious clinical consequences including failure of a muscle flap transfer used to reconstruct a large surgical or traumatic wound, failure of re-implanted digits or limbs or amputation of a limb afler tourniquet application. The result may be a serious functional impairment or in some circumstances death of the patient. Understanding the mechanisms of ischemia/reperfusion and evaluating the efficacy of pharmaceutical interventions may obviate rnany of these serious consequences.

Comparison of different studies with regards to muscle damage secondary to ischemidreperfusion is difficult due to the use of different models, use of various methods to produce ischemia and different duration ofischemia with or without subsequent reperfusion. In addition, several different methods are used to evaluate muscle damage. Harris et al. (Harris, Leiderer, Peer, et al. 1996) evaluated skeletal muscle of hamsters after varying duration of ischemia to determine changes in several parameters including vesse1 diameter, macromolecular leakage, leukocyte adherence, functional capillary density and ce11 nuclei necrosis. This study revealed that 1 to 2 hours

of ischemia resulted in the accumulation of leukocytes but this did not result in increased
muscle damage after 2 hours of reperfusion. After 3 hours of ischemia, the rnean endothelial thickness increased as did the number of non viable cells. After 4 hours of ischemia, rnacromolecular leakage and decreased functional capillary density were noted in addition to the previous findings. Afier 5 hours of ischemia, almost 50 % of the capillaries demonstrated no reflow. (Hams 1996). Other studies evaluating ischemic skeletal muscle histologically (using light or electron microscopy) concur with these findings and report no histological abnormalities afier 2 hours of ischemia (Blebea, Kerr, Shumko, et al. 1987; Harris 1986; Jennische & Hansson 1986; Patterson & Klenerman 1979; Scully, Shannon, & Dickersin 1961). Most studies agree and report that muscle necrosis is evident histologically afier 7 to 12 hours of ischemia (Blebea 1987; Harris 1986; Patterson 1979; Scully 1961). Histological abnormalities have been shown to appear between 4 and 7 hours of ischemia (Harris 1986; Jennische 1986; Scully 1961). However, confiicting reports exist as ro wnether 4 hours oi isciieiiiia Cuiiuws b u;iria

periods of reperfusion results in histological abnomalities. Dahlback et al. (Dahlback & Rais 1966), using light microscopy, reported moderate to marked degenerative changes in skeletal muscle subjected to 4 hours of ischernia. These changes were thought to appear after 2 hours or more of ischemia and to worsen after reperfusion (Dahlback 1966). In contrast to these results, Patterson (Patterson 1979) reported minimal ultrastructural abnormalities and Scully, (Scully 1961) reported minimal histological abnormalities after 4 hours of ischemia. Blebea et al. (Blebea 1987) found no changes in histochemical staining of canine gracilis muscle flaps aAer 4 hours of ischemia and 1 hour of reperfusion. However, 6 and 8 hours of ischemia followcd by reperfusion resulted in a high percentage of muscle necrosis (Blebea 1987). These authors documented a worsening of the muscle injury in the reperfused muscle flaps when compared to the non reperfused flaps. These findings support reperfusion injury as a source of muscle damage in addition to the ischemic damage itself. An interesting finding was the increased muscle necrosis at the level whcre the major vascular pedicle entered the gracilis muscle. This was also reported to be supportive of reperfusion injury as it was the site that was exposed the most to oxygenated blood nt the time of reperfusion (Blcbea 1987). In contrast to this, Labbe et al. (Labbe, Lindsay, & Walker 1987) attributed the increased necrosis in the central core of the gracilis muscle subjected to 4 hours of ischemia and 48 hours of reperfusion to the greater vulnerability of this central region to compressive edema.

2.9 Host Defense Mechanisms

The consequence of oxygen being essential to life is that free radicals are produced in small quantities by al1 body tissues under normal circumstances. Defense mechanisms have evolved to reduce the damage caused by these free radicals. Enzyrnatic and non-enzymatic antioxidants are present in the body. Oxygen radicals will only produce tissue damage if they exceed the antioxidant defenses of the tissues.

2.9.1 Enzyrnatic antioxidants

The first line of defense is the cytochrome oxidase electron transport system in which, oxygen is reduced by four electrons to form water, thus avoiding free radical formation. Under normoxic conditions, approximately 98 % of intra-cellular oxygen is removed by this system (Fig 2.1). The rernainder ofthe Free radicals produced in the rnitochondria (- 2%) leak into the cytosol. The body has adapted by developing systems to scavenge these srnall but significant radicak before they cause cellular damage (Fridovich 1978).

Superoxide dismutase (SOD) is the next b e l of defense. McCord and Fridovich discovered in 1969 that SOD removes the superoxide radical by accelerating its transformation to hydrogen peroxide (McCord & Fridovich 1969). Because SOD enzymes generate H,02, work in conjunction with HzOt they removing enzymes. Two into enzymes: catalase and glutathione peroxidase can convert H202 water. Catalases
gnnpar fc he -rr

---

re!a?ive!y inefficient at low H+O - concentrations. Glutathione peroxidase ..

(GSH) is thought to be the major processor of H,O,. Reduced GSH detoxifies H202 and

is regenerated. No defense mechanism exists to protect directly against the hydroxyl radical.

It is noteworthy that SOD, by inhibiting the accurniilation o ~ 0 ~ ' n o t prevents only

tissue injury from the direct attack by 02'but may also prevent the activation of the superoxide-dependent chemoattractant which prevents further tissue injury caused by the accumulation of activated neutrophils (PvlcCord, Wong, Stokes, et al. 1980; Petrone
1980).

2.9.2 Non-enzymatic antioxidants

Final lines of defense consist of miscellaneous groups of non-enzymatic antioxidant compounds including: ascorbic acid, retinol, a-tocopherol (vitamin E), carotene, cystine, ceruloplasmin and transfemn (Reilly, Scliiller, & Bulkley 199t).

P-

2.10 Free radical scavengers

Free radical scavengers are compounds that remove free radicals and therefore decrease tissue injury. Scavengers Vary in their mechanism of action and the species of free radical that tliey can remove. Allopurinol, deferoxarnine, dimethyl sulfoxide

(DMSO) SOD are a few examples of Eree radical scavengers. AlIopurinol is a and
structural analog of hypoxanthine that specifically inhibits XO. The inhibition results f?om binding of oxypurinol, a metabolite of allopurinol to XO (Spector 1988). In addition to its ability to inhibit XO, evidence suggests that allopurinoi may aiso funcrion

as a scavenger of hydroxyl radicals (Moorhouse, Grootveld, Halliwell, et al. 1987), chlorine radical and hypochlorous acid (Das, Englernan, Clement, et al. 1987). Deferoxamine is a chelating agent that prevents the reduction of Fe" by superoxide and therefore prevents the Haber-Weiss reaction (Halliwell & Gutteridge 1984). In addition to its iron binding capabiiities, deferoxamine has been shown to directly scavenge fiee radicals (Sinaceur, Ribiere, Nordmann, et al. 1984). Dimethyl sulfoxide is a specific and effective scavenger of the hydroxyl radical. Dimethyl sulfoxide is highly diffusible across lipid membranes allowing it to act at intra-cellular sites of free radical production such as the mitochondria. Over the years several drugs such as heparin, corticosteroids, calcium channel blockers and others were advocated for their potential free radical modi@ing effects (Bushell, Klenerman, Davies, et al. 1996; Knight, Zhang, Monison, et al. 1997; Li, Cooley, Fowler, et al. 1995; Salm, Sutter, Marx, et al. 1996; Tauber, Lehr, Ney, et al. 1994; Wright, Kerr, Valeri, et al. 1988; Zavitsanos, Huang, Panza, et al. 1996).

Studies of skeletal muscle ischernia have demonstrated that SOD and catalase (Lee, Cronenwett, Shlafer, et al. 1987; Menger 1992; Reikeras & Ytrehus 1992; Yamamoto, Takenaka, Shibata, et al. 1997), allopurinol (Appell, Duarte, Gloser, et.al. 1997; Asami, Oni, Shirasugi, et al. 1996; Ferrari, Battiston, Casella et al. 1996; Menger 1992; Oredsson, Plate, & Qvarfordt 1995; Oredsson, Plate, & Qvarfordt 1991), dimethyl sulfoxide (Korthuis, Granger, Townsiey et al. 1985), mannitol (Oredsson, Plate, & Qvarfordt 1994; Walker, Lindsay, Labbe et al. 1987) and deferoxamine (Smith 1989C; Zavitsanos 1996) afford protection against skeletal muscle injury as evidenced by

decreased muscle necrosis, decreased tissue edema and improved skeletal muscle function.

2.1 1 Ischernic Precoaditioning and Adenosine

Free radical production is one of niany factors responsible for muscle damage following isclicmidreperfusion. However, it represents an aspect of this syndrome that is amenable to intervention by pharmacologic agents either at the time of reperfusion, or if possible, prior to the ischemic event. An exarnple of the latter is when a planned muscle flap transfer is performed in reconstructive surgery.

Ischemic preconditioning is a technique in which a tissue is subjccted to repeated, sliort periods of ischemia and reperfusion prior to a prolonged pcriod of ischemia (Murry, Jennings, & Reimer 1986). Murry et al. (Muny 1986) first rcported ischemic preconditioning (IPC) in the canine myocardium. In this study, ischemic preconditioning consisted of four cycles of 5 minutes of ischemia each followed by 5 minutes of reperfusion. This technique was found to decrease myocardial infarction by 75 % following a 40 minute ischemic insult (Murry 1986). Results of studies performed later revealed that a single short period of ischemia and reperfusion prior to the major ischemic insult was sufficient to induce myocardial protection against ischemia (Liu, Thomton, VanWinkle, et al. 1991; Mizumura, Nithipatikom, & Cross 1995; Schulz, Rose, & Heusch 1994). Mounsey et al. (Mounsey, Pang, Boyd, et al. 1993) were the first to demonstrate the appiicaibiiiry of iscnemic preconuiiioning in skciciai iiiu3cit: (pidiic

latissimus dorsi flaps) using three episodes of alternating 10 minutes occlusion and reperfsion prior to a 4 hour period of ischemia. This study demonstrated a 25% increase in skeletal muscle flap survival. Pang et al. (Pang 1995) then showed that ischemic preconditioning was effective in decreasing ischemic damage in porcine gracilis muscle flaps. In this model, three periods of 10 minutes of ischemia and reperfusion prior to a 4 hour ischemic event and 48 hour reperfsion period resulted in a 44 to 62% decrease in muscle infarction (Pang 1995). In contrast to the reports in cardiac muscle, a single (Mounsey 1992; Pang 1995) or double (Mounsey 1992) period of ischemia and reperfusion was not successful in reducing skeletal muscle infarction. Jerome et al. (Jerome, Akimitsu, Gute, et al. 1995) also demonstrated the attenuation of capillary noreflow in canine gracilis muscle flaps following IPC. Along the same lines, Liauw et al. (Liauw, Rubin, Lindsay, et al. 1996) evaluated sequential ischemia in a canine gracilis muscie flap model. In this study, one gracilis muscle flap was made ischernic for 5 hours followed by 48 hours of reperfusion. Then, the contra-lateral gracilis muscle flap was made ischemic for 5 hours and reperfused for 48 hours. The results of this study revealed

a 60% mean reduction in nluscle necrosis associated with sparing of ATP in the second
flap. These results were thought to suggest a protective niechanism acquired afier the first ischemic event (Liauw 1996).

Kitakaze et al. (Kitakaze, Hori, Takashima, et al. 1993) noted that adenosine levels were increased in canine myocardial tissue following ischemic pre-conditioning. Several studies later corroborated this finding and concluded that adenosine appeared to
be the trigger mediator for ischemic pre-conditioning (Pximirsu, Guie, & k r i i i u i s 19%;

Downey, Cohen, Ytrehus, et al. 1994; Pang, Neligan, Zhong, et al. 1997; Papanastasiou, Estdale, Homer-Vanniasinkam, et al. 1999; Urabe, Mura, Iwamoto, et al. 1993). Adenosine is an endogenous nucleotide primarily formed as a degradation product of adenosine triphosphate (ATP). The half life of adenosine is less than I O seconds aAer intravenous injection (Klabunde 1983). In dogs, high concentrations of adenosine are reieased within seconds after the onset of ischemia (Fox, Reed, Meilnan, et al. 1979). Several cardiac studies (Liu 1991; Yao & Gross 1994) and also skeletal muscle studies (Forrest, Neligan, Zhong, et al. 1997; Lee, Schroeder, Shah, et al. 1996; Pang 1997) documented that treatment with exogenous adenosine pnor to the ischemic event mimicked the effect of ischemic pre-conditioning. These studies also revealed however, that treatment with adenosine during ischemia or at the tinie of reperfusion was not effective in augmenting ischemic tolerance of skeletal muscle (Forrest 1997; Pang 1997).

The use of adenosine to precondition tissues rather than perforrning IPC may be advantageous. Administration of adenosine is more practical than IPC for the treatment of o r g n s where vascular access is limited. Also, treatment with adenosine does not have
the disadvantage of lengthening the duration of the procedure as would be required if IPC

was performed. This is likely to be an important factor when lengthy f-ree flap transfers are perfomed.

Adenosine has been administered experimentally as a slow infusion over a period of 8 to 10 minutes just prior to the ischemic event (Forrest 1997; Lee 1996; Yao & Gross
1994). Forrest et ai. (Forrest iYY7j fbiiowe tilt: siow iril'usiuii f clciiijsiiic b 1

minutes of normal perfusion pior to the ischemic event. in this study, several dosage protocols were evaluated for adenosine pretreatment in pig latissimus (0.5 and 2 mg) and gracilis (10 and 20 mg) muscle flaps (Forrest 1997). The results of this study revealed that adenosine at al1 doses tested reduced muscle infarction. However, increased dosage of adenosine did not result in decreased muscle infarction. It was determined that approximately 0.5 mg of intraarterial adenosine was required to precondition a n~uscle flap of about 55 gm for increased ischemic tolerance in the pig (Fonest 1997).

The precise mechanisms of action of ischemic preconditioning and adenosine pretreatment which results in increased ischemic tolerance of tissues are uncertain (Pang 1997). It is thought that adenosine acts via stimulation of adenosine receptors A, (Downey 1994; Liu 1991; Pang 1997; Wang, Drake, Sajjadi, et al. 1997) and A, (Liu, Richard, Olsson, et al. 1994; Wang 1997) as well as activation of K,-p channels (Jerome 1995; Pang 1997; Yao 1994). Myocardial ischemic preconditioning has been demonstrated to preserve ATP levels, reduce metabolite accumulation, restore highenergy phosphate production capacity, improve recovery of cardiac rnuscle function and decrease infarct size (Liu 1991; Murry 1986; Muny, Richard, Reimer, et al. 1990; Urabe 1993). Studies perforrned by Pang et al. (Pang 1997; Pang 1995) revealed that ischemic preconditioning or treatment of skeletal muscle with adenosine prior to ischemia and reperfusion resulted in decreased muscle infarction, a slower rate of energy depletion and decreased metabolite accumulation during sustained ischemia. A lower neutrophilic myeloperoxidase activity was also noted in skeletal muscle treated with adenosine prior to ischerniafreperfusion cornpared to the tirne matciied iscnemic conrroi (Fang i 9971.

In addition to the previously mentioned effects, it has been siiggested that, adenosine possesses an antioxidant activity that coiild result in the inhibition of the

XD/XO system in tissues. Through this mechanism, adenosine could play a role in
decreasing fiee radical production at the time of reperfusion (Pang 1997). Cronstein et al. (Cronstein, Daguma, Nichols, et al. 1990; Cronstein, k a m e r , Weissrnann, et al. 1983; Cronstein, Rosenstein, Kranier, et al. 1985) demonstrated that adenosine, acting via .A2 receptors, potently inhibits superoxide eneration by activated neutrophils. This could therefore result in a reduction of free radical mediated tissue injury. As well, adenosine and ischemic preconditioning may inhibit neutrophil endothelial adhesion and emigration (Akimitsu 1996; Cronstein, Levin, Philips, et al. 1992; Grisharn, Hernandez, & Granger 1989). Magginvar et al. (Maggirwar, Dhanraj, Somani, et al. 1994) investigated the role of adenosine in providing cytoprotection through an incrense in the activity of antioxidant enzymes. The results of this study revealed that stimulation of A, adenosine receptors by pretreating cells with adenosine resulted in a 3 to 3 fold increase in activity of SOD, catalase and glutathione peroxidase. In addition, activation of this specific receptor decreased the degrce of lipid peroxidation in these cells as assessed by malondialdehyde assay (Maggirwar 1994). These results represent indirect evidence that adenosine could, through this mechanism, indirectly decrease free radical mediated reperfusion darnage.

