MECHANISMS THAT JEOPARDIZE SKELETAL MUSCLE … · Mechanisms that Jeopardize Skeletal Muscle...
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MECHANISMS THAT JEOPARDIZE SKELETAL MUSCLE PERFUSION DURING SURGERY By Timothy H. Mak A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto © Copyright by Timothy H. Mak (2013)
Transcript of MECHANISMS THAT JEOPARDIZE SKELETAL MUSCLE … · Mechanisms that Jeopardize Skeletal Muscle...
MECHANISMS THAT JEOPARDIZE SKELETAL MUSCLE PERFUSION DURING SURGERY
By
Timothy H. Mak
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Physiology University of Toronto
© Copyright by Timothy H. Mak (2013)
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Mechanisms that Jeopardize Skeletal Muscle Perfusion during Surgery
Timothy H Mak
Master of Science
Department of Physiology University of Toronto
2013
ABSTRACT
We assessed potential mechanisms that may jeopardize skeletal muscle perfusion
during surgery leading to adverse outcomes including muscle injury and flap hypoxia. In
craniotomy patients, we observed an increase in serum lactate and creatine kinase and urine
myoglobin; indicative of muscle damage. The early rise in lactate correlated with elevated
BMI, suggesting that obesity caused tissue compression and muscle ischemia. In our rodent
model, we investigated the effects of flap preparation and phenylephrine on muscle perfusion by
assessing microvascular blood flow and tissue PO2. Phenylephrine reduced muscle blood flow
by ~20%, yet increased PO2 by ~10% suggestive of decreased O2 metabolism. At baseline,
muscle flap blood flow was reduced by ~50% while PO2 was severely reduced ~80% (~5 torr)
suggesting that flap perfusion was attenuated and O2 metabolism was increased. Phenylephrine
infusion further reduced muscle flap perfusion. These data demonstrate multiple mechanisms
by which muscle perfusion is jeopardized during surgery.
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Acknowledgements
I would like to sincerely thank my supervisor Dr. Gregory Hare for his continued mentorship,
encouragement and guidance throughout my research experience which has provided me with
opportunities to learn in both the clinical and experimental realms. I would also like to thank my co-
supervisor and committee members from the Department of Physiology and Anesthesia: Dr. David
Mazer, Dr. Steffen Sebastian Bolz, and Dr. John Laffey for their mentorship, guidance, insight, and
support throughout our committee meetings. Additionally, I would like to thank our project
collaborators from the Department of Plastic Surgery: Dr. Melinda Musgrave, Dr. James Mahoney,
and Dr. Sami Alissa for their mentorship and support of this research project. I am also grateful for
the mentorship of Dr. Michael Cusimano, and Dr. Marco Garavaglia who trained me in the
neurosurgery operating room for data collection in our clinical research study. I am extremely
fortunate to have such excellent mentors and collaborators throughout my research program.
I would like to thank the members of my research team, Dr. Sami Alissa who performed the free
flap surgery in our experimental protocols, and Dr. Elaine Liu, and Dr. Albert Tsui who trained me
in the laboratory. I would also like to thank other members of the laboratory, Dr. Sanjay Yagnik,
Charmagne Crescini, Sharon Klimosco, and Namhee Kim for their friendly encouragement and
support throughout my research program.
I would like to thank the Department of Physiology at the University of Toronto and the
Cardiovascular Science Collaborative Program for my wonderful research program. I am thankful
for the funding of this research project from the Departments of Plastic Surgery and Anesthesia at
St. Michael’s Hospital. I am also thankful for the Dr. Alan W. Conn Graduate Award 2012,
Department of Anesthesia and the UHN Medical Staff Association Volunteer Educational Award
2012 which contributed to the funding of my research program.
Finally I am extremely grateful for my supportive family members and friends who have
supported me throughout my academic career.
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TABLE OF CONTENTS
Abstract ................................................................................................................................... ii Acknowledgements ................................................................................................................ iii Table of Contents ................................................................................................................... iv List of Figures ..........................................................................................................................x List of Tables ......................................................................................................................... xi List of Abbreviations ............................................................................................................ xii List of Contributors .............................................................................................................. xiii
Chapter 1. Overview and Hypothesis
1.0 Overview ............................................................................................................................1
1.1 Hypothesis ..........................................................................................................................4
Chapter 2. Introduction
2.0 Oxygen ...............................................................................................................................5
2.01 The Importance of Oxygen for Mammalian Survival ..................................................5
2.02 Oxygen Pressure Gradient from the Air to the Tissues ...............................................6 2.03 Oxygen Delivery and the Role of Hemoglobin ...........................................................9 2.1 Importance of Cardiovascular System in Regulating Tissue Oxygen Delivery ...............10
2.11 Cardiac Output is Regulated to Optimize Oxygen Delivery to Tissues ....................11 2.12 Importance of Maintaining Blood Pressure and Global Blood Flow ........................13
2.13 Regulation of Blood Flow by the Resistance Arteries ...............................................14
2.2 Methods for Measuring PO2 in Muscle ............................................................................15
2.21 Clark Electrode (Licox) .............................................................................................15 2.22 Electron Paramagnetic Resonance Oximetry .............................................................17
2.23 Oxyphors and O2 Dependent Phosphorescence Quenching ......................................18
2.3 Skeletal Muscle ..................................................................................................................19
2.31 Skeletal Muscle Structure and Function ....................................................................19 2.32 Vascular Organization of Skeletal Muscle Circulation .............................................20
2.33 Regulation of Skeletal Muscle Blood Flow ...............................................................21 2.34 Oxygen Pressures in Interstitial Skeletal Muscle Tissue ...........................................22 2.35 Energy Sources for Skeletal Muscle ..........................................................................23
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2.4 Markers to Evaluate Health of Skeletal Muscle Clinically ................................................24
2.41 Clinical Importance of Serum Lactate .......................................................................25 2.42 Rhabdomyolysis and Muscle Damage .......................................................................26
2.43 High Body Mass Index as a Risk Factor for Muscle Ischemia ..................................27
2.5 Reconstructive Flap Surgery ..............................................................................................28
2.51 What is a Free Flap? ..................................................................................................28 2.52 Potential Causes of Free Flap Failure ........................................................................30 2.53 Rectus Abdominus Skeletal Muscle Flap ..................................................................31
2.6 Vasopressors ......................................................................................................................34
2.61 Phenylephrine, a Specific α1 Agonist ........................................................................34 2.62 α1 Agonist-Receptor Mediated Intrinsic Signaling Pathway ....................................35 2.63 Clinical Debate: Vasopressor Use during Reconstructive Surgery ...........................36 2.64 Clinical Studies ..........................................................................................................37 2.65 Animal Studies ...........................................................................................................40
2.7 The Effect of Temperature on Tissue Metabolism and Perfusion .....................................43
Chapter 3. Methods
3.0 Experimental Design ..........................................................................................................45
3.1 Clinical Study Methods......................................................................................................46
3.11 Study Design ..............................................................................................................46 3.12 Study Population ........................................................................................................46 3.13 Study Protocol ............................................................................................................46 3.14 Data Collection ..........................................................................................................46
3.15 Statistical Analysis .....................................................................................................47
3.2 Rat Experiment Methods ..................................................................................................47
3.21 Animals ......................................................................................................................47 3.22 Surgical Procedure .....................................................................................................48 3.23 Free Flap Reanastomosis Surgery ..............................................................................48 3.24 Arterial Blood Gas and Co-oximetry Analysis ..........................................................49 3.25 Ultrasound Doppler and Arterial Blood Flow ...........................................................49 3.26 Laser Doppler and Microvascular Blood Flow ..........................................................50 3.27 Microsensor G4 Oxyphor and Interstitial Muscle Tissue PO2
Measurements ............................................................................................................50 3.28 Calibration of the Effect of Temperature on the Oxygen Quenching Constant .....................................................................................................................51 3.29 Invivo Calibration of To .............................................................................................52
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3.3 Initial Developmental Protocol ..........................................................................................55
3.4 Experimental Protocols ......................................................................................................59
3.41 Protocol 1: Femoral vs Carotid Blood Flow Ultrasound Doppler Flowmetry ..................................................................................................................59
3.42 Protocol 2: Bilateral Rectus Abdominus Muscle Laser Doppler Microvascular Blood Flow (Developmental model) ................................................ 59 3.43 Protocol 3: Bilateral Rectus Abdominus Muscle G4 Oxyphor PO2 ..........................60
3.44 Protocol 4: Rectus Abdominus Muscle Flap vs Contralateral Control Laser Doppler Microvascular Blood Flow ..........................................................................60
3.45 Protocol 5: Rectus Abdominus Muscle Flap vs Contralateral Control G4 Oxyphor PO2 ..............................................................................................................61 3.46 Protocol 6 Rectus Abdominus Muscle and Flap Temperature Analysis ...................62 3.47 Summarized Experimental Timeline .........................................................................62
3.5 Statistical Analysis .............................................................................................................64
Chapter 4. Results
4.0 Clinical Study – Assessing Skeletal Muscle Perfusion during Craniotomy for
Resection of Brain Tumours ..............................................................................................65
4.01 Patient Blood Pressure and Body Temperature during Surgery .................................65 4.02 Elevated Serum Lactate during Surgery .....................................................................65 4.03 Elevated Creatine Kinase and Myoglobinuria in Some Patients ................................66 4.04 Hemoglobin Levels were Stable during OR and ICU ................................................66 4.05 Body Mass Index Correlated with the Early Rise in Serum Lactate ..........................66 4.06 Arterial Blood Gas and Cooximetry ...........................................................................66
4.1 Protocol 1: Assessing Femoral vs Carotid Blood Flow with Ultrasound Doppler Flowmetry ..........................................................................................................................73
4.11 The Effect of Phenylephrine on Mean Arterial Pressure ............................................73 4.12 The Effect of Phenylephrine on Heart Rate ................................................................73 4.13 The Effect of Phenylephrine on Carotid Blood Flow .................................................74 4.14 The Effect of Phenylephrine on Femoral Blood Flow ................................................74 4.15 Carotid Blood Flow versus Femoral Blood Flow .......................................................75 4.16 Stable Rectal Temperature throughout Experimentation ............................................75 4.17 Arterial Blood Gas and Cooximetry ...........................................................................75 4.18 Electrolyte and Metabolic Data ..................................................................................76 4.19 Protocol 1 Summary ...................................................................................................76
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4.2 Protocol 2: Assessing Bilateral Rectus Abdominus Muscle Laser Doppler
Microvascular Blood Flow (Initial Developmental Model) ..............................................78
4.21 The Effect of Phenylephrine on Mean Arterial Pressure ...........................................78 4.22 The Effect of Phenylephrine on Heart Rate ...............................................................78 4.23 The Effect of Phenylephrine on Bilateral Rectus Abdominus Microvascular Muscle Blood Flow ....................................................................................................79
4.24 Stable Rectal Temperature during the Experiment ....................................................80 4.25 Arterial Blood Gas and Cooximetry ..........................................................................80 4.26 Electrolyte and Metabolic Data .................................................................................80 4.27 Protocol 2 Summary ..................................................................................................80