2.12 hIodels for Skeletal Ischernia

Several models of ischemia have been developed to evaluate RI in skeletal muscle. Hind Iimb ischemia conditions are ofien creaied by appiicaiiuii o r a iuuiiiiqei.

This technique has been criticized due to the potential for partial biood flow to be maintained during tourniquet placement. However, Klenerman et al. (Klenerman & Crawley 1977) reported a blood flow of less than 1% during tourniquet ischemia. A major disadvantage reported with the use a tourniquet to create ischemia is the direct trauma caused to the tissues which results in more severe and lasting darnage to the muscle lying beneath the tourniquet than the muscle distal to it (Patterson 1979). Other studies have created ischemia in specific muscles such as the cremaster, gracilis and rectus fernoris muscles (Faust, Chiantella, Vinten-Johansen, et al. 1988; Hoballah, Mohan, Schipper, et al. 1996; O'Farrell, Chen, Seaber, et al. 1994; O'Farrell, Chen, Seaber, et al. 1995). The vascular anatomy of these muscles is not ahvays ideal for establishing consistent ischemia followed by reperfusion. Kuzon et al. (Kuzon, Walker, Mickte, et al. 1986) developed a mode1 of controlled ischemia and reperfusion using a modification of the isolated canine gracilis muscle flap described by others (Duran & Renkin 1 974; Emerson, Parker, & Jelks 1 974; Kille & Klabunde 1984). The gracilis muscle is classifieci as a type II muscle (Mathes & Nahai i98 1). Type II muscles are vascularized by one or more dominant pedicles entering at the origin or insertion of the muscle as well as minor pedicles entering the muscle belly (Mathes 1981). If elevated bascd on the dominant pedicle alone, type II muscles will survive since the angiosomes vascularized by the ligated minor pedicles will be vascularized via choke anastomoses (Mathes & Nahai 1982). This type of muscle is ideal for studies of ischemia and reperfusion because it allows isolation of the muscle on a single vascular pedicle. Kuzon et al. (Kuzon 1986) found no significant difference in blood flow, oxygen uptake, glucose uptake and lactate release foiiowing dissection of rhe muscic rlap arid iigaiiii cjf the

minor vascular pedicle. The control (dissected but non ischemic) muscle flap was stable for 9 hours at body temperature and remained unaffected by reperfusion of the contra lateral ischemic muscle flap. This study also revealed that 2 hours of ischemia is completely reversible with 4 hours of reperfusion but that 7 hours of ischemia resulted in irreversible changes even after reperfusion (Kuzon 1986).

2.13 Skeletal Muscle Ischemia

Ischemia, if maintained long enough, has been shown to result in irreversible skeletal muscle injury. However, reperfusion of ischemic tissue results in further tissue
damage. Blebea et al. (Blebea 1987) documented histochemically worsening of initial

ischemic canine skeletal muscle injury after reperfusion. In contrat, Belkin et al. (Belkin, Brown, LaMorte, et al. 1988) investigated skeletal muscle infarction using triphcnyltetrazolium chloride reduction assay and concluded that the duration of the ischemic period rather than the reperfusion itself was the determinant of skeletal muscle injury. However, the results of other studies also support that reperfusion of ischemic skeletal muscle is responsible for a significant worsening of the injury (Dahlback 1966; Menger 1992; Oredsson 1991; Oredsson, Qvarfordt, & Plate 1995). Several studies have investigated the effects of ischemia and reperfusion on skeletal muscle and have postulated that free radical production at the time of reperfusion was likely involved in the process. Most studies investigating reperfusion injury have associated free radical production and skeletal muscle damage using indirect methods.

Walker et al. (Walker 1987) compared normal reperfusion to controlled reperfusion with reduced concentrations of oxygen in a canine gracilis muscle flap mode1 subjected to 5 hours of ischemia followed by 48 hours of reperfusion. These authors found that controlled reperfusion significantly reduced skeletal muscle necrosis as assessed by nitroblue tetrazolium dye studies (Walker 1987). Subsequent experiments reveaIed that the addition of fiee radical scavengers (catalase, SOD and mannitol) further decreased muscle necrosis when compared to untreated reperfused muscle flaps (Walker 1987). In these experiments, the fiee radical scavengers were administered at the time of reperfusion rather than prior to the ischemic event. The rationale for this treatrnent regimen was to simulate the clinical situation in which pre-iscliemic therapy would not be possible (Walker 1987). The sparing effect of hypoxic reperfusion was also confirmed in a study performed by Korthuis et al. (Korthuis 1989).

Reperfusion of skeletal tissue following a period of ischemia has been shown to result in increased vascular penneability (Korthuis, Granger, Townsley et al. 1985; Korthuis 1988). This increase in vascular permeability rcsults in the formation of tissue edema and increased total water content of the tissue. In certain muscles, this increasc in local edema if severe c m result in an increased compartmental pressure which in turn may diminish arterial inflow to a muscle cornpartment. The exact pathophysiologic process which results in the increased vascular permeability remains uncertain but endothelial damage initiated by fiee radicals is thought to be involved.

Korthuis et al. (Korthuis 1985) used a canine gracilis muscle flap mode1 to evaluate the effect of various free radical scavengers on vascular permeability postischemia. The osrnotic reflection coefficient and isometric capillary pressure were used
to assess changes in capillary permeability. The increased vascular permeability

produced by four hours of ischemia followed by hvo hours of reperfusion was reduced significantly by the administration of SOD,catalase, allopurinol and DMSO. The administration of antihistamines (cimetidine and diphenhydrarnine) did not attenuate the increase in vascular permeability when compared to the untreated control group. Thcse findings suggest that histamine, which is known to cause increased vascular permeability

in skeletal muscle is not involved in the changes in vascular pemeability created by

ischernidreperfusion (Korthuis 1985). The authors concluded that free radicals were responsible for the increased vascular pernieability caused by ischemia and reperfusion (Korthuis 1985). In another experiment (Appel1 1997), aIlopunnol and vitamin E, administered to mice subjected to a hindlimb tourniquet for 90 minutes and then reperfused, were found to reduce oxidative stress, capillary permeability, and endothelial disturbances. Allopurinol was recommended prior to application of tourniquet to reduce oxidative changes (Appel1 1997). Several other studies, using vanous ischemidreperfusion models, have also docutnented the beneficial effect of various fiee radical scavengers in decreasing vascular permeability in skeletal muscle (Ferrari 1996; Oredsson 1991 ; Seyanla 1993; Yamamoto 1 997).

The beneficial effect of allopurinol in ischemic and reperfused muscle has

provided support for the theory thac nu 1s an irnporiai~i r~ieiair reperfsii of

.-fi

iiijrj:

(Asarni 1996; Korthuis 1985; Oredsson 1991; Smith 1989A). Smith et al. (Smith 1989A), measured the XO activity in non-ischemic and ischemic rat muscles, and then supplemented rats with a tungsten-nch, moIybdenum-deficient diet to accomplish XO depletion. Another group of rats was suppIemented with oxypurinol to determine if it would protect the muscle against increased vascular permeability caused by reperfusion injury. Increased XO levels were found in the ischemic muscle compared to the control group. Inhibition of XO with oxypurinol and depletion of XO levels tvith a tungstensupplemented diet both provided protection [rom increased permeability induced by ischemia and reperfusion. These findins were believed 10 support the role of XO in the pathogenesis of ischemia and reperfusion injury in skeletal muscles (Smith 1989A). Another study (McCutchan, Schwappach, Enquist, et.al. 1990) supporting these results, cavenger (dimethylthiourea), tungsten found that treatment with either an H202 supplementation or allopunnol before reperfusion increased gastrocnemius muscle peak tetanic force of contraction and decreased muscle H102production in rats subjected to hindlimb ischemia and reperfusion (McCutchan 1990). These results were thought to suggest that XO-denved H,02 contributes to reperfusion injury and that allopurinol iniproved post-ischemic miiscle function (McCutchan 1990).

Ferrari et al. (Ferrari 1996) used a mode1 of ischemic rat paws to evaluate endothelial permeability (muscular edema) by measunng compartmental pressures and lipid peroxidation metabolites (malondialdehyde) with and without allopurinol therapy.
A 11--..Ar-i Al A l l U ~ U l l l ~ Un e r n n e o A Ubbib-bu

e n J n t h ~ 1 ; n l n n ~ a h i l i hy ~ ~~ t
-~AUW.*A--A-.

=--.---------

nnt rediice

lipid peroxidation.

The authors explained these findings by the fact that XO is present mainly in endothelial

cells. Allopunnoi would therefore have inhibited XO at the endothelial level and thus inhibited the production of a certain subset of free radicals. This could explain the diminished endothelial permeability. Free radicals derived fi-om other pathways such as leukocytic production were thought to be responsible for the increased lipid peroxidation both in the allopurinol treated and the control group (Ferrari 1996).

Lcukocytes have been proposed as a source of fiee radical production and their role in reperfusion injury has been studied extensively in several tissues. It is postulated

that leukocytes have the sarne deleterious effect in ischemic and reperfused skeletal
muscle as in other tissues since leukocyte depletion prior to reperfusion results in attenuation of rnicrovascular and parenchymal cell dysfunction. Korthuis et al. (Korthuis

1988) used a canine gracilis muscle flap mode1 of ischemia to demonstrate the role of
leukocyes in reperfusion injury. ARer 4 hours of ischemia, flaps were reperfused with either whole blood or leukocyte-depleted blood. Reperfusion of flaps with leukocyte deficient blood largely prevented the increased vascular permeability seen in the group reperfused with whole blood (Korthuis 1988). An experiment evaluating reperfusion injury in ischemic hindlimbs OC rats found that administration of dirnethylmyleran

(DMM) seven days before the ischemic event to obtain a selective leukopenia
Ineutropenia), improved depolarization of resting transmembrane potential difference

after reperfusion compared to controls (Yokota, Minei, Fantini, et al. 1989).

Administration of SOD and catalase had the same effect as DMM. These findings were said to irnplicate leukocyte-generated free radicals as mediators of rcperfusion injury in post-ischemic muscle (Yokota i Wj.

Although these findings support the role of leukocytes in reperfusion of ischemic skeletal muscle, no objective assessment of leukocyte accumulation in reperfused tissue had been published. Smith et al. (Smith 19893) designed a study in which the main objective was to quantitatively assess neutrophil accumulation in ischemic and reperfused skeletal muscle. The authors used an assay for the neutrophil enzyme myeloperoxidase. During the four-hour period of ischemia, the niyeloperoxidase enzyme levels did not increase. However, a 26 fold increase in enzyme activity was noted in the reperfused muscle aAer 1 hour of reperfusion indicating a large influx of neutrophils into the tissue.
A concurrent decrease in reduced glutathione was noted whilc the SOD and CAT levels

rneasured one-hour aAer reperfusion remained unchanged. These results confirmcd that neutrophils accumulate in large numbers in tissues subjected to ischemia and reperfusion. Also, these findings were thought to once again support that ischemia/repetfusion injury may be in part due to free radicals generated by activated leukocytes (Smith 19898). Later, iri vivo rnicroscopy techniques allowing direct evaluation of microcirculatory injury in ischemic reperfused skeletal muscle confirmed using a hamster model that reperfusion elicited marked enhancernent in Ieukocyte rolling and leukocyte adherence and was accompanied by significant leakage of rnacromolecules from capillaries (Menger 1992). In a rat cremaster muscle model similar findings revealed that ischemia followed by reperfusion is associated with a reduction in capillary perfusion, a nse in leukocyte adherence to venular endotheliurn and a reduction in muscle viability (Pemberton, Anderson, & Barker 1994). Administration of SOD and allopurinol was effective in preventing leukocyte r o i h g and aunerence; fnis was <nougiii io suppvri i k iui uf itee

radicals produced at the tirne of reperfusion in attracting leukocytes to the site of injury (Menger t 992).

Carden et al. (Carden 1990) evaluated whether prevention of neutrophil adherence or specific neutrophil depletion would attenuate the microvascular dysfunction seen in post-ischemic skeletal muscle. The authors wanted to determine whether neutrophils must adliere to the endothelid wall to cause endotheiial ce11 injury especially because of

the previous implication of leukocyte adherence to the endothelial wall in the no reflow
phenornenon. Specific neutrophil depletion was perforrned using a polyclonal antineutrophil serum. Prevention of neutrophil adherence was achieved by admjnistering monoclonal antibody directed against adherence proteins. Ischemia and reperfusion were performed in a canine gracilis muscle model. Results revealed that both prevention of neutrophil adherence and neutrophil depletion prevented the increase in vascular permeability noted in the control groups. The authors concluded that neutrophils play an important role in post-ischemic endothelial dysfunction and that adherence of neutrophils is essential for the development of increased vascular penneability (Carden 1990). Muscle necrosis was also evaluated following blockade of adhesion mulecules of neutrophils (Petrasek, Liauw, Romaschin, et al. 1994). In contrast to the results of the previously mentioned study, this investigation revealed that prevention of neutrophil adherence alone did not result in a significant decrease in muscle necrosis. However, prevention of neutrophil adherence and fasciotomy resulted in the least muscle necrosis

and was significantly less than if fasciotomy was perfomed alone (Petrasek 1994).

Based on the findings of these studies, it appears that white blood ceIl depletion, inhibition of leukocyte adherence and administration of free radical scavengers al1 result in attenuation of reperfusion injuy in skeletal muscle. These findings suggest that leukocytes are a source of free radical production in ischemic/reperfused skeletal muscle. These results represent once again indirect evidence of the role of free radicals in reperfiision injury.

Measurement of free radicals in vivo is hanipered by their short half-lives. Several methods have been developed ta measure Free radical activity using indirect measures. The main approach has consisted of measuring the products of lipid peroxidation. Lipid peroxidation is a chah reaction initiated by oxygen free radicals, resulting in oxidative deterioration of polyunsaturated fatty acids. A number of lipid derivatives can be assayed when attempting to document oxidative stress. One of the first methods developed involved the measurement of malondialdehyde using the thiobarbituric acid reaction (Ohkawa, Ohishi, & Yagi 1979). This test also measures other aldehydes and is known as TBA-reactant substance determination. Malondialdehyde (MDA) is a controversial but widely used indicator of lipid peroxidation. Chopineau et al. (Chopineau, Sommier, & Sautou 1994) used MDA levels
as an indicator of free radical production in a rabbit mode1 of tourniquet ischemia. Lipid

peroxidation metabolites were noted to be increased 1 minute after release of the tourniquet, but did not Vary dunng the ischemic period (Chopineau 1994). Seyama (Seyama 1993) measured MDA in rat skeletal muscle before and after a period of 2 hours

o;isc~eniia as

&fiz i hi repc&s.vii. f

T . - -&.A.. J I: 11115 J L U U

,lm, L Cv L U L U I J U --.,nolacl C ~ U UIUL

thq+& A n d
A . L L I ~ .

levels were not different after ischemia alone but were significantly increased after reperfusion when compared to the preischemic and postischemic levels. In addition, this experiment evaluated the effect of free radical scavengers (allopurinol or SOD and catalasc) on MDA levels and revealed suppression of MDA levels after treating with free radical scavengers (Seyarna 1993). Supporting these findings, is a study performed by Asami et al. (Asami 1996) that treated canine gracilis muscle flaps with allopurinol prior to reperfusion. This resulted in decreased measured levels of xanthine, uric acid, MDA aiid CPK (Asami 1996). Ferrari et al. (Ferrari 1996) found an increase in MDA levels afler varying petiods of ischemia in a rat hind paw model of ischemia. However, in contrast to the results of Asami et al. (Asami 1996) and Seyarna et al. (Seyama 1993), pretreatment with allopurinol in this model did not significantly decrease the MDA levels although it did improve compartmental pressures (Ferrari 1996).

Malondialdehyde is the end product of a long chain of reactions and does not appear to be specific to free radical mediated lipid peroxidation. For this reason, other researchers have used the identification and quantification of conjugated dienes to assess lipid peroxidation (Lindsay, Walker, Mickle, et al. 1988). Conjugated dienes have been called the chemical signature of oxygen free radical mediated injury (Romaschin, Rebeyka, Wilson, et al. 1987). These molecules are formed fiom the attack of the ce11 membrane free fatty acids by Cree radicals. Lindsay et a!. (Lindsay 1985) have documented an increase in hydroxyconjugated dienes within minutes of reperfusion of canine gracilis muscle flaps subjected to 3 and 5 hours of ischemia and followed by 3 hours ofreperfusion. Tiiese findings aiiiiuugii siiii iii diii vidolic~ USB f dicn!

production were thought to be direct evidence of lipid peroxidation at the time of reperfusion in canine skeletal muscle. Hams et al. (Harris 1986) evaluated the production of conjugated dienes after vanous periods of ischemia, Their results suggested that conjugated dienes accumulated in tissues at the time of reperfusion after 7 and 7 hours of ischemia. The skeletal muscle subjected to only 2 hours of ischemia was able to normalize the level of conjugated dienes present within 60 to 90 minutes afler reperfusion. However, in the muscle subjected to 7 hours of ischemia the increased levels of conjugated dienes persisted at approximately 2.5 times the levels of the control muscle flaps (Harris 1986). These findings correlated with wide spread necrosis of skeletal tissue in the 7-hour ischemic group. The authors concluded that conjugated dienes are in fact produced after ischemia and reperfusion of skeletal muscle but hypothesized that the production and metabolism was dependent on the duration and reversibility of the damage to the tissue (Harris 1986).

A reimplantation mode1 using rabbit tibialis antenor muscle was developed to assess histological changes and muscle function after 5 and 8 hours of ischemia in control, SOD-treated and DMSO-treated groups (Feller, Roth, Russel, et al. 1989). The group treated with SOD following five hours of ischemia had essentially normal Enction and histological appearance after 5 weeks but no protection was seen in the eight-hour ischernic group. Dimehylsulfoxide (DMSO) had no beneficial effect after five hours of ischemia but improved function and histological abnornlalities were noted afler eight hours of ischemia when compared to the controls (Feller 1989). The authors conclude that although increased preoperative iscnemic iniewais Uecrcast: iiit: ciiaiice fur scccs&.i:

reimplantation, free radical scavengers administered prior to reperfusion may improve the outcome (Feller 1989). The findings of this study may also suggest that different types of free radicals (superoxide versus hydroxyl) are responsibie for tissue damage depending on the duration of the ischemic period.

Ischemia of skeletal muscle results in depletion of energy stores (Harris 1986; Pang 1995). Cronenwett et al. (Cronenwett, Lee, Shlafer, et al. 1989) evaluated the role of free radicals in reperfusion injury using a rat h i r i d h b tourniquet model. Five minutes before a three-hour ischemic period followed by 19 hours of reperfusion, either saline or

SOD and catalase were administered to the rats. The evaluation of calcium uptake by
sarcoplasmic reticulum was used to evaluate sub-cellular muscle function. In the ischemic-saline group, the calcium uptake was reduced by 48 % compared to the nonischenlic controls. Rats treated with SOD and catalase showed a less severe reduction (27 %) in calcium uptake. These results were thought to implicate free radicals in abnonal calcium transport in reperfused muscle (Cronenwett 1989). Hypotheses for the decreased calcium uptake noied in a similar study performed by the same group (Lee 1987) included a defect in ATPase function as well as permeability changes due to lipid peroxidation.

The studies mentioned up to this point have used indirect measures such as increased vascular permeability (edema), increased level of lipid peroxidation metabolites, decreased antioxidant enzyme level or increased XO activity to assess free
t raical~ cue ifi s z c ~qrcsy;;sib!,o radical production. hese sturiies suggest ihai k :

for tissue injury based on the fmdings that the administration of Free radical scavengers or antioxidants reduce or prevent reperfusion injury.

Electron paramagnetic rcsonance spectroscopy is a more direct method that can be used to detect fiee radicals. Electron paramagnetic resonance spectroscopy (EPR) in conjunction with spin trapping (ST), has been used extensively to investigate free radical production in ischemic and reperfused myocardium (Arroyo 1987; Bolli, Jeroudi, Lai, et al. 1988; Bolli 1989; Li, Zughaib, Jeroudi, et al. 1993; Kuzuya 1994; Bolli, Zughaib, Li, et ai. 1995; Itoh, Yanagishita, Aoki, et al. 1999).

Only a few studies have used EPR spectroscopy to measure free radical activity in ischemic and reperfused skeletal muscle. DeSantis et al. (DeSantis & Pinclli 1994) measured free radical formation in rat rectus femoris muscle flaps. PBN was injected at different times (before ischemia, after ischernia but before reperfusion and afler reperfusion) and EPR spectroscopy perfonned on the effluent blood. No EPR signal was detected before ischemia (control) or afler 15 minutes of ischemia. PBN radical adducts were detected after 30,60, 120, 180 minutes o f ischemia and also when PBN was injected 10 minutes after resumption of the blood flow (DeSantis 1994). Choudhury et al. (Choudhury, Sakaguchi, Koyano, et al. 1991) measured free radical formation in ischemic and reperfused canine gracilis muscle by perfomling EPR spectroscopy on fiozen muscle tissue samples. Three groups were evaluated: a control group, an ischemic but untreated group and an ischemic group treated with coenzyme Q10, a fiee radical scavenger. n e mean vaiue or C r K uliensiiy
C " m

wits

Cuulid i b ;igifi~zt:jr Iiigh~i :Hc iii

ischemic/untreated group when compared to the control (non-iscliemic) and ischemic/treated groups (Choudhury 1991). As well, rnorphological evaluation of the muscles using a dye exclusion test revealed decreased muscle injury afler treatment with coenzyme Q10. These fmdings were thought to support the role of free radicals in reperfusion injury of skeletal muscle (Choudhury 1991).

In s u m r n q , reperfusion injury occurs when ischemic tissues are reperfused with oxygenated blood. Several studies have provided evidence supporting the role of free radicals in mediating reperfusion injury in severai different organs. Free radicals arc produced through different mechanisms but it is thought rhat the free radicals produced immediately at the time of reperfusion ofischemic tissues are mainly derived from the

XO system. Free radicals derived From activated neutrophils are produced later. Due to
their structure, free radicals are unstable and reactive making thern difficult to idcntify in biological systems. Many studies investigating reperfusion injury have used indirect methods to provide evidence supporting their role in reperfusion injury. These methods rely mainly on the improvernent of a measured parameter after treating one group of experiments with an antioxidant molecule and comparing it to an untreated group.

Several compounds have been shown to attenuate reperfusion injury. Adenosine, a

degradation product of ATP is one such agent. However, the mechanism of action by which adenosine attenuates reperfusion injury has not been completely elucidated. Some evidence suggests that adenosine may have an antioxidant effect. However, this possibility has not yer been investigated.

CHAPTER 3

DETECTION OF FREE RADICALS IN ISCHEMIC AND REPEWUSED CANINE GRACILIS MUSCLE FLGPS BY SPIN-TRAPPING USING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY (ST-EPR).'

BRIGITTE A. BRISSON, DMV', CRAIG W. MILLER, DVM, MVSC,

Diplomate ACVS ', GUOMAN CHEN, P ~ D ' JILL MCCUTCHEON, , DVM, P ~ D ? , EDWARD G. JANZEN, P W .

From the Department of Clinical Studies, Ontario Veterinary College, University of

Guelph, Guelph, Ontario, Canada NlG 2W1.

rom the Department of Pathobiology, Ontario Veterinary College, University of

Guelph, Guelph, Ontario, Canada N 1G 2 W 1.

Dr. Chen's present address is University of Toronto, Department of Botany, Toronto Ontario, Canada.

This study was supported by grants from the Pet Trust Fund and the Natural Sciences and Engineering Research Council of Canada.

--

Accepted for publication in AJVR Professor emeritus

Presented at the Twenty Sixth Annual Symposium of the American College of Veterinary

Surgeons, October 1998, Chicago, Il.

The authors acknowledge Dr Larry D.Haire for his assistance with the evaluation of the

EPR spectra.

Objective - The purpose of this study was to determine whether free radicals are

produced in a model of canine skeletal muscle ischernia/reperfusion and whether these could be detected fiom effluent blood using electron paramagnetic resonance (EPR) spectroscopy. Another objective was to determine whether skeletal muscle damage was detectable by light rnicroscopy in this model. Study Design - Prospective experimental study. Animals - Six healthy mongrel dogs.

Procedure - Under anesthesia, paired gracilis muscles were isolated leaving only the
major vascular pedicle intact, Four hours of complete vascular occlusion was created in one flap; the contra-lateral flap served as a non-ischemic control. Ischemic flaps were then reperfused for 15-minutes. a-Phenyl-N-rert-butylnitrone (PBN), a spin-trapping agent was administered parenterally 1-hour prior to reperfusion. Following reperfusion, effluent blood samples were collected from each muscle flap for EPR spectroscopy. Muscle biopsies were taken for histopathological evaluation. Results - No spin-adduct was detected from control flaps. Radical spin-adducts were detected in al1 six ischemic/reperfused muscle flaps. The principal signals identified were characteristic of oxygen or carbon-centered radicals. Significantly more muscle damage was detected on histological examination of ischernic/reperfused flaps compared with control flaps. Conclusions - Free radicals were detected from the effluent blood in this model using EPR spectroscopy and were not detected irorn perfusecconrroi daps. in adiiion,

pathological changes were detectable in skeletal muscle subjected to 4 hours of ischemia followed by reperfusion.

Clinical Relevance - The described mode1 will be useful for studying the effect of
various antioxidants and free radical scavengers in reducing ischemia/reperfusion mediated skeletal niuscle damage.

INTRODUCTION

Free radicals (e.g. superoxide and hydroxyl) and reactive oxygen species (e.g. hydrogen peroxide) are toxic metabolites of oxygen that are generated during normal cellular aerobic nietabolism. These molecules, if produced in amounts exceeding the protection afforded by normal defense mechanisms, are capable of initiaring lipid peroxidation leading to the production of oxygen and carbon-centered free radicals that may contribute to tissue damage

'.

Free radicals have been implicated in several disease processes, Their role in repcrfusion injury has been studied in several different organs such as the heurt '83, brain
4

, small intestine and skin

"'.

Skeletal muscle rnay be subjected to transient ischemia

followed by reperfusion during a vanety of circumstances including thermal injury, trauma or when muscle flaps are used in reconstructive surgery. Compared with other organs, reperfusion injurj mediated by free radicals in skeletal muscle has been the focus

of relatively few studies

Free radicals are difficult to detect in biological systems due to their short lifetime and their low concentration in tissues. Therefore, most studies investigating free radical formation in skeletal muscle make use of indirect methods to assess reperfusion injury.

Such indirect methods include the measurement of increased vascular permeability 9'14,
muscle xanthine oxidase levels ",'3, leukocyte activation

''and the measurement of

degradation proucts je.g. rnaiondiaidrhydc as a iiiriicaiur u iipiri p~iiiiiidiiii~ii) i "'".

Studies using these indirect methods typically compare an untreated control group to a group treated with a lcnown antioxidant such as allopurinol or superoxide dismutase
(SOD). Because free radicals are not identified directly, differences in the measured

parameter behveen control and antioxidant-treated groups are assumed to be mediated by free radicals.

Electron paraniagnetic resonance (EPR) spectroscopy, altematively known as electron spin resonance (ESR) spectroscopy, is a more direct method of detecting free radicals
16.

Free radicals possess at least one unpaired electron. When placed in a

magnetic field, the unpaired electrons give nse to a typical absorption spectrum. Spintrapping is a technique in which free radicals that are not easily detectable by EPR spectroscopy react with a scavenger or "spin trap" molecule to producc a new, more stable free radical with a longer lifetime, and a spectrum that can be identified by EPR spectroscopy ". Nitrones such as u-phenyl N-iert-butylnitrone (PBN) and 5,5-din~ethyl1-pyrroline-N-oxide (DMPO) are commonly used as spin traps. Spectra obtained using this method, can be analyzed to identify the free radical adducts by use of the hyperfine splitting constants of known spin adducts "118. Electron paramagnetic spectroscopy with or without spin-trapping has been used to detect free radicals in other organs " 1 9 and in
cardiac 'J skeletal muscle 'Oy2'. and

Our primary hypothesis was that fiee radicals would be detected From effluent blood sarnples afler 4 hours of ischemia m d 15 minutes of reperfusion using spintrapping EPR. A secondary hypofhesis was inat nistopatinoiogicai changes wuuiJ bc:

observed in canine skeletal muscle aAer 4 hours of ischernia and 15 minutes of

reperfusion.

MATERIALS AND METHODS

Anima1 Preparation

Six adult, conditioned mongrel dogs weighing between 24 and 34 kg were used for this experiment. Al1 dogs were determined to be free of pre-existing disease on the basis of a physical examination and a complete blood count. The experimental protocol was approved by the Ontario Veterinary ColIege animal care cornmittee and the animais were cared for according to the Canadian Council for Animal Care and Use Guidelines.

The dogs were premedicated using acepromazine maleate"0,OS m g k g

intramuscularly FM]) and oxynorphoneh (0.05 mgikg IM) and anesthesia was induced by intravenous administration of thiopental sodium' (10 rngkg) given to effect. All dogs were intubated and placed on 100% oxygcn at 1.5 L/min. Anesthesia was maintained using isofluraned. Blood pressure was rnonitored using an indirect blood pressure measurcment technique. An arterial catheter was placed in each dog in a dorso-pedal artery to allow blood collection for blood gas evaluation.

Surgical protocol

The surgical sites were clipped and aseptically prepared. Isolated, paired,
nonheparinized itt vivo gracilis muscle preparations were constructed, similar to those
1' described by other investigaton '2.

Briefly, a skin incision was made over the gracilis

muscle. Each muscle was dissected ftom the proximal attachrnent to the pubis and fiom its distar insertion on the medial aspect of the tibia. The muscle was dissected fiee of al1

fascia1 attachrnents isolating the vascular and neural pedicles. The branches of the obturator nerve supplying the gracilis muscle were transected. Any minor vascular pedicles were double ligated and transected. Only the major vascuIar pedicle was leR intact. Arterial and venous occlusion was accomplislied using a Rommel tourniquet around the vascular pedicle. The time of occlusion was recorded and the skin was closed over the ischemic flap. Concurrently, the contra-lateral gracilis muscle was dissected as described previously, but the major vascular pedicle was not occluded. This flap served as a control. The side allocated to the occluded and control flaps was alternated between consecutive dogs.

The ischemic muscle flaps were subjected to four hours of tota1 artenal and venous occlusion. ARer three hours of ischemia, al1 dogs were administered u-phcnyl-N-tertbutylnitrone (PBN)"75 mgAg IV) dissolved in sterile physiological saline, injected into a cephalic vein using a glass syringe and stainless steel needle. One hour latcr (4 hours post ischemia), the Rommel tourniquet was removed from the vascular pedicle of the ischemic muscle. The muscle was observed for evidence of revascularization, thrombosis of the pedicle, colour and arterial pulse. AAer 15 minutes ofreflow, a 20 ml venous sample was taken fiom the vein draining each muscle flap using g l a s syringes

and stainless steel needles. The blood was transferred to heparinized glass tubes, covered
and placed on ice. While the blood was transported for EPR analysis the dimensions of each muscle flap was taken. Three biopsies (1 cm2) were taken from each flap. These were taken approximately 2-4 cm, 6-8 cm and 10-12 cm fiom the proxirnaI end of the muscle tlap. Samples were piaceci in iV%bufiered formaiin ior rouiiric i ~ i s i u p a i i i u i u ~ ~ .