4.3 Protocol 3: Bilateral Rectus Abdominus Muscle G4 Oxyphor PO2 ..................................83 4.31 The Effect of Phenylephrine on Mean Arterial Pressure ...........................................83 4.32 The Effect of Phenylephrine on Heart Rate ...............................................................83 4.33 The Effect of Phenylephrine on Phosphorescence Lifetime and
Muscle PO2 .......................................................................................................................................................................... 84 4.34 Rectal Temperature was Stable throughout the Experimentation ..............................85 4.35 Arterial Blood Gas and Cooximetry ..........................................................................85 4.36 Electrolyte and Metabolic Data .................................................................................85 4.37 Protocol 3 Summary ..................................................................................................85
4.4 Protocol 4: Rectus Abdominus Muscle Flap vs Contralateral Control Laser Doppler Microvascular Blood Flow ..................................................................................88
4.41 The Effect of Phenylephrine on Mean Arterial Pressure ...........................................88 4.42 The Effect of Phenylephrine on Heart Rate ...............................................................88 4.43 The Effects of Surgery and Phenylephrine on Microvascular Blood Flow in
Rectus Abdominus Muscle and Muscle Flap ............................................................89 4.44 Rectal Temperature was Stable throughout the Experimentation ..............................89 4.45 Arterial Blood Gas and Cooximetry ..........................................................................90 4.46 Electrolyte and Metabolic Data .................................................................................90 4.47 Protocol 4 Summary ..................................................................................................90
4.5 Protocol 5: Rectus Abdominus Muscle Flap vs Contralateral Control G4 Oxyphor PO2 .93
4.51 The Effect of Phenylephrine on Mean Arterial Pressure ...........................................93
4.52 The Effect of Phenylephrine on Heart Rate ...............................................................93 4.53 The Effects of Surgery and Phenylephrine on Phosphorescence Lifetime and Muscle and Flap PO2 .........................................................................................94 4.54 Rectal Temperature was Stable throughout the Experimentation ..............................95 4.55 Arterial Blood Gas and Cooximetry ..........................................................................95 4.56 Electrolyte and Metabolic Data .................................................................................95 4.57 Protocol 5 Summary ..................................................................................................95
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4.6 Protocol 6: Rectus Abdominus Muscle Flap and Contralateral Muscle Temperature ......98
4.61 Mean Arterial Pressure Response to Phenylephrine ..................................................98
4.62 Bilateral Muscle Temperature during Experimentation ............................................98 4.63 Muscle Control and Muscle Flap Temperature during Experimentation ..................98
Chapter 5. Discussion
5.0 The Significance of Hyperlactatemia during Craniotomy for Brain Tumour Resection ..........................................................................................................................101
5.01 Clinical Significance of Increased Serum Lactate ...................................................101 5.02 The Potential Source of Increased Serum Lactate ...................................................103
5.03 Body Mass Index as a Risk Factor for Increased Serum Lactate during Craniotomy ..............................................................................................................105 5.04 Mechanism 1: Muscle Compression leading to Muscle Ischemia and Rhabdomyolysis ................................................................................................105
5.1 Development of the Rat Model of Muscle Perfusion ......................................................108
5.11 Establishing the Dose of Phenylephrine for Increased Mean Arterial
Pressure ....................................................................................................................109 5.12 The Effects of Phenylephrine on Mean Arterial Pressure .......................................109 5.13 The Effects of Phenylephrine on Heart Rate ..........................................................110
5.2 The Effects of Phenylephrine on Muscle Perfusion and Metabolism ..............................111
5.21 Mechanism 2: The Effect of Phenylephrine on Muscle Perfusion ....................................... 111 5.22 The Effect of Phenylephrine (α1 agonist) on Muscle Metabolism ..........................114
5.3 The Effects of Surgery and Phenylephrine on Muscle Flap Perfusion ............................116
5.31 Mechanism 3: The Effect of Muscle Flap Preparation and Microvascular Surgery on Muscle Flap Perfusion .............................................................................................116
5.32 Muscle Flap Oxygen Metabolism after Flap Preparation ........................................117 5.33 The Effect of Phenylephrine on Muscle Flap Perfusion ..........................................119
5.4 The Potential Benefits and Harms of Phenylephrine use during Reconstructive Surgery ............................................................................................................................121
5.5 The Effect of Temperature on Muscle Perfusion .............................................................122
5.6 The Effect of Isoflurane on Muscle Perfusion .................................................................123
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5.7 Limitations of the Study...................................................................................................124
5.71 Clinical Study Limitations ........................................................................................124 5.72 Rat Study Limitations ...............................................................................................125
5.8 Future Directions .............................................................................................................126
5.81 Future Directions for the Clinical Study ...................................................................126 5.82 Future Directions for the Experimental Study ..........................................................127
Chapter 6. Summary
6.0 Summary ..........................................................................................................................130 6.1 Key Experimental Findings .............................................................................................132 6.11 Assessing Skeletal Muscle Perfusion and Health during Neurosurgery ....................132
6.12 The Effects of Phenylephrine use on Muscle and Muscle Flap Perfusion ................132
6.13 The Effects of Surgical Free Flap Preparation on Muscle Flap Perfusion during
Reconstructive Surgery ..............................................................................................132
6.14 The Effects of Temperature on Muscle and Muscle Flap Perfusion .........................132
Chapter 7. Conclusion
7.0 Conclusion ........................................................................................................................136 References ...........................................................................................................................138
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List of Figures
Figure 1: The Oxygen Gradient from the Inspired Air to the Mitochondria ..................................8
Figure 2: The Vascular Organization of the Rectus Abdominus Muscle Flap .............................33
Figure 3: Stern Volmer Relationship and Calibration of Temperature Effect on Quenching Constant in G4 Microsensor Oxyphor ........................................................................53
Figure 4 Calibration of To in Euthanized Rats (n = 4) ..................................................................54
Figure 5 Measurement of Mean Arterial Blood Pressure after Two Different Infusion Protocols
of Phenylephrine .....................................................................................................................56
Figure 6 A Consistent Mean Arterial Pressure Response to Phenylephrine was observed in Four Different Experimental Protocols ......................................................................57
Figure 7 Heart Rate Response in Four Different Experimental Protocols ....................................58
Figure 8 Experimental Timeline of Phenylephrine Infusion Experiments ...................................63
Figure 9 Patient Blood Pressure and Temperature during Surgery ..............................................68
Figure 10 Elevated Serum Lactate and Creatine Kinase in Neurosurgical Patients .....................69
Figure 11 Average Serum Lactate, CK, and Hemoglobin during Surgery and in ICU ................70 Figure 12 Positive Correlation between Serum Lactate and Body Mass Index ............................71
Figure 13 The Effect of Phenylephrine on Carotid and Femoral Blood Flow ..............................77
Figure 14 The Effect of Phenylephrine on Bilateral Rectus Abdominus Muscle Blood Flow.....................................................................................................................................82
Figure 15 The Effect of Phenylephrine on Bilateral Rectus Abdominus Muscle Tissue PO2 .....................................................................................................................................87
Figure 16 The Effect of Phenylephrine on Muscle and Muscle Flap Microvascular Blood Flow.....................................................................................................................................92
Figure 17 The Effect of Phenylephrine on Muscle and Flap Tissue PO2 .....................................97
Figure 18 Assessing Temperature in Rectus Abdominus Muscle and Muscle Flaps ...................99
Figure 19 Muscle Compression during Surgery Leads to Muscle Ischemia Followed by Elevated Serum Lactate, CK, and Myoglobinuria ..................................................107
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Figure 20 The Effects of Phenylephrine on Rectus Abdominus Muscle Perfusion ......................................................................................................................................113
Figure 21 The Effects of Free Flap Surgery and Phenylephrine on Muscle Flap Perfusion ..............................................................................................................................120
Figure 22 Clinical Study Summary: Elevated Serum Lactate, CK and Myoglobinuria Characteristic of Muscle Ischemia Induced Muscle Damage Associated with Patient BMI ......133
Figure 23 Bilateral Rectus Abdominus Muscle Perfusion Model Summary ..............................134
Figure 24 Muscle Flap vs Contralateral Control Muscle Perfusion Model Summary ................135
List of Tables
Table 1 Patient Demographics, Characterization of Tumour Pathology and WHO Grade Relative to Lactate and Body Mass Index ................................................................72
Table 2 Arterial Blood Gas and Co-oximetry Data for Craniotomy Patients in the OR and ICU ...................................................................................................................................72
Table 3 Arterial Blood Gas and Cooximetry Data Analysis: pH, PCO2, PO2, Hb, SaO2 at Baseline and Post PE ......................................................................................................100
Table 4 Electrolytes and Metabolic Data Analysis: K+, Na+, Ca+2, Cl-, glucose, lactate, base, HCO3
- at Baseline and Post PE ...........................................................................................100
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List of Abbreviations ABG – Arterial Blood Gas ANOVA – Analysis of Variance ASA – American Society of Anesthesiologists score ATP – Adenosine Triphosphate BMI – Body Mass Index CaO2 - Arterial Oxygen content CPP – Cerebral Perfusion Pressure CK – Creatine Kinase CO – Cardiac Output COPD – Chronic Obstructive Pulmonary Disease CvO2 – Venous Oxygen content DIE - Deep Inferior Epigastric artery DO2 – Oxygen Delivery 2,3 DPG – 2,3 Bisphosphoglyceric acid EKG - Electrocardiography EPR – Electron Paramagnetic Resonance ETC – Electron Transport Chain GPCR – G protein coupled receptor Hb – Hemoglobin HR- Heart Rate ICP – Intracranial pressure ICU – Intensive care unit IP3 – Inositol trisphosphate IP3R – Inositol trisphosphate receptor Kq – Quenching Constant MAP – Mean Arterial Pressure MI – Myocardial Infarction MLCK – Myosin Light Chain Kinase MLCP –Myosin Light Chain Phosphatase NE – Norepinephrine OR - Operating room O2 - Oxygen PaCO2(PCO2) – Partial Pressure of Carbon Dioxide PaO2(PO2) - Partial Pressure of Oxygen Pcr – Phosphocreatine PE – Phenylephrine PIP2 – Phosphatidylinositol 4,5 – bisphosphate PLT – Phosphorescence Lifetime PU - Perfusion Units RBC – Red Blood Cell RM – Rhabdomyolysis SERCA – Sarco/endoplasmic reticulum Ca+2 ATPase SD - Standard Deviation SV – Stroke Volume SVR – Systemic Vascular Resistance TNF-1α- Tumour Necrosis Factor 1 alpha VO2 – Oxygen Consumption WHO – World Health Organization
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List of Contributors
Dr. Gregory Hare (MD PhD) – Department of Physiology and Anesthesia; Primary supervisor of the research project; Committee member; Mentor
Dr. David Mazer (MD) – Department of Physiology and Anesthesia; Co-Supervisor of this research project; Committee member; Mentor
Dr. Steffen Sebastian Bolz (MD PhD) – Department of Physiology; Committee member; Mentor
Dr. John Laffey (MD) – Department of Physiology and Anesthesia; Committee member; Mentor
Dr. Melinda Musgrave (MD) – Department of Plastic Surgery; Collaborator; Mentor
Dr. James Mahoney (MD) – Department of Plastic Surgery; Collaborator; Mentor
Dr. Sami Alissa (MD) – Department of Plastic Surgery; Collaborator; Mentor; Plastic surgeon who performed all free flap surgery procedures in rat model
Dr. Marco Garavaglia (MD) – Department of Anesthesia; Mentor; trained me in collecting data in clinical study
Dr. Michael Cusimano (MD) –Department of Neurosurgery; Mentor; Performed craniotomy and brain tumour resection in patients
Dr. Albert Tsui (PhD) – Department of Anesthesia; Post Doctoral Research Associate who provided expertise in operating the PMOD oximeter and calibration of G4 Oxyphor microsensor probes in experiments that assessed quantitative PO2
Dr. Elaine Liu (MD) – Provided expertise in basic surgical procedures including tracheostomy and the cannulation of the artery and vein for blood pressure and drug infusion respectively
Dr. David Wilson (PhD) – Invented G4 oxyphor microsensor method of assessing quantitative PO2, helped with initial calibration of the oximeters
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CHAPTER 1 OVERVIEW AND HYPOTHESIS 1.0 Overview
Conditions which lead to inadequate tissue perfusion are a major source of morbidity
in patients. Traditionally, medical research and practice has focused the impact of
inadequate perfusion (ischemia) of vital organs, including the brain (stroke) and heart
(myocardial infarction (MI)). Severe adverse clinical outcomes and patient mortality are
much higher if these ischemic events occur to patients undergoing surgery.1, 2 For example,
the mortality associated with perioperative stroke and MI exceed ~50%, suggesting that the
systemic conditions associated with surgery (inflammation, tissue hypoxia, anesthesia), may
contribute to worsened outcomes. 2-4 In my thesis, I have focused on assessing the impact of
surgery, and its associated conditions, on the adequacy of muscle perfusion during surgery.