Artenal pa02, paC02 and pH were determined before the onset of ischemia, immediately after ischemia, following PBN injection and after reperfusion. The dogs were then euthanatized by lethal injection of barbiturate'.

EPR spectroscopy
Each blood sample was extracted twice with 10-15 ml of chloroform. This was accomplished by gently inverting the test tubes containing the biphasic mixture 10 times. The mixture was separated by centrifugation and the organic (bottom) layer was decanted using a pasteur pipette. The chlorofom extract was thcn roto-evaporated (temperature around 25 C) and the residue redissolved in 2 ml of benzene. The benzene solution was concentrated with a nitrogen gas Stream to a volume of 0.5 ml. The benzene solution was transferred to a round EPR cell. After degassing the solution with nitrogen gas for 5 minutes, the samples were analyzed at roorn temperature in a ST4102-ESR cavity with a Bruker ER-200 X-band EPR spectrometer. The EPR instrument settings were as follows: microwave power: 20.5 mW, modulation amplitude: 0.1 mT,time constant: 50 ms, scan range: 10 mT and sweep time: 50 S. Hyperfine splitting constants were determined for al1 spectra that appeared to originate from biologically derived free radicals ".

Histology The fixed sarnples were ernbedded in paraffin and six pm thick sections prepared on a sledge microtome. The slides were stained using hematoxylin and eosin. One

investigator (JM) blinded to both the treatment group and biopsy site, analyzed the sections.

A scoring scheme was devised to evaluate the histological samples. Each sarnple was evaluated for 8 histological parameters each graded from O to 3 for a possible total score of 24. Scores were given as follows: O = normal, 1 = mild histological abnormalities, 3 = rnoderate histologicaI abnormalities, 3 = severe histological abnormalities. Histological parameters included: interstitial edema, intra-cellular ederna, thrombosis, necrosis, inflammation, muscle fiber integrity, nucleus integrity and connective tissue integrity.

Statistical Analyses

Al1 statistical analyses of the histological scores were perfonned using a commercially available software programs*'s. Univariate analysis was applied to al1 variables to ensure that the data met the assumptions of normalityh. A square root transformation was applied to al1 data to allow parametric evaluation of the histological sum data. Pararnetric data were analyzed using ANOVA for a mixed model'. An F statistic was used to test for significant differences between treatments (perfused or ischemic/reperfused) and biopsy sites (proximal, middle and distal) for the total histological score. A completely randomized (CRD) plot expeiimental design was split used. Results were considered significant at P < 0.05.

RESULTS

Al1 flaps had one major vascular pedicle located just proximal to the middle of the
muscle belly. The minor vascular pediclels) entered distally in the muscle. Two fiaps had two minor pedicles whereas the other 10 had only one. Al1 flaps had good arterial pulses and bled From cut surfaces a h four hours of ischemia and 15 minutes of reperfusion. AI1 cantrol flaps rernained well perfused and no change in the colour of the muscle was noted. Although the proximal three quarter to four fifths appeared well reperfused based on colour, the distal one quarter to one fifth remained cyanotic in al! iscliemic/reperfused gracilis flaps.

Al1 dogs maintained a mean arterial blood pressure (MAP) of at leas( 70 mniHg (mean 88.0 mmHg; SEM 2.89 mmHg) throughout the entire experiment. During this experiment, the mean p,O1 was 475.55 m M g (SEM 22.78 mmHg) and the mean p,COz was 54.7 mmHg (SEM 3.34 mmHg). Three of six dogs were placed on positive pressure ventilation during the experiment due to the increased p,COz leveIs.

EPR Spectroscopy
Spin adduct spectra were detected in al1 of the ischemic/reperhsed muscles. No
spin adduct spectra were identified in the perfsed (control) flaps (Fig 3.1). The likelihood of achieving this result under the nul1 hypothesis was calculated at 2 % (P =
0.02 one-tailed). Detemination of the hyperfine splitting constants of each of the spectra

obtained from the ischemicireperr'usd iiap revcaid rtiaiii sigiials ~ l i i i i ~ ~ ~ fk2t i i

mixture of carbon and oxygen-centered radical adducts (triplet of doublet)

l7

(Table 3.1).

In addition, an unknown radical was also identified in the effluent blood of the ischemic
and reperfused flap of dog 1 (Fig 3.2). We are currently unable to determine the structure of this unknown nitroxide radical. A signa1 consistent with tert-butyl hydronitroxide was noted in dogs 3 through 6 (Fig 3.3). This free radical adduct is thought to originate as a degradation product of the hydroxyl radical adduct of PBN 726. Large deflections consistent with the presence of manganese ions 27Z8 were noted in two spectra (dogs 7 and 5) after ischemia and reperhsion (Fig 3.4) and also in the spectra of a control sarnple (dog 6).

Histology
Tliere were significantly more histological changes in the ischemic group than in the control group (P = 0.034) when the total histological scores were compared (Fig 3.5). Although there was a trend for the proximal and distal biopsy sites to have more severe histological changes than the middle biopsy site, no statistical difference was detected behveen biopsy sites for the total histological scores (Fig 3.6).

DISCUSSION

Free radical spin adduct signals were detected in al1 of the ischemic/reperfused muscles and no free radical signals were detected in the perfused or control flaps. These findings confirrn that spin-trapping with EPR spectroscopy is useful for detecting fiee radicals from effluent blood aAer reperfusion of ischemic skeletal muscle. This also confirms that detectable levels of fiee radicals are produced after 4 hours of ischemia and

15 minutes of reperfusion in the canine gracilis muscle flap and that free radicals are not
produced in detectable concentrations aAer muscle flap dissection alone.

The measurement of the hyperfine splitting constants for the EPR spectra obtained in this study revealed signals characteristic of carbon or oxygen-centercd radical adducts in al1 of the blood samples from the ischernic and reperfused muscle flaps ". The ranges for hyperfine splitting constants of oxygen and carbon-centered free radical adducts overlap which prevents us from being able to differentiate these iising this method

""'.

It

is believed that the same mixture of carbon or oxygen-centered radical adducts of PBN was detected in al1 6 dogs (Table 3.1). The variation in the a" and apH values (Table 3.1) is likely due to the low intensity of the ESR signals. The range in the aNandasHvalues (Table 3.1) correspond well with these from controlled chernical systerns 30'31.The hyperfine splittings from Table 3.1 match best with primary alkyl, secondary alkyl, alkoxyl radical adducts or a mixture of these adducts ". Al1 of these are likely due to lipid peroxidation products such as ethyl or pentyl from p-scission of larger alkoxyl lipid

radicals 12. It is noteworthy that in other canine ischemia~reperfusion systems oxygen and carbon-centered radical adducts of PBN have also been detected 3.728.31-35

No signal indicating the presence of free radicals was detected in the control flaps. These findings support the hypothesis that free radicals are not produced in significant concentrations in perfused gracilis muscle, even after fl ap dissection and transection of the minor vascular pedicles and nerve branches. A signal consistent with ter/-butyl hydronitroxide was noted in dogs 3 through 6 (Fig 3.3). This free radical adduct product

is thought to originate as a degradation product of the hydroxyl radical adduct of PBN

and is not of direct biological origin 7,26. An unknown free radical adduct of PBN was detected in the ischemic and reperfused flap of dog 1. This adduct is characterized by a hydrogen splitting constant which is much greater than the nitrogen splitting constant.

This free radical adduct has not yet been identified. Interestingly, in some of Bolli et al's
systems, another unknown adduct was detected Large peaks suggestive of

manganese ions were also noted in two spectra from ischemic and reperfused blood (dog 2 and 5) as well as in the spectra from a control sample (dog 6) in which no free radical adducts were present. These are thought to be normal ions that may be detected in blood using EPR spectroscopy.

Choudhury et al.'

used EPR without spin-trapping to detect fiee radicals fiom

ischemic and reperfused canine gracilis muscle flaps. In that study, the muscle itself was analyzed using a flat EPR cell. The spectra obtained were only analyzed quantitatively and the radicai aaducts were not iaentir'ieci by usc oi'iiypeihe npiiiiiiig ~ i G i i T i i ~%c .

use of a round EPR cell in our study allowed us to puri@ and concentrate our samples, which allows for stronger and clearer spectra. AIso, the use of blood rather than the muscle is less invasive and more applicable to clinical cases. To the author's knowledge, no studies have reported the use oFEPR spectroscopy for the detection of free radicals from the effluent blood of canine skeletal muscle.

Histological findings after ischemia and reperfusion of skeletal muscle have been described in the literature but remain controversial. Thzse reports are difficult to interpret and c m o t easily be compared as different animal and muscle models were used with variable ischemic (+/- reperfusion) duration. The results of several reports indicate that microscopy is unreliable in demonstrating muscle damage after ischeniia and reperfusion. In contrast to the findings of Dahlback et al.36who found that 4 hours of ischernia followed by reperfusion resulted in moderate to marked degenerative changes in skeletal muscle, Blebea et al. 37,like others 3"39 found that histoiogical damage was minimal afier only 4 hours of ischernia. In contrast to these last results, our study reveded a high level

of significance behveen the treatment groups (occluded vs perfused) with regard to the
degree of histological damage (P c 0.05). A significant difference in histological evidence of muscle damage between sites in muscle flaps has been reported in previous studies. Blebea et al. 37 reported that afler 6 hours of ischemia and 1 hour of reperfusion histological abnormalities were more pronounced at the level where the major vascular pedicle enters the canine gracilis muscle. The authors' exphnation for these findings was that the muscle at that Ievel was more likely to be reperfused than muscle located more
p ~ i proximaiiy or distaiiy ana inerefore possi'oiy morc aCecieri by i I i ~ i ~ d ~ i if ificc

radicals. These authors hypothesized that the muscle in the more proximal and distal portions was more likely to suffer from thrombosis or "no reflow" compared to more centraliy located muscle and would therefore be protected from reperfusion. That study also revealed that ischemia without reperfusion caused significantly Iess damage to the skeletal tissue. In Our study we noted grossly that the distal one fifth to one quarter of the muscle appeared to remain cyanotic after reperfusion. We attributcd this finding to decreased perfusion possibly due to the distribution of the transected rninor vascular pedicle or to thrombosis and "no reflow". However, we did not find significant histological evidcnce of increased thrombosis or damage in the distal portion of the muscle. Spasm of the anastomoses between vessels originating from the major vascular

rm pedicle and vessels originating f o the minor vascular pedicle could have been
responsible for the change in colour in the distal portion of the ischemic/reperfused flaps. Unlike previous studies, Our statistical results suggest a trend in which there is increased damage in the proxiinal and distal portions of the muscle when compared to the central region but these results were not statistically significant. These findings could be duc to decreased reperfusion of the cxtremities of the gracilis muscle related to the distance froni the vascular supply, increased trauma to these portions of the muscle flaps secondary to the dissection and possibly to the no-reflow phenomenon.

Frorn our results, we conclude that bio1ogically derived free radicals are produced in canine skeletal muscle aAer 4 hours of ischemia and 15 minutes of reperfusion. The spin trap adducts of these free radicals can be detected from the effluent blood of canine skeietai muscie using spin-trapping LrK speciroscopy.

---

Tit: Cret: radical

aduci~

identified in this study were likely derived fkom potentially destructive superoxide and hydroxyl radicals. These free radicals do not appear to be produced in detectable concentrations after muscle flap dissection alone. In addition, light microscopy confimxd damage in the ischemic/reperfused muscle flaps that was not present in the non-ischemic, perfused control. These findings are compelling direct evidence that free radicals are involved in ischemidreperfusion OC canine skeletal muscle and that spintrapping EPR is an important tool for the study of this injury process. It is hoped that the results of this study will lead to a better understanding of the mechanisms and potential for prevention of ischemidreperfusion injury in muscle.

FOOTNOTES

"travet;

Ayerst Laboratories, Montreal, Quebec, Canada.

Numorphan; DuPont Phanna, Misissauga, Ontario, Canada. Pentothal; Abbott Laboratories, Montreal, Quebec, Canada.
d

Aerrane; Janssen, Toronto, Ontario, Canada.

' PBN; Aldrich, Wisconsin.

f

Euthansol; Schering-Plough Animal Health, Pointe-Clair, Quebec, Canada. SASJSTAT Release 6.12; SAS Institute, Cary, NC.

PROC UNIVARIATE, Release 6.12; SAS Institute, Cary, NC.

' PROC MIXED, Release 6.13, SAS Institute, Cary, NC.

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studying fiee-radical mediated liver injury. Philos Tram R Soc Lond B Biol Sci 1985: l7:633-45.

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20. Choudhury N, Sakaguchi S, Koyano K, et al, Free radical injury in skeletal muscle ischemia and reperfusion. J Surg Res 1991;5 1:392-8.

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muscle reperfusion edema. J Surg Res 1989;47:389-396.

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method. In: Floyd R, ed. Free Radicals and Cancer. New York: Marcel Dekker, Inc, 1982; 397-422.

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28. Itoh S. Yanagishita T, Aoki S, et al. Generation of Free radicals and the darnage done to the sarcoplasrnic reticulum during repcrfiision injury following b i e f ischemia in the canine hem. Jpn Circ J 1999;63:373-8.

29. Janzen E, Towner R, Haire D. Spectral mixtures of various radical spin adducts may occasionally be distinguished by very small differences in g value, e.g. PBN-alkyl vs PBN-alkoxyl. Free Radic Res Cornm 1987;3:357-64.

30. Haire D, Oehler U, Krygsman P, et al. Correlation of radical structures with EPR spin adduct parameters: Utility of the H, C and N hyperfine splitting constants of aminoxyl adducts of PBN-nitroxyl-C for three-parameter scatter plots. J Org Chern 1988;53:453542.

3 1. Janzen E7Oehler U, Haire D, et al. ENDOR Spectra of Arninoxyls. Conformational study of alkyl and aryl spin adducts of deuterated alpha phenyl-N-tert-butylnitrone (PBN) based on proton abbd beta-C hyperfine splittings. J Am Chem Soc 1986; 108:6858-63.

32. Halliwell B, Guttendge J. Lipid peroxidation: a radical chain reaction. In: HalIiwell

B, Gutteridge J. eds. Free Radicals in Biology and Medicine. New York: Oxford; 1989;

188-266.

33. Bolli R, Jeroudi M, Lai E, et al. Demonstration of Free radical generation in "stunned"
myocardium of intact dogs with the use of the spin trap u-phenyl N-tert-butyl nitrone. I Clin Invest 1958;82:476-85.

34. Li X, Zughaib M, Jeroudi M, et al. Demonstration of free radical generation in the "stunned" myocardium in the conscious dog and identification of major differences between conscious and open-chest dogs. J Clin Invest 1993;92:1025-41.

35, Bolli R, Zughaib M, Li X, et al. Recurrent ischemia in the canine heart causes
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36. Dahlback L, Rais O. Morphologie changes in striated muscle following ischemia. Acta Chir Scand 1966; 131:430-40.

37. Blcbea J, Kerr J, Shumko J, et al. Quantitative histochemical evaluation of skeletal muscie ischemia and reperfusion injury. J Surg Res l987;43:3 1 1-321.

38. Scully R, Shannon J, Dickersin R. Factors involved in recovery fiom expenmental

skeletal muscle ischemia produced in dogs. Am J Pathol 1961; 39:721-737.

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TABLE LEGEND

Table 3.1. EPR spectral data for the PBN spin adducts extracted from the
ischemic/reperfused muscle ilaps. The spectra were recorded at room temperature and

the hyperfine splitting constants are listed in millitesla (mT).

Table 3.1

Dog 1

aN (mT)

aBH (mT)

Radical Type Carbon or oxygen Unknown Carbon or oxygen Manganese ion Carbon or oxygen tert-butyl hydronitroxide Carbon or oxygen tert-butyl hydronitroxide Carbon or oxygen tert-butyl hydronitroxide Manganese ion Carbon or oxygen tert-butyl hydronitroxide

Reference 17

1.46 1.35

0.3
5.45

1.44

0.26 8

17 27 17 26 17 26 17 26 27 17 26

1.42 1.48

0.27 1.7 0.3 1.71 0.26 1.68 8

1.43 1.47

1.43

1.48

1.44 1.48

0.3 1.68

Figure 3.1. Typical EPR spectmm (obtained From extraction in benzene) from the effluent blood of a control (perfused) gracilis muscle flap. No deflections are seen in the nonnal baseline. Bar equals 2.5 millitesla (mT).

Figure 3.2. EPR spectnim of PBN radical adducts (in benzene) detected from effluent blood of a gracilis muscle flap after 4 hours of ischemia and 15 minutes of reperfusion (dog 1). A carbon or oxygen-centered free radical adduct with hyperfine splitting constants of: a" 1-46 mT; apr'= 0.3 mT is assigned in this spectrum (a). An 1.35 mT;

unknown free radical adduct of PBN with hyperfine splitting constants of: a"

aliH=5.45 mT is also present in this spectrum (b). Bar equals 2.5 millitesla (mT).

Figure 3.3. EPR spectra of PBN radical adducts (in benzene) detected from effluent blood of gracilis muscle flaps afler 4 hours of ischemia and 15 minutes of reperfusion (dogs 3 , 4 and 6). Ali these spectra contain a signal characteristic of rertbutyl hydronitroxide (a). Carbon or oxygen-centered free radical adducts are also seen in these spectra (b). Bar equals 2.5 millitesla (mT).

Figure 3.4. EPR spectra of PBN radical adducts (in benzene) detected in the effluent blood of gracilis muscle flaps aAer 4 hours of ischemia and 15 minutes of reperfusion (dogs 2 and 5). Both spectra contain large deflections suggestive of the

presence of manganese ions (a). Carbon or oxygen-centered free radical adducts are also seen in these spectra (b). Bar equals 2.5 millitesla (mT).

Figure 3.5. Mean +/- SEM for the summed histological scores according to treatment group. Ischemia and reperfusion resulted in a significant increase in histological score (*P < 0.05).

Figure 3.6. Mean +/- SEM for summed histological scores according to biopsy sites. No significant differences were seen behveen biopsy sites with regards to histological scores. However, a trend for increased histological scores in the proximal and distal biopsy sites was noted.

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Summed Histological Scores According to Treatrnent

Group

O Control

Ei Occluded

Treatment Group

Figure 3.6

Summed Histological Scores According to Biopsy Sites

O Control 61 Occluded

Proximal

Middle
Biopsy Sites

Distal

CHAPTER 4

Y DETECTION OF FREE RADICALS BI SPIN TR4PPING

ELECTRON PARAMAGNETIC RESONANCE SPECTRQSCOPY (ST-EPR) IN ISCHEMIC AND REPERFUSED CANINE GRACILIS MUSCLE FLAPS AFTER ADENOSINE PRE TREATMENT."

BRIGITTE A. BRISSON, D M V I ,CRAIG W. MILLER, DVM, MVSC,

Diplomate ACVS ', GUOMAN CHEN, PhD1,JILL MCCUTCHEON,

DVM, P ~ D ' ,EDWARD G. JANZEN, PhD1'.

From the Department of Clinical Studies, Ontario Veterinary College, University of

Guelph, Guelph, Ontario, Canada N1G 2W 1.

'From the Department of Pathobiology, Ontario Veterinary College, University of

Guelph, Guelph, Ontario, Canada N1G 2W 1.

Dr. Chen's present address is University of Toronto, Department of Botany, Toronto Ontario, Canada.

Yonnatted for submission to AJVR Professor emeritus

This study was supported by grants from the Pet Trust Fund and the Natural Sciences and Engineering Research Council of Canada.

ABSTRACT

Objective - To determine whether adenosine can attenuate fiee radical production and
histological evidence of muscle injury in ischemic/reperfused canine muscle flaps using spin trapping electron paramagnetic resonance spectroscupy (EPR) and light microscopy.

Study Design - Prospective experimental study.

AniniaIs - Nine mongrel dogs.

Procedure - Under general anesthesia, paired gracilis muscles were isolated leaving only
the major vascular pedicle intact. Local intraarterial treatment with saline was performed in one flap. Ten milligram of adenosine (equivalent volume) was injected into the contralateral flap of each dog. Complete vascular occlusion was thcn created for four

(PBN) was administered parenterally hours in both flaps. u-Phenyl-N-tert-butylnitrone

1-hour prior to reperfusion. The flaps were then reperfused for 15-minutes. Following

reperfusion, samples of effluent blood were coilected from both muscle flaps and processed for scanning by EPR spectroscopy. Muscle biopsies were taken for histopathological evaluation.

Results - EPR spectra of strong intensity were obtained in 5 pairs of samples. The
principal signais identified were characteristic of oxygen and carbon-centered fiee radical adducts. The intensity of the spectra were compared and 24% attenuation was noted in the adenosine treated samples. The other 4 pairs of spectra were too weak for evaluation. HistologicaI evaluation did not reveal a difference between treatment groups.

Conclusions - The results of this study revealed that adenosine appears to reduce free
radical production in this mode1 oI'ischernia/reperfusion;however, it Soes not appear to

attenuate histological abnormalities, Further studies on a larger number of dogs are

needed to assciss the outcome of skeletal inuscle treated with adenosine.

INTRODUCTION

Free radicals produced aRer ischemia and reperfilsion of skeletal muscle have been linked to tissue damage resulting in increased vascular permeability, ce11 death and tissue necrosis '". However, most of the curent evidence implicating free radicals in this process is indirect. The conclusions of such indirect studies are generally based on improved muscle function or morphology after treatment with known antioxidants or free radicar scavengers. Spin trapping electron paramagnetic resonance (ST-EPR) spectroscopy is a more direct method of detection and identification of free radicals. A previous study using ST-EPR " confirmed the presence of Cree radical adducts in the effluent blood of a canine gracilis muscle flap model of ischemia and reperfusion. The principal free radical adducts identified were consistent with a mixture of oxygen and carbon centered species ". These free radical adducts were most likely derived from superoxide and hydroxyl free radicals produced at the time of reperfusion of tissues. It
was also deternlined using this model, that ischemia followed by a short period of

reperfusion resulted in significant histological abnonnalities when cornpared to isolated but continuously perfused control flaps '.

Ischemia and reperfusion of skeletal muscle may occur in a clinical situation when free vascular muscle flaps are used in reconstructive surgery, at the time of reimplantation of severed digits or limbs, when a tourniquet is used intra operatively and subsequently reIeased or aAer removal or lysis of vascular thrombi. Intensive research efforts have been directed towards the identification of pharmacological agents that would improve

ischemic tolerance of tissues such as skeletal muscle. Ischemic preconditioning consists of subjecting a tissue or organ to rcpeated, short periods ofischemia and reperfusion prior to a prolonged period of ischemia '. Ischemic preconditioning has been shown to attenuate capillary no reflow, decrease reperfusion injury and skeletal muscle necrosis Adenosine appears to be the principal mediator of ischemic preconditioning
"13.

High

concentrations of adenosine are released within seconds after the onset of ischernia IJ. Several cardiac studies 1 5 ~ 1 6 and also skeletal muscle studies 12q17.1%evealed treatrnent that with sxogenous adenosine pnor to an ischemic event mimicked the effect of ischemic pre-conditioning.

The precise mechanisms of action of ischemic preconditioning or pre-treatrnent

with adenosine which result in increased ischcmic tolerance of tissues are unknown ". However, studies have demonstrated tliat adenosine acts via stimulation oladenosine A,
lo,l2,ls,lY

and A, "." receptors as well as activation of KAT,channels ".Ib

and results in a

slower rate of energy rnetabolism and metabolite accumulation during sustained ischemia

".

If has also been suggested that adenosine may act as an antioxidnnt and inhibit the

xanthine dehydrogenaselxantliine oxidase (XD/XO) system in ischemic tissues resulting in decreased free radical production at the time of reperfusion ". As well, it is thought that via its action on A, receptors, adenosine may modulate superoxide anion generation

by activated neutrophils which would therefore limit Free radical mediated tissue injury '"

".

Magginvar et al. ?'' have demonstrated that pre treating cells with adenosine resulted in

a 2 to 3 fold increase in activity of superoxide dismutase (SOD), catalase and glutathione

peroxidase whicn resuirea in aecresed cciiuiar iipid peiu~jdiiiiii.Tc, h w k d g c , i

study has evaluated free radical production

in ischemic reperfused skeletal muscle

pretreated with adenosine.

The objective of this study was to evaluatc the ability of adenosine to attenuate

fiee radical production in the canine gracilis muscle mode1 of ischemia and reperfusion using spin trapping EPR spectroscopy. A further objective was to determine if pre treating ischemic reperfused muscle flaps with adenosine would decreasc histological abnormalities noted aAer ischemia and repcrfusion.

MATERIALS AND METHODS

Animal Preparation
Nine adult, conditioned mongrel dogs weighing between 24 and 33 kg were used for this experiment. Al1 dogs were determined to be free of pre-existing disease on the basis of a physical examination and a complete blood count. The experirnental protocol was approved by the Ontario Veterinary College animal care cornmittee and the animals were cared for according to the Canadian Council for Animal Care and Use Guidelines.

The dogs were premedicated using aceprornazine maleate"O.05 mgikg intramuscularly [[MI) and oxyrnorphoneb (0.05 mgkg IM) and anesthesia was induced by intravenous administration of thiopental sodiumc (IO mgkg) given to effect. All dogs were intubated and placed on 100% oxygen at 1.5 Llmin. Anesthesia was maintained using isofluraned. Blood pressure was nionitored using an iridirect blood pressure measurement technique'. An arterial catheter was placed in each dog in a dorso-pedal artery to allow blood collection For blood gas evaluation. The dogs were placed on positive pressure, if needed, to maintain p,CO, levels between 35 and 45 mmHg.

Surgical protocol
The surgical sites were clipped and aseptically prepared. Isolated, paired, nonheparinized Ni vivo gracilis muscle preparations were constructed, similar to those Briefly, a skin incision was made over the gracilis described by other investigators ''v'~. muscie. Each muscie was aissecteci from ifie proxitiiai aiiaiiiiieiii i i k phk ~u76kiii

its distal insertion on the medial aspect of the tibia. The muscle was dissected free of al1 fascia1 attachments isolating the vascular and neural pedicles. The branches of the obturator nerve supplying the gracilis muscle were transected. Any minor vascular pedicles were double ligated and transected. Only the major vsscular pedicle was leR intact. The femoral artery was isolated 3 to 4 cm proximal and distal to the major vascular pedicle. The dissection was performcd concurrently (step by step) in the paired (left and right) muscle flaps. The control flap was treated first to prevent any effect caused by the adenosine on this muscle flap. The vasculature located within 3 , j ccntimeters proximal and one centimeter distal to the major vascular pedicle was inspected. Any small arterial branches other than the major vascular pedicle were double ligated and transected to prevent treatment of tissues othec than the gracilis muscle. The femoral artery was temprarily occluded 0.5 cm distal to the major vascular pedicle using

a bulldog clamp. This ailowed unique perfusion of the isolated gracilis muscle by the
solution to be injected. First, five rnilliliters of sterile saline were infused in the fernoral artery (1 to 1.5 cm proximal to the major vascular pedicle) of the control flap using a 27 gauge needle. The solution \vas injected slowly over a period of 8 to I O minutes.

Following the injection, the bulldog clamp was removed and al1 tissues allowed normal
perfusion for 10 minutes. Afer that time, arterial and venous occlusion was accompIished by tightening a Rommel tourniquet placed around the major vascular pedicle of the control flap. The time of occlusion was recorded and the muscle LemporariIy covered with a sterile gauze sponge soaked in sterile saline. The other muscle flap was then treated using the same protocol described for the control flap.
However insreau osaiinr, ihis cap bras iiciiied wiih I iig f3dGi5iiikf diI:cd

h Z:CT;I!C

saline for a total volume of 5 ml. Following local adenosine treatment, 10 minutes of normal perfusion was allowed and the major vascular pedicle was then occluded using a Rommel tourniquet. The time of occlusion was recorded and the skin was closed over the ischemic flaps. The side allocated to the saline treated and adenosine treated flaps was alternated between consecutive dugs.

The ischemic muscle flaps were subjected to four hours of total arterial and venous occlusion. After three hours of ischernia, al1 dogs were administered a-phenyl-iViert-butylnitrone (PBN)' (75 mgkg IV) dissolved in sterile physiological saline, injected into the cephalic vein using a glass syringe and stainless steel needle. One hour later (4 hours post ischernia), the Rommel tourniquet was removed from the vascular pedicle of the saIine and (20 minutes later) the adenosine treated ischemic muscle. The muscles were observed for evidence of revascularization, thrombosis of the pedicle, colour and arterial pulse. After 15 minutes ofreflow, a 20 ml venous sample was taken from the vein draining each muscle flap using glass syinges and stainless steel needles. The blood
was transferred to heparinized glass tubes, covered and placed on ice. While the blood

was transported for EPR analysis, three biopsies (1 cm2)were taken from each flap.

These were iaken approximately 2-4 cm, 6-8 cm and 10-12 cm fiom the proximal end of

the muscle fiap. Samples were placed in 10% buffered formalin for routine
histopathology.

Arterial paO,, paCO, and pH were determined before the onset of ischemia, immediately afler ischemia, prior to PBN injection and aiter PBN injection. The dogs were then euthanatized by lethal injection of barbiturateh.

EPK spectroscopy
Each blood sample was extracted twice with 10-15 ml of chloroform. This was accomplished by gently inverting the test tubes containing the biphasic mixture 10 tirnes. The mixture was separated by centrifugation and the organic (bottom) layer was decanted

using a Pasteur pipette. The chloroform extract was then roto-evaporated (- 25

OC)

and

the residue redissolved in 2 ml of benzene. The benzene solution was conccntrnted with a
nitrogen gas Stream to a volume of 0.5 ml. The benzene solution was transferred to a round EPR cell. After degassing the solution with nitrogen gas for 5 minutes, the samples were analyzed at roorn temperature in a ST4102-ESRcavity with a Bruker ER-

200 X-band EPR spectrometer. The EPR instiument settings were as follows: microwave
power: 20.5 mCV, modulation amplitude: 0.1 mT, time constant: 50 ms, scan range: 10

mT and sweep time; 50 S. Hyperfine splitting constants were determined for al1 spectra
that appeared to originate from biologically derived tiee radicals
17.

His tology

The fixed samples were embedded in paraffin and six pm rhick sections prepared
on a sledge microtome. The slides were stained using hematoxylin and eosin. One
investigator (JM) bIinded to bo th the treatment group and biopsy site, analyzed the sections.

A scoring scheme devised to evaluate the histological sarnples in a previous

experiment was used '. Each sample was evaluated for 8 histological parameters each gaded fiom O to 3 for a possible total score of 24. Scores were given as follows: O = normal, 1 = mild histological abnormalities, 2 = moderate histological abnormalities, 3 = severe histological abnormalities. Histolagical parameters included: interstitial edema, intra-cellular edema, thrombosis, necrasis, inflammation, muscle fiber integrity, nucleus integrity and coiuiective tissue integrity.

Statistical Analysis
The EPR spectra were evaluated. When the spectra were strong enough to allow measurements to be made, the intensities of the saline and adenosine treated spectra were compared for each dog. The height of each of the deflections consisting of a single free radical adduct was measured in centimeters as described by others 3.2s*2". A mean height was calculated for each adduct by averaging the same number of measurements from each spectrum. The mean height of the spectrum generated in the saline-treated control flap was considered to be 100 percent of the height. The percentage of attenuation (percentage difference) was calculated in the adenosine treated group by dividing the mean height of the spectrum generated in the adenosine-treated flap by the mean height of the spectrum in the untreated control flap. Mean attenuation attained with adenosine was calculated by averaging the percent attenuation calculated for each pair of flaps.