Although perfusion of skeletal muscle may be considered of less vital importance than brain
or heart perfusion, nevertheless, inadequate muscle perfusion can also lead to adverse
clinical outcomes including: (1) muscle weakness and pain, (2) rhabdomyolysis during
prolonged neurosurgery, 5-13 and (3) muscle flap failure during reconstructive surgery.14-19
Accurate assessments of muscle perfusion (serum biomarkers, blood flow, and tissue PO2)
are important to evaluate the health of skeletal muscle during surgery.12 These studies
suggest that intraoperative muscle ischemia may lead to tissue hypoxia accompanied by the
presence of anaerobic muscle metabolism and elevated serum lactate. Prolonged
deprivation of adequate muscle perfusion can lead to subsequent muscle damage and
necrosis characterized by the release of muscle enzymes (CK) and myoglobin into the blood
stream. Early detection and identification of the cause and onset of inadequate muscle
perfusion is important in order to correct conditions that lead to inadequate systemic
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perfusion. Evidence of inadequate muscle perfusion may also signify inadequate perfusion
of other organs including the intestine, liver and kidneys. Many factors can contribute to
compromised muscle perfusion during surgery and we review some possibilities in this
thesis: (1) muscle compression, (2) vasopressor use (phenylephrine (PE)), (3) free flap
surgery, and (4) temperature.
Muscle compression induced ischemia is a potential mechanism that can contribute
to positional rhadomyolysis observed in patients undergoing craniotomy. Rhabdomyolysis
is a serious condition in which the breakdown of muscle cells release cellular components
(creatine kinase and myoglobin), which can be toxic, resulting in organ dysfunction,
particularly the kidney5-7. Indeed, in our recently published study, we observed a novel
correlation between patient body mass index and an early increase in serum lactate in 18
neurosurgical patients undergoing brain tumour resection. This data supports the hypothesis
that heavy body mass caused muscle compression leading to inadequate muscle perfusion
and anaerobic lactate production. Some of these patients also exhibited an elevation in
serum creatine kinase and myoglobinuria suggestive of muscle breakdown. In our clinical
model, we further assessed the factors that may influence skeletal muscle perfusion during
craniotomy12.
The use of vasopressors to restore blood pressure is another mechanism that may
jeopardize muscle perfusion during surgery. For example, PE is an α1 adrenergic
vasoconstrictor which acts at the level of the resistance arteries. Vasopressors are commonly
utilized to treat intraoperative hypotension.20 The potential cost of this approach is
vasopressor induced ischemia due to constriction of the resistance arteries. Indeed, this topic
has initiated a recent clinical debate; the use of phenylephrine as a primary means of treating
intraoperative hypotension at the cost of limiting tissue perfusion during surgery has been
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questioned. 21-25 Our data provides new evidence of altered muscle perfusion and
metabolism with infusion of phenylephrine, in a dose dependent manner.
In addition, clinical practice of plastic surgeons uniformly reject the use of
vasopressors to treat systemic hypotension due to the potential negative impact on flap
perfusion. 26, 27 This opinion is strongly enforced despite the publication of reviews of recent
clinical studies which suggest that there is no correlation between the use of vasopressors
and flap complications and failure. 22-25 While microsurgeons continue to warn against the
use of vasopressor during reconstructive surgery, few data actually link this treatment with
flap failure in clinical or experimental models. Thus, we pursued a translational
investigation of muscle free flap perfusion. In a rodent model we assessed rectus abdominus
skeletal muscle and free muscle flap perfusion and evaluated the impact of surgery, infusion
of phenylephrine (an α1 agonist), and temperature on tissue perfusion as assessed by
measuring microvascular blood flow and muscle tissue PO2. We observed evidence of
inadequate muscle perfusion in both our clinical and experimental models as will be further
elaborated within this thesis.
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1.1 Hypothesis
GENERAL HYPOTHESIS: SKELETAL MUSCLE PERFUSION IS JEOPARDIZED DURING SURGERY Sub-hypotheses: to delineate factors that jeopardize skeletal muscle perfusion during surgery. i) Muscle compression leads to inadequate muscle perfusion and muscle ischemia during surgery.
This hypothesis was derived from the clinical observation that serum lactate increased
frequently in patients undergoing craniotomy for brain tumor resection. A prospective
observational study was designed to assess clinical factors that might lead to increased
serum lactate including, length of surgery, body mass index (BMI), administration of
mannitol.
ii) Phenylephrine, an α1 agonist will lead to severe resistance artery constriction and impair skeletal muscle perfusion.
This hypothesis was derived to assess the impact of phenylephrine administration on
skeletal muscle perfusion in an anesthetized rat model. It will provide important control data
with which to compare the ongoing results in skeletal muscle free flap perfusion.
iii) Surgical manipulation and skeletal muscle free flap preparation will impair muscle flap perfusion This hypothesis was established to determine the impact of skeletal muscle free flap
preparation and phenylephrine on microvascular blood flow and tissue PO2.
iv) Skeletal muscle perfusion will be influenced by temperature in our clinical and experimental models This hypothesis will address the observation that tissue metabolism and oxygen
consumption are influenced by temperature and may effect the muscle perfusion and PO2.
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CHAPTER 2 INTRODUCTION
2.0 Oxygen
2.01 Importance of Oxygen for Mammalian Survival
Oxygen was first present in our environment as a function of the evolution of plant
photosynthesis which initiated over 500 million years ago. It was first characterized by
Schelle, Lavoisier and Priestley over 200 years ago. Priestley was the first to link the
production of oxygen by plants to mammalian survival.28, 29 We now understand that oxygen
is vital to the survival of mammalian organisms including humans, as it is necessary to
generate biological energy necessary for the cellular processes of life that are essential for
organ function and survival. Cellular energy in the form of adenosine triphosphate (ATP) is
required by cells to perform essential activities such as membrane transport, growth, cellular
repair, and maintenance processes as well as other facultative functions such as contraction
and motility.28, 30 In the presence of oxygen, aerobic metabolism involving glycolysis, krebs
cycle and the electron transport chain (ETC) occurs to yield a net production of 36 ATP per
glucose molecule, a highly efficient production of energy. Oxygen serves an important role
as the final electron acceptor of the ETC in the mitochondria of cells and is converted into
water to generate ATP via oxidative phosphorylation. However, in the absence of oxygen
during hypoxia, anaerobic metabolism takes place and a net yield of only 2 ATP is
generated per glucose molecule with lactate produced as a byproduct. Cells that are hypoxic
over prolonged periods of time will eventually become dysfunctional and die due to
inadequate ATP production. 30, 31 Adequate oxygen delivery is essential to preserving organ
function and compromised oxygen delivery may result in tissue hypoxia, inadequate ATP
generation, organ failure and death.28, 30, 31 Inadequate tissue oxygen delivery can occur in a
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number of pathological conditions including environmental hypoxia (high altitude), organ
ischemia (stroke, MI), trauma, and surgery and acute blood loss-anemia. These conditions
are associated with mortality due to inadequate oxygen supply.2-4, 31-34 Elevated serum
lactate levels resulting from inadequate oxygen delivery are very late, but are a significant
indicator of inadequate tissue perfusion. 12, 35-41 In some cases, such as critically ill or trauma
patients, a prolonged increase in serum lactate is indicative of reduced patient survival.35, 36
The presence of increased lactate is a balance of increased production and or reduced
metabolism or consumption. Some tissues such as the brain may use lactate as a biological
fuel. 40, 42 Thus, understanding the clinical significance of a transient rise in serum lactate is
complex. This thesis will explore the phenomenon of a transient rise in lactate which has
been observed during neurosurgery. Central to this thesis, the deprivation of oxygen to
skeletal muscle tissue will result in muscle breakdown and necrosis characteristic of
rhabdomyolysis and muscle flap failure.7, 9, 12 This represents a focused look at the adequate
oxygen delivery to muscle, which may not be critical for organism survival, but may have
important implications for reducing patient morbidity and event free patient survival. It
contributes a piece of the puzzle in the overall picture of mammalian survival in which
oxygen is necessary for the production of cellular energy in the form of ATP to maintain
cellular function and organism survival.