Al1 statistical analyses of the histological scores were performed using a

commercially available sohvare p r ~ g r a m ' *Univariate analysis was applied to al1 ~~. variables to ensure that the data met the assumptions of normality'. Pararnetric data were analyzed using ANOVA for a mixed modelk. This modei was used to test for significant differences between treatment groups (saline and adenosine) and biopsy sites (proximal, middle and distal) for the total histological score. A Tukey-Kramer test was applied to compare the total histological score of each biopsy site. A completely randomized (CRD) split plot experimental design was used. Results were considered signifiant at P < 0.05.

RESULTS

AAer four hours of ischemia, the control muscles appeared grossly more cyanotic (darker coloured) than the adenosine treated flaps. Based on improved colour and the presence of a good pulse, al1 muscles appeared to be well reperfused after the tourniquets were released. After reperfusion, a difference in colour between the adenosine and saline treated fiaps was no longer noticeable. However, in al1 flaps the distal one quartet to one fiflh appeared to remain cyanotic after 15 minutes of reperfiision. All flaps bled from cut surfaces at the time of biopsy; bleeding was decreased at the distal biopsy site. Al1 dogs maintained a mean arterial blood pressure ( M M ) of at least 70 rnmHg (mean MAP 85.1 mmHg; SEM 2.38) throughout the entire experiment. Durin this experiment, the mean p,02 was 469.5 mmHg (SEM 9.03 mmHg) and the mean p,C02 was 43.5 mmHg (SEM I .29 mmHg).

EPR Spectroscopy
Signals characteristic of fiee radical adducts were identified in the EPR spectra of
five pairs of effluent blood samples (Dogs 1, 2,4,5 and 9) (Fig 4.1). The signals in three

pairs of spectra (dogs 3, 7 and 8) were too weak to allow measurement of splittiiig

) constants. However, in two of these three spectra (dogs 3 and 7, weak signals indicative
of the presence of Iow concentrations of free radical adducts were evident in both treatment groups. The s p e c t m fiom dog 6 contained a signal characteristic of an unknown copper ion complex (Cu2-)which would have obscured the si p a l s from spin trap adducts had they been present.

Free radical adducts were present in both the control and adenosine treated samples in al1 five dogs in which strong spectra were noted. The intensity of the signals varied f o dog to dog and was measured in these five pairs of spectra. The measured rm intensity of the signals was weaker (by 20 to 53 %) in the effluent blood from the adenosine treated flaps when comparsd to the saline treated flaps in 4 of 5 dogs (Fig 4.2). In one dog the intensity of the adenosine treated flap was mildly stronger (4%) than the saline treated sarnple. Pre treating gracilis muscle flaps with adenosine resulted in a 24 %
mean attenuation (range - 4 to 53 %) in the intensity of the spectra (Table 4.1).

Determination of the hyperfine splitting constants (HFSC) of the five pairs of spectra with strong intensity revealed signals characteristic of a mixture of oxygen and carbon centered free radical adducts (triplet of doublet) in al1 spectra (Fig 4.2). The HFSC varied slightly: as = 1.4 - 1.49 mT and + " ,
= 0.23

- 0.32 mT (Table 4.1). A second

specrrum characteristic of rert-butyl hydronitroxide (doublet of triplet: as = 1.48 mT, 5%'

= 1.7 mT)was

present in both the saline and adenosine treated spectra of dog 9 (Fig 4.2).
= 1.31 mT

A 1:l: 1 triplet with a HFSC of a"

was also present in the control sample of

dog 9 (Fig 4.2). This triplet represents a di-iert-butyl nitroxide spin trap adduct. A ditert-butyl nitroxide radical adduct was not evident in the sarnple from the adenosine treated flap. Signals suggestive of manganese ions were noted in both spectra from two dogs (dog 4 and 5) (Fig 4.3). Free electron centers within the quartz ce11 wall (colour center artifacts) were noted in the spectra of dogs (4,5 and 7) (Fig 4.3).

Histology
A difference was not found between histological scores of the saline and
adenosine treated groups (P > 0.05) (Fig 4.4). Because no significant difference was detected between the treatment groups, the scores from both groups were pooled for cornparison of the biopsy sites. A trend for the proximal and distal biopsy sites to show less histological abnormalities than the middle biopsy site was noted. However, statistical analysis only revealed a significant difference between the rniddle and the distal biopsy sites (P < 0.05) (Fig 4.5). The summed histological scores were mildly highcr in the adenosine treated groups than in the saline treated group in six of the nine dogs evaluated. However, dogs 3, 4 and 5 had higher histological scores in the saline treated flaps than in the adenosine treated flaps (Fig 4.6).

DISCUSSION

Spectra characteristic of Free radical adducts in which the intensity of the spectra allowed HFSC to be rneasured werc obtained in five pairs of muscle flaps. Thcse spectra demonstrated fiee radical adducts in both the saline and adenosine treated samples with attenuation of the intensity of the EPR spectra in the adenosine treated flaps when cornpared to the respective control flaps. These results suggest that prcconditioning canine gracilis muscle flaps with local adenosine prhr to a period of ischemia can reduce Free radical production. However, pre treating muscle flaps with adenosine does not completely abolish free radical production.

EIectron paramagnetic resonance spectroscopy is primarily a qualitative tool. Howevcr, several studies have analyzed EPR spectra in a quantitative manner by using

the height of the spectra as a measure of its intensity and compared the spectral intensities
of different treatment groups 3.29n'9. Others have used the area under the absorption spectrum; the intensity is directly proportional to the area ". In the present study, we performed peak to peak intensity measurements (using the first derivative presentation) and compared the results of the saline and adenosine treatment groups for the five dogs with sufficiently strong spectra. This was deerned to be an adequate method to compare spectral intensities since both the treated and control flaps were located in the same dog.

In addition, the width of the spectral lines, another factor that controls intensity, was
similar for the saline and adenosine treatrnent groups. The calculated intensity of the

spectra from the adenosine treated flaps was weaker than that of the saline treated flaps in

4 of 5 dogs. In one dog however, the adenosine spectrum was 4 % stronger than that of the saline treated flap. Decay of free radical adducts in the saline sample may have been responsible for these results due to the longer delay prior to EPR evaluation of the saline samples. The mean intensity of spectra in the adenosine treated group was decreased by
24 % conipared to the control group. It is well h o w n that due to the instability of free

radical adducts, their concentration in sarnples decreases over time 32.33. In this study, the samples from each dog (saline or adenosine treated) were processed for EPR concurrently and EPR spectroscopy was perfoned on each sample within minutes of eacli other (control always first). However, due to the two-stepped repertsion, the control sample did have a longer waiting period (20 minutes) prior to EPR spectroscopy than the adenosine sample. Despite this, the adenosine treated spectra were generally weaker than the spectra from the saline treated group supporting a tnie attenuation adenosine.

Previous studies have suggested that adenosine may prevent skeletal muscle injury throuh an antioxidant effect. Based on the results of the present study, adenosine appears to have antioxidant properties resulting in attenuation of the intensity of the EPR spectra. Despite this, free radical production was diminished but not completely abolished in the present study. We therefore hypothesize that the antioxidant effect of adenosine may not be the only mechanism of action by which this dnig reduces postischemic damage in skeletal muscle. Another possibility, which must be considered, is that higher doses of adenosine than the ones utilized in this study are required to further attenuate Eree radical production in canine skeletal muscle. The dose of adenosine used in

the present study was based on the recornmendations made by Forrest et al. " in a swine
model.

Hyperfinc splitting constants could not be measured in three pairs of spectra (dogs

3,7 and 8). In two of these spectra (dogs 3 and 7), deflections were noted that appeared
stronger than the normal baseline suggesting low concentrations of free radical adducts however confident hyperfine splitting constant and intensity measurements couid not be made. Due to technical difficulties encountered during this study, the samples from dogs

3, 7 and 8 were not evaluated as promptly (delay of 40 to 120 minutes) as the samples
from the remainder of the dogs. Howcver, the delay in performing EPR spectroscopy was the same for both of the adenosine and saline treated paired sarnples. We believe that free radical adducts were Iikely present in these samples but that the increased tirne elapsed between sampling and analysis resulted in decay of the free radical adducts producing EPR spectra of weak intensity.

Ischemic preconditioning and adenosine pretreatmeni have been reported to reduce skeletal muscle infaction sa''.'7. No difference was found between the total histological scores in the adenosine treated group compared to the saline treated groups

(P > 0.05) in the present study (Fig 4.4). When the total histological scores were
evaluated on a per dog basis (Fig 4.6), we noted that three dogs (dogs 3,4 and 5) had higher histological scores in the control flaps indicating less darnage in the adenosine treated flap than in the saline treated flap. In the other 6 dogs, the adenosine treated

samples had mildiy but consistentiy nigher hisioiogicai scores (Fit: 4.6) The rruiiiiiuu iz

scores and the srnall number of sarnples preclude a meaningfi statistical differentiation between treatrnent groups. The power of this study with regards to detecting histological differences was approximately 50%. It is possible that a treatment effect could have been noted if a larger number of dogs had been included in the study. It is also possible that the histological differences may have been more evident afier several hours of reperfusion. No obvious reason can explain the differences between dogs 3,J and 5 and the other 6 dogs used in this study.

Adenosine has been shown to decrease neutrophil function and leukocyte adhesion 3J.'s. Leukocytes were only noted in a few histological samples in which both treatment groups were represented. We hypothesize that the free radicals noted using

EPR spectroscopy in our study were those produced through the XO system at the time of
reperfusion. The attenuation in intensity of the EPR signal seen in this study could be related to the energy sparing effect of adenosine resulting in decreased transformation of

XD to XO ". This could result in less fiee radical production early in the reperfusion
period. Further studies are required to determine hypoxanthine and XO levels in these skeletal muscle flaps. In this study, the muscle flaps were only reperfused for 15 minutes. 1t is possible that if the reperfusion penod had been longer, increased vascular permeability, neutrophil invasion and muscle infarction would have been noted in the saline treated group. It is interesting to note that the previously mentioned studies in which skeletal muscle injury was improved with adenosine, evaluated skeletal muscle injury after 48 hours of reperfusion ' ' v ' ~ .Furthemore, the studies reporting decreased siceietai muscie iniarcrion .-.. wiii.1azriusiiic peiTedilliClii ~ c r 21: pcrfr;;;~! i;i s.:.+iac c
Il 17

models. It is possible that species differences could explain the difference seen between the results of the present study and the previousiy mentioned studies. Further studies evaluating histological damage in skeletal muscle flaps treated with adenosine after various penods of reperfusion should be performed.

In a previous study ',no difference between histological scores at each biopsy sites was noted. However, a trend for increased histological abnormalities was found in the proximal and distal biopsy sites when compared to the middle biopsy site '. This was thought to be secondary to increased trauma and susceptibility of the penpheral regions of the muscle, decreased reperfusion of the extremity of the flaps and further ischemic damage or the no-reflow phenomenon. In the present study, ri trend for the middle biopsy site to show more histological abnonnalities than the proximal and distal biopsy sites was seen. However, this was statistically significant only between the middle and distal biopsy sites (P < 0.05). This could be the result of decreased reperfusion in the distal biopsy site resulting in less Free radical mediated reperfusion injury. Decreased perfusion of the distal portion of the gracilis muscle flap codd be caused by vasoconstriction of the choke anastomoses responsible for perfusion of the distal portion of this muscle after transection of the minor vascular pedicIe(s). Another reason for decreased reperfusion of the distal portion of the flaps could be the "no reflow" phenomenon; however histological findings supporting this were not noted. We cannot explain why the pattern of histological abnorrnalities in the present study appears to have a different distribution
than that seen in our previous experiment ". We can only speculate that treatment of the

muscle tlaps with saline or adenosine may nave airerea fie paiien or"i1istoiugicai

damage. In addition, the increased statistical power related to the largcr number of biopsy samples may be responsible for this difference.

Hyperfine splitting constant analysis was performed when the intensity of the spectra allowed. This revealed the presence of a mixture of oxygen and carbon centered free radical adducts in al1 5 pairs of samples. This type of adduct was also present in al1 the ischemic/reperfused flaps in a previous stiidy ". The variation in the splitting constant values obtained in this sludy is consistent with that reported in controllcd chernical systems 3"'7 and in previous experiments J,38. These free radical adducts are thought to be derived from lipid peroxidation of ceIl membranes caused by superoxide and hydroxyl free radicals J9. rerr-Butyl hydronitroxide adducts wcre also reported in previous studies
4.3Y.

This free radical adduct has been shown to derive from the very unstable aiid reactive

hydroxyl radical rnolecule ". The 1:1:1 triplet identified in this study bas previously been described by others as di-rerr-butyl nitroxide and is thought to represent a degadation

production of tert-butyl hydronitroxide "O. Thcse findings support the theor); of the decay of very reactive spin trap adducts within the samptes. Peaks suggestive of mananese

ions have been identified previously in effluent blood from canine skeletal muscle ' and
the copper ion complex is also thought to be h m biological origin. These ions are
believed to be of no significance to this study. Free electron centers also named colour centers, are free electrons caught in the wall of the quartz ceIl used to perform EPR spectroscopy. These have been documented in the literature '' and were also present in previous work '. Efforts should be made to use EPR cells that do not contain electron centers to aid in the evaluation of the specrra.

The adenosine treated flaps appeared grossly less cyanotic than the saline treated fiaps afler ischemia but prior to reperfusion. Other investigators have reported a lower energy depletion of skeletal muscle treated with adenosine prior to ischemia '2"7"8. In the present study, no evaluation of energy depletion was made. We therefore can only speculate that this may have been related to a better conservation of energy and accumulation of less degradation products in the adenosine treated flaps.

In conclusion, the present study revealed that adenosine when administered prior to the period of ischemia appears to reduce free radical production in ischemic and reperfused canine muscle flaps. However, pre treating fiaps with adenosine did not result in complete attenuation of free radical production. In addition, local, pre ischemic adenosine treatment did not result in improved histological appearance after 15 minutes of reperfusion when compared to the saline treated group. Wide variation and small sample size made interpretation of data difficult. Future studies should compare muscle

injury after different periods of reperfusion in skeletal muscle pretreated with adenosine.
In addition, studies should be perforrned to determine the effect of various doses of adenosine in the canine model.

FOOTNOTES

"travet;
b

Ayerst Laboratories, Montreal, Quebec, Canada.

Numorphm; DuPont P h m a , Mississauga, Ontario, Canada.

' Pentothal; Abbott Labaratories, Montreal, Quebec, Canada.

d

Aerrane; Janssen, Toronto, Ontario, Canada.

' Djnamap; Critikon, Tampa, FL.

f

Adenocard 3mgml; Fujisawa Canada, Inc., Markham, Ontario, Canada

V B N ; Aldrich, Wisconsin.
Euthansol; Schering-Plough Animal Healtli, Pointe-Clair, Quebec, Canada.

' SAS/STAT Release 6.12; SAS Institute, Cary, NC.

' PROC WIVARiATE, Release 6.12; SAS Institute, Cary, NC.

"ROC MIXED, Release 6.17, SAS Institute, Cary, NC.

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3. Choudhury N, Sakaguchi S, Koyano K, et ai. Free radical injury in skeletal muscic

iscliemia and reperfusion. J of Surg Res 1991;5 1342-8.

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injury in ischemic myocardium. Circulation lB6;74: 1124-36.

6. Mounsey R, Pang C, Forrest C. Preconditioning: A new technique for improved

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8. Pang C, Yang R, Zhong A, et al. Acute ischemic preconditioning protects against

skeletal muscle infarction in the pig. Cardiovasc Res 1995;29:782-8.

9. Urabe K, Miura T, Iwamoto T, et al. Preconditioning enhances rnyocardid resistance

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12. Pang C, Neligan P, Zhong A, et al. Effector Mechanism of adenosine in acute

ischemic preconditioning of skeletal muscle against infarction. Am J Physiol 1997;273:R887-95.

13. Papanastaiou S, Estdale S, Homer-Vanniasinkam S, et al. Protective effect of

preconditioning and adenosine pretreatment in experimentaf skeletai muscle reperfusion
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14. Fox A, Reed G, Meilman H, et al. Release of nucleosides from canine and human

hearts as an index of'prior ischemia. Atn J Cardiol 1979;43:52-8.