2.02 Oxygen Pressure Gradient from the Air to the Tissues
The air in the atmosphere is composed of 21% oxygen, 78% nitrogen and smaller
portions of other gases such as carbon dioxide, argon and helium.43 At atmospheric pressure
(760mmHg), the partial pressure of oxygen is approximately 160 mmHg. As air enters the
lung and alveoli the partial pressure of oxygen is offset by the acquisition of dissolved water
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and carbon dioxide. [PAO2 = FIO2 (PATM – PH2O)-PaCO2/RQ, where PAO2 is the partial
pressure of oxygen in the alveolus, FIO2 is the fraction of inspired oxygen, PATM is the
atmospheric pressure, PH2O is the partial pressure of water, PaCO2 is the partial pressure of
CO2 in artery, and RQ is the respiratory quotient] These gases reduce the partial pressure of
oxygen in the alveolus. An oxygen gradient cascade exists in which oxygen travels from a
high partial pressure in the alveolus (~100 mmHg) into the blood where the early conduit
arterial PO2 is near ~95-98 mmHg.43 Exchange of oxygen from the vasculature to the tissue
occurs at the level of the microcirculation comprised primarily of capillaries. Novel
quantitative methodology (phosphorescence quenching) has demonstrated that the gradient
of oxygen partial pressures decreases rapidly as oxygen moves away from the hemoglobin
in the red blood cell (RBC). 44-48 Studies in the mammalian brain indicate that oxygen
moves along its pressure gradient from the RBC (~ 60 mmHg) to the tissue (PO2 ~25-40
mmHg).28, 44, 46, 49, 50 Under physiological conditions, the cell membrane provides very low
resistance to oxygen which flows freely into the intracellular compartment (10-20 mmHg)
where it is utilized by the mitochondria (5-15 mmHg) as the final electron sink in the
process of ATP production via oxidative phosphorylation.28, 43, 47 Thus, oxygen follows a
concentration gradient from the air to the microvasculature and into the intracellular
compartment (Figure 1).
8
Figure 1 The Oxygen Gradient from the Inspired Air to the Mitochondria. Oxygen from the air (160mmHg) enters the lungs (alveoli) (100mmHg) and into the arterial blood (95-98mmHg) where it is transported to the tissues of the body by hemoglobin. At the microvasculature the PO2 ranges from 30-60 mmHg. Oxygen follows the gradient into the tissues (25-40mmHg), cells (10-20mmHg), and finally the mitochondria (5-15 mmHg), where it serves as the final electron acceptor in the electron transport chain and is converted to water in the process of ATP production
9
2.03 Oxygen Delivery and the Role of Hemoglobin
The majority of oxygen in the blood is carried by hemoglobin in a highly efficient
manner, as 99.8% of oxygen combines with hemogloblin of the RBCs and 0.2% of oxygen
is dissolved in the blood plasma. Hemoglobin consists of 4 globular proteins (2 α and 2 β
subunits) with 4 heme groups. The heme group is an iron porphyrin compound that is
essential for oxygen binding to the hemoglobin molecule, thus each hemoglobin molecule
can bind up to 4 oxygen molecules and blood oxygen capacity is directly proportional to Hb
level. Hemoglobin binds oxygen to become oxyhemoglobin in a cooperative manner in
which the binding of oxygen to one of the heme groups increases the affinity for subsequent
oxygen binding due to a conformational change in hemoglobin.51 This is indicated by the
sigmoid shape of the oxygen dissociation curve composed of the association and
dissociation segments. Several factors can influence the dissociation curve including pH,
2,3 bisphosphoglyceric acid (2,3 DPG), temperature, and PCO2. An increase in acidity
(decreased pH), 2,3 DPG, temperature, or PCO2 will result in a right shift on the oxygen
hemoglobin dissociation curve leading to lower affinity for oxygen. The release of O2 from
Hb is favored in situations when O2 is needed, such as in skeletal muscle during exercise.
Conversely, a decrease in acidity (increased pH), 2,3 DPG, temperature, or PCO2 will result
in a left shift on the oxygen hemoglobin dissociation curve leading to increased affinity for
oxygen, such as at the lungs. Each gram of hemoglobin can carry 1.39 ml of oxygen. 43, 51
Oxygen saturation is the ratio of the amount of oxygenated hemogloblin to the total
hemogloblin in 100 ml of blood and arterial blood and venous blood is 95-98% and 60-80%
saturated with oxygen respectively. At the lungs, the partial pressure of oxygen is high and
the affinity for oxygen is great and thus oxygen loading occurs and hemoglobin is 98%
10
saturated, however at the tissues the partial pressure of oxygen is low and oxygen
dissociation occurs and the hemoglobin saturation is 75%. Thus, on average, the tissues
extract and use 25% of the oxygen from hemoglobin during resting conditions. As the major
function of hemoglobin is the transport of oxygen from the lungs to the other tissues in the
body, it is clinically measured as an indicator of oxygen content [CaO2 = 1.39HbSO2+
0.003PO2] in patients during surgery. Oxygen content in the blood is a sum of the oxygen
content in the solution in addition to the oxygen carried by the hemoglobin and thus is a
determining factor of adequate oxygen delivery (DO2) [DO2 = CaO2 x CO].43, 51 This
formula emphasizes the importance of hemoglobin and cardiac output (Section 2.11) in
determining oxygen delivery/supply. The oxygen demand is the amount of oxygen required
to sustain the metabolic requirements of all body tissues. The total oxygen delivery must be
equal to the total oxygen demand for homeostasis to be maintained, failure for oxygen
delivery to meet oxygen demand can result in organ damage and failure.31, 34 O2
consumption (VO2) [VO2 = CO (CaO2-CvO2)]51 is the amount of oxygen actually used by
the tissues and is generally equal to the oxygen demand during normal conditions. In this
thesis, we will examine the effects of phenylephrine administration on skeletal muscle
oxygen consumption and metabolism, an estimate by changes in blood flow and tissue PO2.
2.1 Importance of the Cardiovascular System in Regulating Tissue Oxygen Delivery
The cardiovascular system, comprising of the heart and the vasculature is required
for the delivery of oxygen and vital nutrients as well as the removal of metabolic waste
products (carbon dioxide, serum lactate).52, 53 The heart functions to pump blood to the rest
of the organs and tissues in the body through the large conduit arteries such as the carotid
and femoral arteries. From there, hemoglobin enters the microcirculation comprising of the
11
smaller arterioles (resistance arteries), the capillaries, and the venules. The resistance
arteries are muscular vessels that control organ specific blood flow and oxygen delivery to
the tissues by the regulation of microvascular tone via vasoconstriction and vasodilation.
The capillaries are a network of vessels that are one cell thick and allow for the exchange of
nutrients and wastes between the tissues and the blood. Oxygen diffuses through the
capillaries into the tissues and to the mitochondria of the cells, while carbon dioxide diffuses
out of the tissues and into the blood. Finally, the venules and veins are the capacitance
vessels that store blood volume and carry the deoxygenated blood and metabolic wastes
back to the heart. At any point in the systemic or pulmonary circulation physiological
shunts exists by which arterial blood can travel directly from the conduit artery to the venule
thus bypassing the microvasculature. This leads to hypoxemia (low blood O2) in pulmonary
circulation and tissue hypoxia if it occurs in the systemic circulation. Key regulators of
tissue perfusion in the cardiovascular system include the cardiac output permitted by the
heart and the regulation of local organ blood flow at the level of the resistance arteries also
influenced by the autonomic nervous system. Thus, tissue perfusion is regulated at different
levels of the cardiovascular system. In understanding the regulation of adequacy of tissue
perfusion, clinicians often assume that adequacy of conduit artery PO2 (radial artery arterial
blood gas) correlates with specific tissue PO2.
2.11Cardiac Output is Regulated to Optimize Oxygen Delivery to Tissues
The cardiac output (CO) is the amount of blood that is pumped out by the heart per
minute and it is equivalent to the sum of all blood flow to the tissues in the body. The
average resting cardiac output in men is a function of body weight and is measured to be
near 70 ml/kg/min or about 5.0 L/min.54 As emphasized, a key regulator of tissue perfusion
12
is the cardiac output permitted at the level of the heart. CO is defined by the heart rate and
stroke volume [CO = HR x SV]. Heart rate can be increased by β1 adrenergic stimulation
which increases overall contractility. The β1 signaling pathway is complex and will not be
an emphasis of this thesis. Stroke volume can be increased by an increased preload (left
ventricular end diastolic volume), the degree of stretch on the ventricles prior to contracting,
and reduced by the afterload, the aortic pressure which hinders the ejection of blood from
the ventricles. When cardiac output is increased reflective of increased heart rate and/or
stroke volume, tissue oxygen delivery may also increase. For instance, CO can be greatly
increased at times of increased oxygen demand such as physical exercise. However, during
situations in which CO is decreased, such as β blockade and cardiac arrest, oxygen supply
can be severely impaired as demonstrated by studies in our laboratory. Ragoonanan et al
(2009) have studied the effects of β blocker metoprolol on cerebral tissue oxygen tension
after acute hemodilution in rats and reported reduced oxygen delivery to the brain.49
Additionally, Yu et al (2013) have examined microvascular brain perfusion in a pig cardiac
arrest model and observed a severe decline in brain tissue PO2 associated with ventricular
fibrillation.50 Traditional physiologists have emphasized that it is the tissues requirement
for oxygen that ultimately regulates cardiac output and specific tissue blood flow. 54, 55 This
end purpose of the cardiovascular system has led us to focus on measures of adequacy of
tissue perfusion (lactate and tissue PO2 in our models). Therefore, the oxygen supply
permitted at the level of the heart is an important determinant of oxygen delivery.
13
2.12 Importance of Maintaining Blood Pressure and Global Blood Flow
Blood pressure is the force that the blood exerts against the walls of blood vessels.
The pumping action of the heart generates blood pressure which generates blood flow.
Poiseuilles Law defines blood flow: F = π∆Pr4/8ηl , where ∆P is the pressure difference
between the ends of the vessel, r is the radius of the vessel, l is the length of the vessel, and
η is the viscosity of the blood. The mean arterial pressure (MAP) is the average pressure in
the arteries as is defined as the cardiac output multiplied by the systemic vascular resistance
[MAP = CO x SVR] and is the driving force of global blood flow. Increases in cardiac
output and/or systemic vascular resistance will lead to an increase in MAP. A pressure
gradient exist that drives blood flow from a high pressure at the aorta toward a lower
pressure within the arterioles and capillaries, with the lowest pressure at the vena cava. It is
generally assumed that increased perfusion pressure correlates to increased tissue perfusion.
However, if taken to the extreme example, severe constriction of resistance arteries will
increase MAP but eventually limit microvascular blood flow and tissue perfusion. Thus, it
has been argued that using vasopressors to increase MAP may actually impair perfusion in
some vascular beds. Clinicians use MAP as an indicator of adequate perfusion in the
operating room and use vasopressors to treat intraoperative hypotension by increasing MAP
with the goal of increasing perfusion. Thiele et al (2011) recently describe this approach as
a “tangible bias” which describes our tendency to favour treating a parameter that we can
see (MAP) without a full understanding on the impact of what we cannot see (tissue
perfusion).21 In other words, favoring less important but immediately measureable variables
such as mean arterial blood pressure (MAP) over more important but less measureable
tissue oxygen delivery (DO2) as indicators of adequate perfusion.21, 56 Nevertheless,
14
vasopressors are commonly used to raise MAP to maintain cerebral perfusion pressure
[CPP = MAP - ICP] which is vital to patient survival during surgery. Although treating
severe hypotension by increasing MAP is assumed to be reflective of improved tissue
perfusion, we will demonstrate that this is not always the case. Different organs receive
different amounts of blood flow depending on the metabolic needs of the specific organ.
For instance, carotid blood flow to the brain is greater than the femoral blood flow to the
femoral muscles at rest because the brain requires greater amounts of oxygen and has a
higher metabolism than resting skeletal muscle. Improved cerebral perfusion by increased
MAP may not be reflective of improved skeletal muscle perfusion. In this thesis, we will
examine whether or not an increase in MAP correlates to increased skeletal muscle
perfusion in a model of skeletal muscle and muscle flap perfusion.
2.13 Regulation of Blood Flow by the Resistance Arteries
Resistance arteries are 10um-100um thick consisting of endothelium and smooth
muscle. By virtue of possessing vascular smooth muscle, these small vessels actively
regulate organ specific blood flow and oxygen delivery to tissues. Both intrinsic and
extrinsic mechanisms determine the degree of smooth muscle activation and vascular tone
(vasoconstriction) in the resistance arteries and thus affect organ blood flow. The intrinsic
mechanisms include endothelial derived factors, and smooth muscle myogenic tone.
Extrinsic regulation includes innervation by a variety of autonomic nerves and locally
produced hormones, and tissue metabolites 57 (ie. sympathetic nerves (norepinephrine [NE])
and other circulating hormones (vasopressin)) that act outside of the blood vessel. Synthetic
pharmacological drugs that are not produced by the human body under normal physiological
conditions such as phenylephrine, an α1 agonist, are also clinically and physiologically
15
relevant in respect to vascular tone and the maintenance of perfusion. Therefore the
regulation of local organ blood flow occurs at the level of the resistance arteries dictated by
vasoconstricting and vasodilating stimuli occurring through intrinsic and extrinsic
mechanisms that regulate microvascular tone.
2.2 Methods for Measuring PO2 in Muscle 2.21 Clark Electrode (Licox) In the early 1950s, Leland Clark developed the Clark electrode which consumes
oxygen in a redox reaction to generate an electric signal indicative of oxygen
concentration.28, 58, 59 The Clark electrode consists of a platinum or gold cathode where
oxygen is reduced and a silver anode that reacts with KCl to generate electrons.58 The
electrons will flow from the anode to oxygen at the cathode. A Teflon membrane separates
the electrodes from the reaction chamber and is permeable only to oxygen. Oxygen will
diffuse through the Teflon membrane and become reduced at the cathode according to the
following reaction: (O2 + 4 electrons +2 H2O 4OH-)60 This reduction reaction allows
subsequent electrons to flow and generates an electrical signal that is proportional to oxygen
concentration. In summary, the Clark electrode measures current generated from the cathode
and electrode immersed in electrolyte solution interacting with oxygen, which is
proportional to the activity of oxygen.60 The Clark electrode is the basis of oxygen
measurements in arterial blood gases and has been applied to clinical medicine to measure
tissue PO2 directly.
LicoxTM is an example of a device that uses the principles of the Clark electrode to
assess oxygen levels in the clinic.58, 61, 62 This device can simultaneously measure tissue
oxygen and temperature and is commonly used to assess brain and other tissue PO2. It has
16
been used in patients with severe traumatic brain injury.61 The Licox probe consists of a
polarographic cathode and anode immersed in electrolyte solution that is separated from the
tissue by a polyethylene membrane. Current is measured by the Licox probe which is
linearly proportional to tissue PO2.58 In addition, temperature is measured by a
thermocouple within the probe. The Clark electrode (Licox) is used in some tertiary
neurotrauma centers to assess brain tissue oxygen levels.58 In assessing brain oxygen levels,
PO2 during normal conditions can range between 25-50mmHg (30mmHg ideal), however
during ischemia brain PO2 can range from 8-12 mmHg and brain PO2 levels less than 2
mmHg is associated with brain cell death. Although a low brain PO2 is associated with
worse outcomes including death, no study to date has demonstrated that therapies which
improve brain tissue PO2 can improve survival.
The use of Licox has also adapted to monitoring microsurgical flap PO2 in cases of
reconstructive surgery.60, 63 Kamolz et al (2002) have assessed 60 free tissue transfers over
a period of 3 years with the Licox Catheter PO2 microprobe and reported that it is an
accurate monitoring system for all types of flaps. Licox was able to detect circulatory
changes and flap failure with no false positives or negatives.63 During cases where a failed
arterial pedicle occurred, PO2 was observed to drop rapidly.63 Additionally, when venous
insufficiency occurred PO2 was observed to drop slowly.63 Furthermore PO2 with in all
failing flaps was observed to drop below 10 mmHg.63 Therefore Licox is a useful tool to
assess tissue PO2 in both the brain and muscle flaps in the clinical setting. Disadvantages of
using an electrode method for measuring tissue PO2 include that it is invasive, causes tissue
damage, is affected by local blood clots, only measures a small area of brain/muscle tissue
and can also suffer from motion artifacts.64 Recent review of the Licox probe revealed that
17
the probe has a tendency to under-read oxygen tension which is more pronounced at higher
temperature.58
2.22 Electron Paramagnetic Resonance Oximetry Electron Paramagnetic Resonance (EPR) oximetry is another technique to measure
highly sensitive and reliable oxygen concentration in tissues by applying magnetic field
gradients to isolate EPR signals from multiple invasive probes of an implantable
resonator.59, 64-66 Small crystalline oxygen sensing probe(s) such as lithium phthalocyanine
(LiPc) are implanted into the organ/tissue site of interest (ie. brain, heart, tumour, etc).64-66
The LiPc probes are inert and can be left within the tissue site over a period of months
without causing significant complications, however this method is not used clinically.66 An
external loop resonator is placed over the LiPc probes and EPR spectra are recorded with an
EPR spectrometer.65 Oxygen is paramagnetic and produces a line-width broadening
resulting from the spin spin interaction between oxygen and the LiPc probe.64, 66 The
recorded line widths of the EPR spectra are linearly correlated with the partial pressure of
oxygen.64, 65 Thus, EPR oximetry can provide a means of an accurate assessment of tissue
oxygen tension determined through changes in EPR spectral line width.66 The advantage of
EPR oximetry is that it is a non-invasive, repetitive, and highly accurate method to assess
tissue oxygenation.64-66 It has been applied to research in numerous of fields and has the
potential for clinical use in studying oxygen in the heart, brain, and tumours. A limitation of
EPR oximetry is that the signal intensity of EPR spectra decreases when PO2 increases or
when LiPc probe size is reduced.65 Another significant disadvantage of this method is that
EPR oximetry measures a mixture of tissue PO2 and capillary PO2, whereas other methods
such as G3/G4 phosphorescence quenching method measures tissue PO2 specifically.28, 47
18
2.23 Oxyphors and O2 dependent Phosphorescence Quenching
Intravascular and interstitial measurements of tissue PO2 can be measured using G2
and G4 Oxyphor and oxygen-dependent quenching of phosphorescence.28, 46-48 When the
phosphorescent probe is excited by pulse of light, it emits phosphorescence over a course of
tens-to-hundreds of microseconds. The lifetime (τ) of the phosphorescence decay is
inversely proportional to the partial pressure of oxygen (PO2) in the environment according
to the Stern-Volmer relationship. [1/ τ = 1/ τ0 + Kq[PO2]], where τ0 is the phosphorescence
lifetime when PO2 is 0, Kq is the quenching constant, and PO2 is the partial pressure of
oxygen.46, 50 In the presence of oxygen, the oxygen will quench the excited electron and
reduce the phosphorescence resulting in a low phosphorescence lifetime. Thus a low
phosphorescence lifetime is correlated to high PO2. Conversely, a high phosphorescence
lifetime is indicative of low tissue PO2. Oxygen measurements by phosphorescence are
independent of the local probe concentration, since the decay lifetime serves as the
measurement signal and not signal intensity. G4 Oxyphor can be used in direct tissue PO2
measurements as a part of an insertable microsensor in muscle and flap tissues. The signals
of the probes are calibrated under physiological pH and temperature and shown to provide
quantitative, selective and absolute measurements of PO2 in vivo. The G3/G4
phosphorescence quenching method is a reliable method to measure quantitative tissue PO2
as other methods of measuring tissue PO2 such as EPR oximetry measures a mixture of
tissue and capillary PO2.28, 47 Therefore, our experiments will utilize the G4 oxyphor method
to assess quantitative interstitial PO2 in skeletal muscle tissue.