15. Liu G, Thornton I, VanWinkle D, et al. Protection against infarction afforded by

preconditioning is rnediated by A l adenosine receptors in rabbit heart. Circulation 1991;84:350-6.

16. Yao Z, Gross G. A cornparison of adenosine-induced cardioprotection and ischemic

preconditioning in dogs. Efficacy, t h e course, and rote of KATP channels. Circulation

t994;89: 1239-36.

17. Forrest C, Neligan P, Zhong A, et al. Acute adenosine treatrnent is effective in augmentation of ischemic tolerance in muscle flaps in the pig. Plast Reconst Surg
1997;99:172-82-

18. Lee T, Schroeder Ir C, Shah P, et al. Preconditioning with ischemia or adenosine

protects skeietal muscle from ischemic tissue reperfusion injury. J Surg Res 1996;63:2934.

19. Wang S, Drake L, Sajjadi F, et al. Dual activation of adenosine A l and A3 receptors mediates preconditioning of isolated cardiac myocytes. Eur J Pharmacol 1997;320:211-8.

20. Liu G, Richard S, OIsson R, et al. Evidence that the adenosine A3 receptor may

mediate the protection afforded by preconditioning in the isolated rabbit heart. Cardiovasc Res l994;28: 1057-6 1.

21. Cronstein B, Rosenstein E, Kramer S, et al, Adenosine: A physiologie modulator of

superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on hiiman neutrophils. J Immun01 1985; 135: 1366-7 1.

22. Cronstein B, Daguma L, Nichols D, et al. The adenosinetneutrophil paradox resolved: human neutrophils posses both A l md A2 receptors that promote chemotaxis and inliibit superoxidc generation respectively. J Clin Invest 1990;85: 1 150-7.

23. Cronsteiti B, Kramer S, Weissmann H,et al. Adenosine: A physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983; 158: 1 160-77.

24. Magginvar S, Dh,uiraj D, Somani S, et al. Adenosine acts as an endogenous activator of the cellular antioxidant defense system. Biochem Biophys Res Commun l994;2O l:5O8- 15.

75. Kumn W, Walker P, Mickle D, et al. An isalated skeietal muscle mode1 suitable for

acute ischemia studies. J Surg Res 1986;41:24-32.

26. Wright J, k a k i C, Belkin M, et al. Post-ischemic hypothermia diminishes skeletal

muscle reperfusion edema. J Surg Res 1989;47:389-96.

27. Janzen E, Davis E. Detection of free radicals by spin-trapping using the nitroxyl

method. in: Floyd R. ed. Free RadicaIs and Cancer. New York: Marcel Dekker, Inc. 1982; 397-422.

28, Sen S, Phillis J. ESR evidence of attcnuation of hydroxyl radical generation in rat

cerebral ischernia-reperfusion mode1 by oxypurinol. Annals N Y Acad Sci - Paper Edition

1 994;7X 1409-1 1,

39. Itoh S, Yanagishita Tl Aoki S, et al. Generation of free radicals and the damage done
to the sarcoplasmic reticulum during reperfusion injiiry following b i e f ischemia in the canine heart. Jpn Circ J 1999;63:373-8.

30. SAS user's guide: version 6, fourth edition volume 2. Cary, NC: SAS Institute, Inc.
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31. Weil J, Bolton J, Wertz J. Signal intensities and spin concentrations. Appendix E,

kxperimentai considerations. in: weii j,Boiion j,W ~ r i z ,e h . Eie~iiiiir l i i i i i & ~ j )

resonance. Elementary theory and practical applications. New York, NY: John Wiley & Sons, Inc. 1994; 493-500.

32. Kotake Y, Janzen E. Decay and fate of the hydroxyl radical adduct of alpha-phenyl-

N-tert-butylnitrone in aqueous media. J Am Chem Soc 1991;I 13:9503-6.

33. Janzen E, Kotake Y, Hinton R. Stabiliries ofhydroxyl radical spin adducts o f f BNtype spin traps. Free Radic Bi01 Med 1992; 12: 169-73.

34. Cronstein B,Levin R, Philips M, et al. Neutrophil adherence to endothelium is

enhanced via adenosine Al receptors and inhibited via A2 receptors. J Immunol 1992; I48:2201-6.

35. Grisham M, Hemandez L, Granger D. Adenosine inhibits ischemia-reperfusioninduced leukocyte adherence and extravasation. Am J Physiol f 989;X 7:H1334-9.

36. Janzen E, Oehler U, Haire D, et al. ENDOR Spectra of Arninoxyls. Conformational study of alkyl and aryl spin adducts of deuterated alpha phenyl-N-tert-butyhitrone (PBN) based on proton abbd beta-C hyperfine splittings. J Am Chem Soc 1986;108:6858-63.

37. Haire D, Oehler U,Krygsman P, et al. Correlation of radical structures with EPR spin

adduct parameters: Utility of the H, C and N hyperfine splitting constants of arninoxyl

adducts of PBN-nitroxyl-C for theree-parameter scatter plots. J Org Chem 1988;53:453542.

38. Miller C, Chen G, Janzen E. Detecrion of free radicals in reperfused dog skin flaps

using eiectron paramagnetic resonance spectroscopy: a pilot study. Microsurgery 1999;19:171-5.

39. Halliwell B, Giitteridge J. The chemistry of olrygen radicals and other oxygen-derived species. In: Halliwell B, Gutteridge J, eds, Free radicals in biology and medicine. 2nd ed.

New York: Oxford Press University; 1989; 23-85.

40. Vincent R, Janzen E, Chen G, et al. Spin trapping study in the lungs and livcr of F344

rats after exposure to ozone. Free Radic Res 1996;25:475-88.

41. Bersohn M, Baird J. Electron paramagnetic resonance in insulators. In: Bersohn M, Baird J. eds. An introduction to electron paramagnetic resonance. New York: W.A. Benjamin, Inc, 1966; 140.

TABLE LEGEND

Table 4.1. EPR spectral data (hyperfine splitting constants, type of adduct and intensity of spectra) for the PBN spin adducts extracted from the ischemic/reperfused n~uscle flaps. The spectra were recorded at room temperature and the hyperfine splitting constants are listed in millitesla (mT).

Table 4.1
DOG
1

Treatment aN(mT) ;a

(mT)

Radical Type
Carbon or oxygen Carbon or oxygen Carbon or oxygen Carbon or oxy gen Loo weak to analyze Carbon or oxygen Carbon or oxygen

3
4

0.28 Saline 1.45 0.28 Adenosine 1.43 0.27 1.45 Saline 1.43 0.26 Adenosine spin adducts present but 0.32 Saline 1.47 1.46 0.3 1 Adenosine

Height (cm) 1.58 1.22 1.25 1-3

0.75 0.6

"/U Intensity Decrease 22.8

increased by 4 %
--

Other Paramagnetic Species --

--manganese complex, colour center

nianganese coniplex, colour center copper coniplex colour center

20
28.3

Saline Adenosine

6
7 8

Carbon or o x y ~ e n 0.32 Carbon or oxygen 0.32 overlying copper complex spin adducts present but too weak to analyze Negative 0.28 Carbon or oxygen 1.43 Saline rert-butyl 1.48 1.7 Iiydronitroxide di-rert-butyl nitroxidc 1.31 0.23 Carbon or oxygcn 1.4 Adenosine ter!-butyl 1.48 1.7 hydroni trox ide 1.46 1.49

1.45 1 .O4

----

--

1.8

--

-0.85
--

52.7

FIGUXIE LEGEND

Figure 4.1. Typical EPR spectra (obtained from extraction in benzene) From the
effluent bIood of saline (control) (top) and adenosine (bottom) treated gracilis muscle flaps (dog 1). This spectrum was strong enough to allow determination of hyperfme splitting constants and to compare the intensity (strength) of the saline and adenosine spectra. These spectra contain a carbon or oxygen-centered free radical adduct (a) with a

hyperfine splitting constant of as= 1.4-1.5 mT;%'& 0.3 mT. Bar equals 2.5 millitesla
(mT).

Figure 4.3. Paired EPR spectra obtained frorn extraction in benzene (dog 9). Note the attenuation in intetisity of the spectra characteristic of a mixture of oxygen and carbon-centered free radical adducts (a) in the adenosine treated flap (bottom) when compared to the saline treated flap (top). A signal characteristic of fer{-butyl hydronitroxide is present in both spectra (b). A 1 :1 :1 triplet characteristic of ch-rerr-butyl nitroxide is also present in the spectra froni the saline treated flap (c). Bar equals 2.5 millitesla (mT).

Figure 4.3. EPR spectra (obtained fiom extraction in benzene) from the effluent
bloud of saline (control) (top) and adenosine (bottom) treated gracilis mtiscIe flaps (dog

5). These spectra contain large deflections suggestive of the prssence of manganese ions

(a), A signai characteristic of the presence of an electron center is also noted in these
spectra (b). Bar equals 2.5 millitesla (mT).

Figure 4.4. Mean +/- SEM for the summed histological scores according to treatment group. No significant difference in total histological score was noted between saline and adenosine treated flaps (P > 0.05).

Figure 4.5. Mean +/- SEM for sumrned histological scores according to biopsy site. A trend for increased histological scores in the middle biopsy site compared to the proximal and distal biopsy sites was noted. However, this was only statistically significant wfien the middle and distal biopsy sites were compared (* P < 0.05).

Figure 3.6. Total tiistological score for each muscle flap (saline or adenosine treated) according to dogs. Dogs 3,4 and 5 have more severe histological abnomalities in the saline treated flaps than in the adenosine treated flaps. However, the other six dogs show similar total histological scores in the saline and adenosine treated flaps.

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Mcan (SE) Total Histological Score

Treatmeat Croups

Figure 4.5

Mean (SE) Pooled Total Histological Scores (Saline + Adenosine) According to Biopsy Sites

O Proximal Middle 61 Distal

Biopsy Sites

Figure 4.6

Total Histological Score According to Dog

O Saline

61 Adenosine

CHAPTER 5

5.0 GENERAL DISCUSSION AND CONCLUSIONS

The process of tissue or organ darnage resulting from total or partial ischernia has been the subject of countlcss studies as improvement of the understanding and treatment of ischemic injury is ofwidespread clinical importance. Conditions such as myocardial infarction, stroke, pulmonary ernbo1ism and intestinal volvulus are only a few examples of ischemic events. In animals gastric dilationlvolvulus, intestinal obstruction, and rend transplantation are a few of the instances where ischemic injury may result in catastrophic outcornes. Though re-establishing blood flow is mandatory for the recovery of ischernic tissues evidence indicates that further damage occurs afier blood flow is restored (Blebea 1987; Dahlback 1966; Menger 1992; Oredsson 1991). Several studies conclude that free radicals generated at the time of reperfusion of ischemic tissues are responsible, at Ieast in part, for the injury suffered by these tissues (Grringer 1981; Korthuis 1985; McCutchan 1990; Oredsson 1995A; Oredsson 1994; Smith l989B).

Reperfusion injury in skeletal muscle is of great importance. In people, arterial embolism and thrombosis is a common cause of acute ischemia to the extremities. Reimplantation of limbs and digits c m also result in reperfusion injury of skeletal muscle. The more common use of skeletal muscle flaps for reconstruction of severely injured lirnbs after trauma and oncoiogic reconstmctive surgery has also increased the need to document free radical production using objective measures and to attenuate fiee

radical mediated reperfusion injury. In the clinical setting, repetfusion injury to skeletal muscle can result in direct cellular injury, failure to appropriately reperfuse, and ultimately necrosis of the muscle flap. A plethora of studies have investigated the use of various antioxidant molecules to attenuate fiee radical mediatcd damage in skeletal muscle. However, most studies have used rather indirect methods to assess free radical production. Common techniques involve the demonstration of irnproved muscle function or morphology after treatment with a known antioxidant. Wowever in these studies, the production of fiee radicals at the time of reperfusion has not been documenred. In addition, the models of ischemia investigated often involve the use of rabbit or rnurine models. The results from these studies are therefore less applicable to dogs since differences in free radical producing systems have been noted between species.

In the current study it was hypothesized that free radicals would be generated in ischemic and reperfused canine skeletal muscle flaps and would be detected from the effluent blood using spin trapping EPR spectroscopy. It was also hypothesized that four hours of ischemia and 15 minutes of reperfusion would result in significant histological abnormalities in iscliemic muscle flaps compared to continuously perfused controls. In the second phase of the study, it was hypothesized that local pre-ischemic adenosine treatment would reduce fiee radical production and attenuate histological abnormalities.

The objective of the experiment described in chapter 3 was to determine, if fiee radicals are generated in ischemic/reperfused canine gracilis muscle flaps and whether ueiag spifi & ~ @ g EPR these free radicais couid be derectea from et?iut.rii biuci saillpic;~

spectroscopy. A secondary objective was to determine if skeletal muscle damage could be demonstrated using light microscopy. Effluent venous blood was collected fiom flaps in which the arterial and venous blood supply was temporarily intempted. Spin trapping

EPR spectroscopy was performed on these blood sarnples and the spectra fiom
ischemidreperfused and from continuously perfused (control) flaps were compared. The results of this study demonstrated that free radical adducts were present in al1 of the effluent blood sarnple after four hours of ischemia and 15 minutes of reperfusion and could be detected from effluent blood using EPR spectroscopy. Free radical adducts were not detected in any of the sarnples fiom the control, continuously perfused flaps. These results provide more direct evidence that fiee radicals are generated in ischemic/reperfused canine skeletal muscle. Based on these results the first hypothesis was therefore accepted. These findings confirm the results of the only other study investigating the creation of free radicals in ischemic reperfused canine skeletal muscle using EPR spectroscopy (Choudhury 1991). Detecting free radicals fiom the emuent blood is an improvement over the method descnbed by Choudhury et al. who used homogenized muscle sarnples. When sarnpling effluent blood, the muscle itself is not damaged allowing more accurate serial sarnpling. In addition, this technique removes the risk of artifactual creation of free radicals during freezing and crushing of muscle tissue. Effluent blood samples may also allow use of EPR in a clinical setting in the future.

In contrast to previous reports (Blebea 1987; Patterson 1979; Scully 1961), the
study described in chapter 3 demonstrated significant histological evidence of skeletal muscie injury a3er 4 hours o isciieiiii mi1 Gficcli riiiiitca f rcpcrfsi~ii ;s.h~i,

cornpared to perfused control flaps. Based on these results the second hypothesis was accepted. These results are important because they suggest that pathological changes caused by ischemia/reperfsion in canine skeletal muscle are detectable by relatively simple methods. The histological changes seen in this study correspond with the production of free radicals in the ischemic flaps though they do not prove that the radicals detected caused the observed damage. Based on the reported findings of a previous study using canine gracilis muscle documenting worsening of the initial ischemic injury with reperfusion (Blebea 1987), it is possible to speculate that the histological abnormalities were due to free radical mediated injury. The fact that we were able to produce demonstrable histological damage in this mode1 may help to support the efficacy of pharmaceutical intervention using free radical modulators in the prevention of injury caused by ischemiaheperfusion. Electron paramagnetic resonance spectroscopy in combination with spin trapping could beconle an excellent method for documenting the ability of various pharnlaceuticals to alter free radical production after the reperfusion of ischemic organs or tissues. The suppression of free radicals could be correlated to tissue or organ damage using light microscopy, or other more sophisticated methods, increasing our understanding of the mechanisms of reperfusion injury and the efficacy of preventative therapeutics.

The objective of the second study was to evaluate the ability of adenosine to attenuate free radical production in ischemic/reperfused canine skeletal muscle as described in the first study. Ischemic preconditioning is known to augment the ischemic

. nnn. toierance of tissues (ivi~u-ryirao; iviurry irrv, n,.. rulis

r.*
C

I nnc\

L ~ J ) A . L U C . L W ~ ~uppriu* . ~~L

A A----:--

---,,-..

in

ha thn

...*

mediator of ischemic preconditioning (Akimitsu 1996; Downey 1994; Pang 1997; Papanastasiou 1999; Urabe 1993). The exact mechanism of action of adenosine in this situation remains unknown. Some have hypothesized that adenosine attenuates ffee radical production (Cronstein 1990; Cronstein 1983; Cronstein 1985; Pang 1997) or increases the activity of antioxidant enzymes (Maggirwar 1994). The objective o f the experiment described in chapter 4 was to determine if pre ischemic local adenosine treatment would reduce or prevent the free radical production documented at the time of reperfsion and correspondingly diminish the histological abnorrnalities documented in the initial study. The results of this study suggest that adenosine reduces but does not entirely prevent free radical production at the time of reperfusion. Based on the results of this study showing a 24 O mean attenuation of the spectral intensity of the adenosine h group compared to the control group, the third hypothesis was acceptcd. These results warrant furtlier investigation.