19
2.3 Skeletal Muscle
2.31 Skeletal Muscle Structure and Function
Approximately 40% of human body mass is comprised of skeletal muscle which
primarily functions to contract and generate mechanical force which provides support to the
skeleton and also facilitates the movement of joints necessary for voluntary movement of
the body.67, 68 Skeletal muscle is a highly organized striated multinucleated tissue that can
be broken down into smaller levels of organization.67 The skeletal muscle is comprised of
muscle fascicles which consist of muscle fibers/cells that are composed of myofibrils
consisting of myosin thick and actin thin filaments.68 The arrangement of the actin and
myosin filaments gives the skeletal muscle its striated appearance and the sacromere is the
contractile unit of the skeletal muscle. Skeletal muscle contraction occurs in response to
stimulation by motor neurons at the neuromuscular junction via the release of the
neurotransmitter acetylcholine which binds to receptors on the muscle membrane and
increases sodium permeability stimulating muscle impulses that travel down the t-tubules
and leads to calcium release from the sarcoplasmic reticulum.69-71 The mechanisms
involved in muscle contraction are complex and is not a primary focus of this thesis and
involve the troponin-tropomyosin complex.68 In resting skeletal muscle, tropomyosin is
wrapped around the thin filaments and covers the active sites preventing the binding of
myosin to the active sites on actin.72 However, when calcium is released from the
sarcoplasmic reticulum and binds to troponin, a conformation change occurs in which
tropomyosin shifts exposing the active sites.67, 72 Myosin can in turn bind with actin forming
a crossbridge and pull the thin filaments towards the midline via the power stroke leading to
muscle contraction.72 ATP is required for muscle contraction to occur and facilitates the
20
release of the actin from myosin so the cycle can continue. In the event that ATP supply is
completely exhausted the actin will remain bound with myosin and the skeletal muscles will
remain stiff as observed in rigor mortis. Muscle relaxation occurs as acetylcholinesterase
decomposes acetylcholine and calcium is resequestered back into the sarcoplasmic
reticulum.67, 71 Thus skeletal muscle is a highly organized tissue that enables organ
movement. It has a high metabolic demand during activity and a reduced basal requirement
of O2 at rest.
2.32 Vascular Organization of Skeletal Muscle Circulation
Large conduit arteries carry bulk blood flow and oxygen to the skeletal muscle
tissues. A relevant example to this thesis is the superior and inferior epigastric arteries that
supply the rectus abdominus muscle tissue. We will also be examining blood flow in the
carotid artery and the femoral artery which supplies the brain and femoral muscle
respectively. These arteries branch off into smaller arterioles which regulate organ specific
blood flow and the microvasculature where oxygen delivery and exchange occurs. The
microvascular organization in the skeletal muscle is highly organized and the arterioles in
the skeletal muscle give rise to capillaries that run in parallel with the muscle fibers and
each muscle fiber is surrounded by approximately 3 – 4 capillaries. Different muscles have
different oxidative capacities which determine the degree of capillary to fiber ratio. Slow
twitch red muscle fibers are dense with capillaries and rich in mitochondria and myoglobin
and can carry more oxygen and sustain aerobic muscle metabolism and thus can contract for
long periods of time with small force.67, 68 In contrast, fast twitch white muscle fibers can
contract quickly and forcefully contributing to muscle strength however white muscle
primarily participates in anaerobic metabolism.67, 68 Thus, muscle fibers with higher
21
oxidative capacity have a higher capillary to fiber ratio and greater maximal flow capacity
compared to muscle fibers with low oxidative capacity but high anaerobic capacity. In
resting skeletal muscle, the requirement of oxygen is much less than when the muscle is
contracting and only approximately 25% of the capillaries in the skeletal muscle are
perfused. However, during muscle contraction and active hyperaemia, all the capillaries
surrounding the muscle fibers may be perfused as a result of capillary recruitment defined as
an increase in the number of flowing capillaries around each muscle fiber which is
necessary for optimal muscle perfusion. Therefore the organization of the arteries that
provide bulk flow and the microvasculature that surround the skeletal muscle fibers are
highly organized and necessary to provide skeletal muscle perfusion.
2.33 Regulation of Skeletal Muscle Blood Flow
During resting conditions, approximately 20% of the cardiac output is permitted to
the skeletal muscles in the body and skeletal muscle blood flow is approximately 3 ml/min
per 100g. The regulation of skeletal muscle blood flow is dictated by the balance between
vasoconstrictive and vasodilating stimuli at the level of the resistance arteries.73 Some
possible factors that can have a vasodilatory influence resulting in increased muscle blood
flow include: (1) increases in interstitial potassium (2) increased H+ production, and (3)
nitric oxide and prostaglandins.73 The mechanisms of action of these vasodilating substances
are complex and will not be a focus of this thesis. Of greater significance are the
mechanisms of increased vascular tone via vasoconstrictors which may reduce skeletal
muscle perfusion. Sympathetic adrenergic nerves innervate the skeletal muscle vasculature,
and the release of norepinephrine as a neurotransmitter will stimulate vasoconstriction at the
level of the α1 adrenergic receptors. The precise signalling pathway and mechanism of
22
action of the α1 agonist-receptor pathway is described in section 2.62. High levels of
circulating catecholamines (epinephrine and norepinephrine, both α1 agonists) can also
induce vasoconstriction in skeletal muscle vasculature which may lead to jeopardized
muscle perfusion.74 In fact, a focus of this research project will investigate the
vasoconstrictive effects of a common antihypertensive drug, phenylephrine, a specific α1
agonist, on skeletal muscle perfusion during surgery. During periods of impaired skeletal
muscle perfusion, skeletal muscle has been documented to tolerate ischemia for up to 3
hours.75, 76 Irreversible muscle damage resulting from muscle ischemia occurs after 4-6
hours.7 When the resting skeletal muscle is denervated, the blood flow to the skeletal muscle
can be expected to increase due to the resultant effect of reduced vascular tone. Thus,
sympathetic stimuli from the sympathetic nerves, cathecholamines, or pharmacologic agents
can all lead to vasoconstriction and dramatically reduce muscle blood flow. As a result of
decrease blood flow to the skeletal muscle, the muscle will increase oxygen extraction and
partake in anaerobic metabolism for ATP production when oxygen is exhausted. In the
event that muscle perfusion is not restored due to excessive administration of
vasoconstrictors, muscle necrosis may occur77.
2.34 Oxygen Pressures in Interstitial Skeletal Muscle Tissue
There have been many advances in technologies associated with the measurement of
interstitial oxygen measurements in tissues, particularly resting skeletal muscle tissue. The
creation of G3/G4 oxyphors and O2 dependent quenching of phosphorescent method
described in section 2.23 have enabled accurate and quantitative assessments of interstitial
skeletal muscle tissue PO2 superior to previous methods of measuring tissue oxygen.46 In
the past, Whalen et al used clark electrodes with small tips to record PO2 values in the cells
23
of guinea pig gracilus muscles with measured PO2 values of 0-5 torr.78 Although this was an
initial attempt to assess muscle tissue PO2, the effects of anesthesia on muscle PO2 was
underappreciated. Other studies have recorded skeletal muscle PO2 values of around 31.4
torr 79 (intravascularly), 19 torr80 (intravascularly), and 26.8 torr81 (interstitially).
Richardson et al used NMR method to measure oxygenated versus deoxygenated myoglobin
in muscle to estimate muscle PO2 and reported muscle PO2 value of 34 torr.82 It is unknown
if different regions of skeletal muscles on the body have differing interstitial PO2 values,
however the differences are expected to be very small. As newer technologies developed,
Wilson et al measured interstitial PO2 in awake and anesthetized rats and reported a muscle
PO2 of 46.2 torr in awake rats and a muscle PO2 of 36.9 torr in rats under isoflurane
anesthesia with G3 oxyphor.47, 48 Resting nonoperated rectus abdominus muscle interstitial
tissue PO2 data obtained in our experimental protocols measured PO2 values ranging from
around 30-40 torr using the microsensor G4 oxyphor phosphorescence quenching method.
Therefore advancements have been made to investigate quantitative levels of oxygen in
skeletal muscle tissue, and our data reports interstitial skeletal muscle PO2 values in the
range of 30-40 torr in rats under the effects of isoflurane. To our current knowledge, no one
has assessed interstitial muscle flap PO2 with the G4 oxyphor method. Our data suggests
that PO2 in muscle flaps is very low at around 5 torr. This reduction in flap PO2 may result
from surgical manipulation and preparation of the muscle free flap.
2.35 Energy Sources for Skeletal Muscle
Oxygen is necessary as the final electron acceptor in the electron transport chain for
the production of 36 ATP through aerobic respiration in skeletal muscle. In the absence of
oxygen, anaerobic metabolism takes place in which a net yield of 2 ATP and lactate is
24
produced. ATP is used as an energy source to supply skeletal muscle contraction.68 When
the energy sources become depleted, muscle cells must regenerate ATP from ADP.
Phosphocreatine (PCr) is another high energy molecule in the skeletal muscle that can act as
a reservoir for energy.68, 83, 84 In the event that ATP is sufficient, the muscle enzyme
creatine kinase (CK) can phosphorylate creatine and form phosphocreatine (PCr) that stores
excess energy in its phosphate bonds.83 When ATP supply is exhausted it becomes ADP,
and phosphocreatine can be used to replenish ATP supply by transferring its phosphate to
the ADP molecule to create ATP again.68, 84 Therefore phosphocreatine is used to restore
ATP levels in the skeletal muscle when the energy supply is low. Creatine kinase is an
important enzyme that facilitates this reversible reaction. The role of creatine kinase as a
clinical marker will be discussed in section 2.4.
2.4 Markers to Evaluate Health of Skeletal Muscle Clinically
Experimentally, skeletal muscle perfusion can be assessed by measuring
microvascular blood flow and interstitial PO2 in rats. However in the operating room,
certain markers can be used as indicators of inadequate muscle perfusion and assess muscle
health during surgery through simple arterial blood gas and urine sample analysis. Serum
lactate which is produced as a byproduct of anaerobic metabolism in the skeletal muscle is
an early indicator of inadequate skeletal muscle perfusion. The normal range of serum
lactate ranges between (0.3-1.3 mmol/L), and elevation of serum lactate beyond the normal
range of serum lactate are of concern.40 Creatine kinase is an enzyme that can also serve as
a marker of skeletal muscle damage when it is leaked into the blood stream in response to
muscle damage. Normal ranges of creatine kinase range between (45-260U/L), elevation of
CK beyond the normal range may be indicative of skeletal muscle damage and CK values
25
>5000U/L are correlated to acute renal failure in patients.6 Myoglobin is an oxygen carrier
in the skeletal muscle similar to hemoglobin in the blood, however it is also another toxic
component that is released into the blood stream when muscle cells lyse. Myoglobinuria
gives patient urine a reddish brown colour and can be toxic to the kidney leading to acute
renal failure.5, 10, 11, 85 Taken together, elevated levels of serum lactate, creatine kinase, and
positive myoglobinuria are suggestive of muscle damage. Our clinical study will investigate
these markers of muscle perfusion in a population of neurosurgical patients undergoing
brain tumour resection.
2.41 Clinical Importance of Serum Lactate
Increased blood lactate level has been correlated with a higher mortality rate and an
elevated risk of developing multiple organ failure in critically ill patients. 35, 36 Furthermore
a rapid clearance of lactate from the blood during the treatment for the critical illness is
correlated with greater survival and better outcome than patients who did not clear their
lactate load.39 Lactate level has also been examined as a predictor of outcome also in
surgical patients. In 2004, Meregalli et al,86 conducted a prospective study on 44 patients,
hemodynamically stable, undergoing non-cardiac surgery and admitted postoperatively to a
general ICU. In their results, the survivor’s blood lactate levels decreased significantly with
time (1.6±0.9 mmol/l vs 2.9±1.7 mmol/l, p=0.012), but levels remained stable in the
nonsurvivor group, supporting the hypothesis that lactate might be a reliable marker in
predicting poor outcome in the early phase after high risk surgery. All tissues can produce
lactate under anaerobic conditions, but only tissues with active glycolysis produce excess
lactate from glucose under normal conditions and release it into the bloodstream. At rest
lactate is produced from skeletal muscle (25%), skin (25%), brain (20%), red cell (20%),
26
and gut (10%). The liver is the primary site of lactate clearance (60%) and the kidneys
metabolize approximately 20 to 30% of daily lactate. The balance between release into the
bloodstream and hepatorenal uptake maintains plasma lactate at about 1 mmol/l. 41, 87, 88
Lactic acid accumulation can occur due to excessive tissue lactate production or
from reduced clearance in conditions of normal hepatic and renal function.89 A continuing
lactic acidosis means that there is continuing production of lactate that exceeds the liver and
kidney’s capacity to metabolize it. This may be due to very excessive production (eg
convulsions, ischemia) with a normal liver at one extreme, or at the other extreme due to
increased production in associated with greatly impaired hepatic capacity to metabolize it
(eg due to cirrhosis, sepsis, hypoperfusion due to hypovolemia or hypotension, hypothermia,
or some combinations of these factors). Historically it has been described that lactic acid
can be abnormaly high in presence of hypoxia reflecting inadequacy of oxygen to support
aerobic metabolism.90 The imbalance between oxygen demand and oxygen availability
results in tissue hypoxia, an impairment of mitochondrial oxidative capacity with
accumulation of pyruvate and the generation of lactate. This is one of the most common
cause of lactic acidosis. Other etiologies of hyperlactatemia are rare.37
2.42 Rhabdomyolysis and Muscle Damage
Rhabdomyolysis is a clinical syndrome resulting from severe skeletal muscle injury
(direct trauma, compression, ischemia, compartment syndrome) causing release of toxic
intracellular contents from myocytes into the circulatory system. 10, 11, 91 Clinically,
rhabdomyolysis is associated with significant elevation in serum CK and myoglobin.
Although traumatic muscle injury remains the most obvious cause of rhabdomyolysis, other
27
medical causes have been reported92 and these are estimated to be more common than
traumatic causes.91 In particular, prolonged operative times, surgical position and
immobility are main risk factor for developing perioperative muscle ischemia and
rhabdomyolysis. 5-7, 85, 93 A direct increase in local tissue/vascular pressure related to
positioning can cause muscle hypoxia/ischemia.92 The risk of rhabdomyolysis is particularly
high when other concurrent risk factors such as peripheral vascular disease, diabetes, high
body mass (body weight more then 30% above ideal body weight), extracellular dehydration
or hypoperfusion are present. 12, 94-96 Lagandré et al. concluded that surgery longer than 4
hours is associated with an increased risk of rhabdomyolysis.97 In a group of 30 patients
undergoing elective craniotomy, Poli et al. found an elevation in CK and the duration of the
surgery was the only factor clearly related to this elevation.7 In a recent review9, De
Tommasi and Cusimano underlined how rhabdomyolysis is highly under-recognized in
neurosurgery and that in their experience obese patients undergoing long neurosurgical
procedure should be closely monitored for CK level, myoglobinuria and acidosis. Our newly
published data (Garavaglia et al 2013) supports this observation as will be discussed in
future sections.12
2.43 High Body Mass Index as a Risk Factor for Muscle Ischemia
Ideal body weight is most commonly calculated from the body mass index (BMI)
and the Centers for Disease Control and Prevention defined overweight as a BMI of 25 to 30
kg/m2 and obesity as a BMI more than 30 kg/m2. Obesity has been linked to increased
morbidity and mortality in patients undergoing several types of surgical procedures. 98, 99
However the specific effect of increased body mass on patient outcome after neurosurgery
has not been well understood.100 The only data available from a literature review showed
28
that obesity increases the risk of infection after spine surgery.101-104 Further research is
necessary to understand the contribution of obesity or increased BMI to significant
perioperative complications and its effect on the outcomes of patients undergoing
craniotomy. A novel association between BMI and serum lactate rise will be described in
this thesis.
2.5 Reconstructive Flap Surgery
2.51 What is a Free Flap?
Coverage of anatomical tissue defects is an important and frequent job of the
reconstructive surgeon. Clinical scenarios where tissue is lost through a variety of
mechanisms including trauma and tissue resection for tumours (benign and malignant) is an
everyday occurrence in the clinical realm. As part of the reconstructive process, surgeons
must consider both the function and form of the loss in an attempt to provide a robust,
accurate, and durable reconstruction.
A flap is a general term used to describe a piece of living tissue that is transferred to
another site where a defect exists in order to provide coverage with the premise of like
replacement. The simplest of flaps involve leaving a large enough base of the flap attached
to provide a blood supply through a plexus of vessels; ensuring the tissue remains alive.
(Figure 2B) The limitations of these flaps are based on the size and geometry to provide
local coverage of defects. This concept is further developed with the term pedicled flap that,
by virtue of having a named and more mobile blood supply, can provide local as well as
regional coverage of defects. Again, the limitations of these types of flaps depends on the
size, reach of the named blood supply and the geometrical orientation of the tissue at the site
29
of inset to ensure adequate arterial inflow and venous outflow. Compromise of any of these
aspects may compromise flap survival.
For some defects, microvascular surgical techniques are widely used to harvest and
move vascularized tissue in clinical scenarios where patients have significantly large,
remote and functional tissue loss. Free tissue transfer or free flaps, can be composed of a
variety of living tissues (bone, muscle, bowel, subcutaneous tissue with skin) and are
harvested with a dedicated blood supply. This tissue flap is then transferred to a new site
where microsurgical techniques are used to re-anastamose the flap vasculature to a new
recipient donor vasculature.
Because of the method of harvest, free flaps are denervated tissue believed to have no
intrinsic sympathetic tone. It is postulated that at the time of harvest, these flaps may be
maximally vasodilated.105 Free flaps are also subject to a period of ischemia prior to re-
anastomosis and have compromised lymphatic drainage rendering them sensitive to fluid
shifts compared to healthy tissue 106. All of these properties point to the complexity of this
method of tissue reconstruction and implicate several different avenues for potential flap
success or failure.
As the art of microsurgery has been perfected over time, success of free flaps has
largely been attributed to the skills of the surgeon. However management of the patient
during these procedures including control of central hemodynamics, regional blood flow
(and ultimately flap blood flow) and volume status have been proposed to play key roles.107
Importantly, these procedures can be long in duration, involve patients with compromised
status, involve a variety of anatomical areas and may see more than one clinician involved
in the intraoperative and post operative care. Important factors like intraoperative
hypotension, management of blood loss with fluid resuscitation and use of vasoactive agents
30
have all been proposed as potential factors influencing microsurgical mileau and potentially
the flap. Compromise in vascular status of the flap by any means may result in flap necrosis
or complete flap loss.
2.52 Potential Causes of Free Flap Failure
Flap losses are usually seen within the first three postoperative days.60 Several
factors can contribute to flap failure during reconstructive surgery and certain patients with
comorbidities may be prone to flap complications. For instance, patients with diabetes or
hypercholestemolemia have a higher risk of microvascular disease and atherosclerosis
which may affect flap outcome during surgery. Microvascular disease can affect healing
and neovascularation of the flap. Another factor that may be associated with flap
complications is obesity, which is generally associated with greater amounts of adipose
tissue which complicates dissection of the vascular pedicle and anastomosis. The skills of
the surgeon is an important factor which can determine the outcome of the flap
characterized by the quality of anastomosis and length of ischemia time as an inadequate
anastomosis may result in free flap failure.
Vascular thrombosis has an occurrence rate of around 8-14% and is the most
common cause of free flap complications and failure, particularly venous thrombosis.19, 60,
108, 109 In a rectrospective study that involved 1000 flaps, Pohlenz et al (2012) noted that
venous thrombosis and hematoma were the most common causes of free flap failure.19
Kruse et al (2011) also attributed most of the flap failure cases to venous thrombosis in a
study with 81 radial forearm flaps.108 Selber et al (2012) performed a study with 4956 flaps
and also concluded that thrombotic events are a major risk factor for flap failure.109 Herold
et al (2011) have reported that increased platelets and leukocytes were associated with flap
31
complications characteristic of clotting and infection.110 Laboratory parameters of
thrombosis and bleeding (activated protein C, fibrinogen, factor VIIIC, von willebrand
factor, and VWF antigen) were elevated in patients with complications in free flap surgery
and thus can serve as a clinical indicator to predict flap outcome.14 Pattani et al (2010)
performed a literature review and identified intraoperative factors that are associated with
free flap failure. Excessive intraoperative fluid administration of >7L, significant medical
comorbidity, nitrate use, bronchodilator treatment, irradiated recipient site, prolonged
operative time > 10 hours, and involvement of greater than one microvascular surgeon was
associated with increased incidence of free flap failure.15 They state that no clinical
evidence exists to support the role of hypotension, vasopressors, colloids, and anticoagulant,
and nitrous gas in flap failure. Suprisingly, old age, smoking, diabetes, and obesity did not
appear to correlate with flap complication and failure in their study. In summary, many
factors can lead to flap failure, and vascular thrombosis is the most common. To ensure
quality of free flaps in our experiments, we administered heparin saline to the flap to prevent
thrombosis and checked for blood flow clinically with microscope and experimentally with
laser doppler flowmetry.