The results of the EPR spectroscopie evaluation in this study were not as clear as those from the study described in chapter 3. The results may have been confounded because, unlike the first study, some experiments (6 of 9) were performed in pairs. The simultaneous experiments resuIted in longer delays between sample collection and processing in addition to increasing the tirne required to purify samples pnor to performing EPR spectroscopy. The average tirne required to transport, puri@ and concentrate samples fion1 one dog was approximately 1 %-2hours. With paired experiments, 3 to 4 hours were sometimes required prior to performing EPR evaluation.

Of tnese sampies, ine second pair cvaiuaieci cuiibi~ieiiil gficraied ~ c M c EFX siF!s. r

In some instances, both pairs of samples generated weak EPR spectra, In one instance

(dog 3), an additional delay of 40 minutes was encountered behveen sampling and processing for EPR. However, in both studies, when only one experiment was performed and the ischemiclreperhsed samples were processed without delay, strong EPR signals were always obtained. Based on the results of the study described in chapter 4, it is considered that the wcakness of some EPR spectra was related to the delay in performing

EPR evaluation. In future studies, the experiments should be performed one at a time and
an effort should be made to avoid any delays in processing the samples for EPR spectroscopy.

When spectra of mcasurable intensities were present in both the saline and adenosine treated groups (Si9 dogs), weaker signals were noted in 80% (4 of 5 dogs) of the spectra fiom adenosine treated flaps when compared to the saline treated flaps. A mean attenuation of 24 % was noted. Unfortunately, the spectra obtained in this experiment were not alI strong enough to compare their intensity objectively. Adenosine
has a half-iife of approxirnately 6 to 10 seconds (Klabunde 1983). Therefore, reperfusion

of thz saline treated flap after 4 hours ofischemia is unlikely to have been affected by the adenosine injected in the contralateral vasculature prior to the ischemic event. In addition, the experiment was designed in a way that would not have allowed cross contamination of the saline treated flaps by adenosine.

Adenosine has been reported to decrease skeletal muscle infuction (Fomest 1997; Pang 1997). The results of the smdy descnbea in cnapler 4 diJ nui cui~fiiiiiilie faiili

hypothesis that pre treating canine gracilis muscle flaps with adenosine would result in improved histological scores when compared to the saline treated flaps. Variation in histological scores and a small sample size precluded meaningful conclusions based on histological scores in this experiment. Given this, the fourth hypothesis cannot be accepted. The studies in which adenosine was found to improve infarction in skeletal muscle were not performed in a canine model. In addition, skeletal muscle injury was evaluated after 48 hours of reperfusion. It is possible that the difference in outcome for the adenosine treated flaps noted in our study was the result of an early evaluation of skeletal muscle damage. The injury noted in the adenosine treated flaps could hypothetically have been reversible and if evaluated 24 to 48 hours later would have appeared less severe when compared to the saline group. In contrast, it is also possible that not al1 the skeletal muscle injury was noticeable at the time point evaluated in our study. Adenosine has been reported to decrease neutrophil adhesion and emigration (Akimitsu 1996; Cronstein 1992; Grisham 1989) and potently inhibit superoxide generation by activated neutrophils (Cronstein 1990; Cronstein 1983; Cronstein 1985). Therefore, the saline treated flaps could potentially have developed more tissue injury related to leukocyte invasion if these had been reperfused for a longer period.

In conclusion, free radicals are generated at the time of reperfusion of canine skeletal muscle flaps subjected to a penod of ischemia of four hours. The study presented in chapter 3 was the first to demonstrate that free radical adducts can be detected in effluent blood of canine skeletal muscle using spin trapping EPR spectroscopy. In addition, this study reveaied that four nours of iscnemia and i 5 rninuics o rcpzi usiuii

results in significant histological abnormalities of skeletal muscle architecture. Finally, the results of the study presented in chapter 4 suggest that preischemic, local adenosine treatment attenuates free radical production in ischemiclreperfused canine skeletal muscle. However, this warrants fhrther investigation.

These findings support further investigations evaluating the use of antioxidant molecules to attenuate free radicaI production at the time of reperfusion of skeletal tissues. The successful use of effluent blood rather than the tissue itself for the dctection

of' free radicals resulted in a less invasive procedure. The use of effluent blood sampling is a clinicdly relevant technique. In addition, the techniques used in this study could be
usefl for evaluation of reperfusion injury in other organs or species.

5.1 FUTURE AREAS OF RESEARCH

The clinical application of preischemic conditioning using adenosine or other pharmaceutical dnigs that augment the ischemic tolerance of tissues such as skeleta1 muscle lies in their ability to lengthen the critical ischemic period tolerated by the tissues. In the case of skeletal muscle, it means that a longer period of ischemia could be sustained prior to reperfusion without compromising flap survival. This would allow more time for surgeons to perform complicated reconstnictive procedures and would likely decrease flap morbidity and flap failure.

Further experiments are needed to more critically document the effect of time between blood collection and spectroscopic evaluation on the intensity of the EPR spectra. Although adenosine did not appear to iinprove skeletal muscle injury noted histologically after 15 minutes of reperfusion, evaluation of skeletal muscle injury aRer different pcriods of reperfusion could help detetmine if adenosine treatment improves the final outcome of canine skeletal muscle flaps. Evaluation of vanous doses of adenosine should also be investigated in the canine rnodel. The fact that adenosine did l~owever appear to reduce free radical production in this study wmants further investigation.

The development of this mode1 will also be usehl for future studies evaluating other Free radical scavengers or antioxidant molecules. The evaluation of mixtures of free radical scavengers to obtain complete suppression of free radical production should be perfonned. After suppression of free radical production, one could then perforrn a

systematic exclusion of certain dmgs aiming to identiQ clinically useful molecules. The use of effluent blood rather than tissues has enormous benefits for use in a clinical situation due to its less invasive nature. Electron paramagnetic spectroscopy could potentially be used to dernonstrate fiee radical production in several clinically important situations. Finally, by following muscle flaps over a longer period post operatively, ST-

EPR spectroscopy could be useful in determining the role of vanous free radicals in
muscle flap failure.

CHAPTER 6

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CHAPTER 7
APPENDICES
7.1 APPENDIX - Determination of byperfine splitting constants

Hyperfine splitting constants (HFSC) (like tlngerpnnts) allow the identification of the fiee radical adducts present in a spectrum. Based on the HFSC measured From a spectrum, published references can be used to determine the identity of a particular free rdical adduct. The detemination of hyperfine splitting constants in this work was performed in accordance to previously piiblished data (Janzen & Davis 1982).

Hyperfine splitting constants are calculated based on the spacing between lines within the spectrum. These can be measured in gauss (G) or in millitesla (rnT). Ten G = 1 mT. For spectra without perfect resolution as the ones obtained in the present studies (chapters 3 and 4), the distance behveen the minima and maxima are measured and averaged. The nitrogen (as) and beta hydrogen (a,,") splitting constants are of greatest importance when evaluating the spectra of nitroxyl spin adducts. For example, with a spectrum consisting of a triplet of doublet, typical of those detected in the study described

in chapters 3 and 4 the larger splitting would represent the nitrogen splitting constant and
be measured between the first and third, second and fourth, third and fifth, and fourth and sixth peaks (Fig 7.1). As mentioned earlier, these measurements were performed at the level of the minima and maxima for a total of eight measurements. These ineasurements were subsequently averaged. In the sarne triplet of doublet s p e c t m , the hydrogen hyperfine splitting constant was represented by the smaller splitting and calculated by

performing measurernents behveen the first and second, third and fourth, and fifth and sixth peaks (Fig 7.1). These measurernents were also perfonned at the level of the minima and maxima for a total of six measurernents and are subsequently averaged. In this type of spectrum, the nitrogen HFSC is always larger than the hydrogen HFSC.

The second type of free radical adduct identified in these studies is considered to be a doublet of triplet (Fig 7.2). In these spectra, the nitrogen HFSC is srnaller than that and of the hydrogen HFSC but larger than 0.5 aPH the apHHFSC is represented by the spacing between peaks 1 and 3 , 2 and 5, and 4 and 6. Again, 6 measurements were perfonned and then averaged. The spacing between peaks 1 and 2 , 2 and 4 , 3 and 5, and 5 and 6 represents the nitrogen splitting. The 8 rneasurernents were also averaged.

The calculated HFSC can then be used to determine the identity of the free radical adduct detected in the spectrum. The splitting constants of different free radical ndducts have been published (Buettner 1987; Janzen, Towner, & Haire 1987) and these tables were used to deterrnine the identity of the free radical adducts present in the spectra. These revealed that al1 the spectra obtained from ischemic and reperfused muscle flaps in the study described in chapter 3 contained carbon or oxygen centered free radical adducts (triplet of doublet) with splitting coiistants of: a " 1.42 - 1.46 mT;%"
= 0.26 - 0.3

rnT.

As well4 of 6 spectra also contained an adduct characteristic of tert-butyl hydronitroxide (doublet of triplet) with splitting constants of: as = 1.47 - 1.48 mT;apH = 1.68 - 1. i l rnT. An unknown adduct was also detected in the spectrum of the occluded sarnple of dog one
(as = 1.35 mT,%H = 5.45 rnT). The latter splitting constants were measured as follows;

a "

spacing beiween peaks 2 and 1,3 and 2.5 and 4, and 6 and 5; aeH= spacing between

peaks 4 and 1 , s and 2, and 6 and 3.

Evaluation of the spectra obtained from ischemic and reperfused muscle flaps in the study described in chapter 4 revealed that 5 pairs of spectra were strong enough to perform determination of HFSC. AI1 these spectra contained carbon or oxygen centered

frec radical adducts (triplet of doublet) with splitting constants of: a "

1.40 - 1.49 mT;

aD1' 0.23 - 0.32 mT. As well 1 pair olspectra (dog 9) also contained an adduct = chuacteristic of rert-butyl hydronitroxide (doublet of triplet) with splitting constants of: as
=

1.48 mT;a," = 1.7 mT. A 1: 1: I tnplet was also detected in the spcctrum of the

control sample of dog 9 (a"

1.3 1 mT). The latter splitting constant was determined by

measuring the spacing between peaks 1 and 2, and 2 and 3.

Figure 7.1 Determination of hyperfine splitting constants for carbon or oxygen centered free radical adducts (triplet of doublet) a = :

---

Figure 7.2 Determination of hyperfine splitting constants for teri-butyl hydronitroxide free radical adducts (doublet of triplet) apH= -

---

APPEFiDIX 7 2 2 Histological scores for each histological paranieter and each biopsy site for the ischemic and reperfused muscle ..
flaps.

r
6

Occluded

Disial

O
1
1
1

Occluded
Occludrd Occludcd Occluded - Occludcd
-- -

Proximal
Middle Distal

O 1
1 2 1 1
2 I

3
O O

2 2 2
3 2
1

2
1

1
1

O
0

9
S

Proximal

1 1

Middle 1 1 Occluded 1 Distnl . 1 - - -Occluded ~ r o x i m a ~

i 2
2
1 O 1 O

1 1 2

1
1

O O

2 O 1
O 1
I

26

2 3
2 1 3 1
1 1

1
1

O O

7 11
1 1
1

2
O 1

I O 1

1
1

Occluded Occluded Occluded Occluded Occluded

- - -

Middle Distal Proximal

Middle

O
O

O 1
O

0.5

Distal

1 O

1 1 1

2 1 1 1 1.5 1

1 I

1 1 2

1
1

I
1 1 0.5

O O O

7 7
1

27

O
O

I l l 6 6
4 7

1 2 3

I
1 O 23

0.5
1 16

O O O
1

2
134 6.89
124 20.67 7.71

1 6
iMean
Staiida.rd Deviation

0.89 0.76
0.18

15 0.83 0.79

Siandilrd Error - of Mean

-izq

95 . 0.53 . - O- 85 0.20

25 1.39 - 0.98 0.23

18.5 1 .O3 0.70

0.16

1.28 1 24 O..O2

0.89
0.44 0.10

0.06 O.24
0.06

3.80 0.30

3.15

7 3 APPENDIX - DATA FROM EXPERIMENT 2 .

APPENDIX 7.3.1 Histological scores for each histological parameter and each biopsy site for the saline treated niuscle flaps.

APPENDIX 7.3.2 Histological scores for each histological parameter and eacli biopsy site for the adenosirie treated muscle flaps.

Standard Deviation

Standard Error of Mean

0.70 0.56 .0.11

-

0.67 0.5 0.10

0.46 0.69 0.13

0.97 0.50 0.10

0.37 0.53 0.10

1
1

1.03 0.49 0.09

0.57 0.41 0.08

1
1
1

--*

--<

4.78

14.33
195 -.--

n O

-.
(

? 40 .-

0.46

1.32

Appendix 7.3.3 Statistical analysis of signal intensity data from experiment #2

Statistical Analysis

A paired r-test was perfomed on signal intensity data fiom spectra in experiment
# 2 (chapter 4). Data from dogs 1,2,4,5,9 were inciuded in the analysis. Data was log-

transformed prior to analysis. The results are as follows:

Parameter
1

Control (saline)
I

Adeoosine

Variance

0.022
l

0.018

7 1

Observations
Degrees of freedom
I

5
4

1
I

5
3

t-stat

2.388

Antilog of mean
di fference

Ioterpretation

The above analysis provides evidence for a significant decrease in the signal intensity of muscle flaps treated with adenosine compared with control (saline treated) muscle flaps (P < 0.04), The antilog of the mean difference provides a ratio of the means

of the adenosine and control groups and provides an approximation of the percentage
signal attenuation of flaps treated with adenosine (ix. %). A confidence interval 36

constructed for this parameter would represent a two-tailed test and is therefore not appropriate for this data.

Only 5 of 9 dogs wcre used for this analysis due to poor signal intensity and contamination with trace elements in the spectra of the remaining 4 dogs. Statistics was not performed on the data provided in the thesis due to the M u r e of these 4 dogs to provide spectra that could bc analyzed. However this analysis further supports the conclusion from the descriptive evaluation that adenosine treatment resulted in a significant attenuation of EPR signal intensity of ischemidreperfused muscle laps.

7.3.4 APPENDIX - DOG 1 EPR spectra obtained from saline (top) and
adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 2 EPR spectra obtained from saline (top) and adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

'Ti

7.3.4 APPENDIX - DOC 3 EPR spectra obtained from saline (top) and adenosine (bottom) treated flaps, Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 4 EPR spectra obfained from saline (top) and adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 5 EPR spectra obtained from saline (top) and adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 6 EPR spectra obtained from saline (top) and

adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 7 EPR spectra obtained from saline (top) and

adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 8 EPR spectra obtained from saline (top) and
adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.3.4 APPENDIX - DOG 9 EPR spectra obtained from saline (top) and
adenosine (bottom) treated flaps. Bar = 2.5 millitesla (mT)

7.4 APPENDIX - Gracilis muscle flap after dissection

Gracilis N m e

Major VascuIar Pedicle

7.5 APPENDIX - Diagram of biopsy sites in gracilis muscle flap

Major

Cracilis llaps during rcpcrl'usion. Isclieinic 1 rcperlliscd tlap on the leii and control 1 perl'iised llap on the righl

Ischemic 1 reperfused muscle tlap

Controll perfused muscle flap

7.8.1 APPENDIX - Histologicul sections control flap 10X (top) ancl 20X (bottoni)

7A.2 APPENDIX - Histological sections iscliemic/repcrSiiscd Llap 10S (top) and 20X (l~ottom)