2.53 Rectus Abdominus Skeletal Muscle Flap
The bilateral rectus abdominus muscles provide abdominal flexion and support the
intra-abdominal contents and is commonly used as a muscle flap for breast reconstruction
surgery. The rectus abdominus muscle flap is innervated by the intercostals nerves and is
supplied by the superior epigastric artery and deep inferior epigastric artery branching from
the external iliac arteries.111 (Figure 2A, 2B) In the creation of a muscle free flap, a midline
incision is performed and the superior epigastric artery is ligated. The external iliac artery
32
proximal to the deep inferior epigastric artery is then transected and reanastomosed and
blood flow is checked clinically with a microscope. (Figure 2B)
Zhang et al (1993) have studied the rectus abdominus muscle flap model in sprague
dawley rats extensively and consider the rectus abdominus muscle model to be the first true
myocutaneous model in the rat.111 Their goal was to design a rectus abdominus muscle flap
and rectus abdominus myocutaneous flap model in the rat for future biological,
pharmacologic and biochemical studies. The anatomy of the rectus abdominus muscle in
rats was studied and reported to be the very similar to humans with a consistent double
blood supply and multiple musculocutaneous perforators.111 In fact, the superior and
inferior epigastric vessels that supply the rectus abdominus muscles make an ideal flow
through system. In experiments in which rectus abdominus muscle flaps were transplanted
to the groin, they noted that the muscle flap survival rate over a period of 5 days was
100%.111 Finally, the tissue mass of the rectus abdominus muscle averages around 3 grams
which is enough for experimental tissue assays to assess ischemia. Based on these
experimental findings in this developmental model we decided to use the rectus abdominus
muscle as our skeletal muscle of interest in studying the effects of vasopressors on skeletal
muscle perfusion.
33
Image adapted from Microsurgeon.org (Dr. Rudolf F. Buntic, MD)
Figure 2. The Vascular Organization of the Rectus Abdominus Muscle Flap (A) The Deep Inferior Epigastric Artery and the Femoral Artery proximal to the External Iliac Artery (B) Rectus Abdominus Muscle Flap.
A
B
34
2.6 Vasopressors 2.61 Phenylephrine, a Specific α1 Agonist
Since 1949, Phenylephrine, a common vasopressor has been used in medical practice
as an antihypotensive treatment. Phenylephrine is a highly specific αl agonist that will
trigger the α1 receptor mediated cascade ultimately leading to vasoconstriction, increased
systemic vascular resistance and increased mean arterial pressure (MAP).21, 56, 112 Clinicians
generally use MAP as an indicator of adequate cerebral perfusion (CPP = MAP - ICP) and
perfusion to other peripheral organs in the operating room. Thiele et al (2011) have
emphasized that an increase in MAP does not necessary equal to an increase in organ
perfusion and that it is tissue oxygen delivery that is reflective of organ specific perfusion.21,
112 Therefore in our experiments we will be assessing MAP, microvascular blood flow, and
quantitative tissue PO2 reflective of oxygen delivery to evaluate the effects of phenylephrine
on skeletal muscle perfusion. As recently reviewed, the potential cost of this treatment is a
decrease in oxygen perfusion to peripheral tissues (muscle, gut, and kidney) despite the
effective treatment of an intermediate outcome (MAP).21 The explanation for this vascular
response to phenylephrine may be explained by differences in α1 receptor density in
different vascular beds. For example, in the brain there are fewer α1 receptors, relative to
other tissues such as skeletal muscle.21, 113 This means that stimulation by α1 agonist will
severely vasoconstrict skeletal muscle vasculature and have a lesser effect on the cerebral
vessels. Indeed, Duebener et al (2004) have demonstrated that PE redirects blood flow from
the bowel and skeletal muscle to the brain and the liver in pigs.114 Our lab has also
previously shown that PE infusion leads to increased cerebral blood flow and tissue PO2 in
rats. 115 The current goal of this research project is to evaluate the effects of PE on skeletal
35
muscle perfusion in rats, which has demonstrated impaired skeletal muscle perfusion.
Therefore phenylephrine infusion may lead to an increase in MAP predominantly due to
peripheral muscle vasoconstriction and increase cerebral blood flow and tissue PO2 due to
the relative lack of α1 receptors in cerebral vasculature.115
2.62 α1 Agonist-Receptor Mediated Intrinsic Signaling Pathway
Adrenergic agonist-receptor interactions play an important role in the regulation of
vascular tone and perfusion. The α1 adrenergic receptor is a G protein coupled receptor
(GPCR) that is highly expressed in the microvasculature of skeletal muscle tissues. 21, 116-119
Upon binding of α1 agonist such as phenylephrine the receptor will become activated
coupling to G protein (GqII).116, 117 Phospholipase C will cleave Phosphatidylinositol 4,5
bisphosphate (PIP2) into Inositol (1,4,5) triphosphate (IP3) and diacylglycerol (DAG).118
IP3 will travel throughout the cytoplasm until it binds with IP3R on the sacroplasmic
reticulum of the smooth muscle cell to induce Ca+2 release into the cytoplasm.118, 119
Calcium can also be resequestered back into the sarcoplasmic reticulum via the
sarco/endoplasmic reticulum Ca+2 ATPase (SERCA). Extracellular Ca+2 also play a
significant role as the sarcoplasmic reticulum of the smooth muscle cells are less developed.
The intracellular calcium will interact with smooth muscle calmodulin which in turn
activates the myosin light chain kinase (MLCK).116, 117 MLCK phosphorylates myosin light
chain resulting in smooth muscle contraction and vasoconstriction.120 β2 Adrenergic
signaling can antagonize α1 adrenergic signaling through the adenyl cyclase- cAMP-Protein
Kinase A pathway which in turns activates myosin light chain phosphatase (MLCP) leading
to the dephosphorylation of the MLC20-P into MLC20 and thus results in vasodilation. In
the absence of α1 agonists adequate muscle perfusion is expected assuming normal
36
physiological conditions since there is no α1 induced vasoconstriction of the muscle
resistance arteries. However, once phenylephrine is administered, there may be impaired
blood flow and perfusion due to vasoconstriction of the resistance arteries. In fact, perfusion
may be severely impaired if the muscle tissue was denervated and reanastomosed as
performed during free tissue transfer surgery with muscle flaps. This thesis will examine
the effects of PE on surgically operated skeletal muscle and muscle flaps.
2.63 Clinical Debate: Vasopressor Use during Reconstructive Surgery
Skeletal muscle perfusion may be at risk during reconstructive surgery as muscle
flap complications such as necrosis and flap failure may occur. Phenylephrine is an α1
agonist that has been used during general surgery to increase mean arterial pressure in
hypotensive patients for the last 60 years. The use of vasopressors during reconstructive
surgery to treat hypotension has been recently debated. Although recent retrospective and
prospective clinical studies 22-25 have found no correlation between the use of vasopressors
and flap complication and flap failure during reconstructive surgery, microsurgeons strongly
warn against the use of vasopressors as peripheral vasoconstriction at the level of the
resistance arteries can impair muscle flap perfusion and lead to vasospasm, thrombosis and
flap failure. Animal studies yield controversial results as one study suggests that
phenylephrine may have no effect on flap perfusion,121 while other studies suggest that
phenylephrine may be detrimental. 26, 27 The role of intraoperative and postoperative use of
vasoactive agents in free flap surgery is still controversial and a source of heated debate in
microsurgical circles. Concerns about administration of vasoactive agents during
microsurgery are related to the effects the drugs have on systemic vascular resistance and
blood flow. It has been proposed that drugs which decrease vascular resistance may have
37
more of an effect in normal tissue (adjacent to the flap) than in the flap itself and as such
may result in a “steal effect” in the flap reducing arterial flow into the flap.122 This is
because the tissue and blood supply at recipient site is still responsive to the properties of
the drug because of an intact sympathetic tone. Alternatively, drugs which increase vascular
resistance may cause vascular spasm at the anastamotic site decreasing blood flow for the
same reasons. 123, 124
2.64 Clinical Studies
Clinical studies looking at the role of vasoactive agents is controversial. Monroe et
al.25 (2010) used a case series with chart review of free tissue transfers to document the
frequency of vasoactive agent use in free tissue transfer surgery and compare the incidence
of flap complications and flap survival in patients who did or did not receive intraoperative
vasoactive agents. In this retrospective study, the patients undergoing free flap surgery were
predominantly male (68%) had an average age of 62 years and were having a flap done for
head and neck cancer with the most common flap used being a radial forearm flap. Of the
241 flaps, data was available for 169 (70%). Overall flap survival was reported to be
96.5%. Overall flap complication rate was reported to be 29%. A total of 139 patients
(83%) received a vasoactive agent intraoperatively. The most common used agent was
phenylephrine (33%). In this group there were 4 flap failures (2.9%) and 40 (29%)
complications. In the 30 patients who did not receive intraoperative vasoactive agents, there
were 2 (6.7%) flap failures and 9 (30%) complications. For all the 6 failures, pedicle
problems (arterial or venous) were the underlying pathology.
In reviewing the data, there was a significant age difference between the vasoactive
agent group and the nonvasoactive agent group, but the American Society of
38
Anesthesiologist scores (ASA) were similar indicating similar surgical risks and
comordibities. There is no comment or data on the timing of drug administration, dose or
clinical indications for use. The authors do comment that the timing of vasoactive agent use
was likely temporally far removed from actual microsurgical anastamosis and would have
had little contribution to flap pedicle vasospasm.
Interestingly, in the vasoactive agent group, there was a significant difference in the
dose of phenylephrine between failed and successful flaps with the failed flaps receiving
almost 4 times the amount of the drug. The authors attribute this to one patient who had a
continual infusion throughout the case and subsequently had a flap failure. This points out
two critical aspects which could not be addressed in this retrospective trial: timing of
administration of vasoactive agent during flap harvest or microsurgery and duration of
exposure/dose of phenylephrine received. These factors may be as critical as the drug itself.
Finally there are no comments as to the etiology of the flap type that failed. The
composition and inherent blood supply of the flaps that failed may again point to the
differences these drugs can have depending on the vascular environment. Thirty-eight
percent of the flaps used were radial forearm flaps which are known to be quite robust. In
fact there has been a study that shows that systemic phenylephrine has no vasoconstrictive
effect on radial arteries harvested for coronary bypass surgery 125, rather the increase in
MAP secondary to phenylephrine administration improved blood flow through the artery.
Perhaps a similar phenomenon occurs in radial forearm flaps.
Monroe et al (2011) also conducted a prospective observational study with 169
consecutive patients undergoing head and neck reconstructive surgery that addressed the
timing of administration of vasopressors. Their conclusions confirm the results of the
previous retrospective study where there was no correlation between vasopressor use and
39
flap failure or complications.24, 25 The proportion of patients experiencing early flap failure
was 4.4% (4/90) in the intraoperative vasopressor group versus 2.5% (2/79) in the
nonvasopressor group.24
Chen et al. (2010) also performed a retrospective study reviewing 187 consecutive
patients undergoing 258 free flaps for breast reconstruction.22 A total of 102 patients (140
flaps) received intraoperative ephedrine or phenylephrine. They found no differences in
rates of reoperation, complete flap loss, partial flap loss, or fat necrosis. Further, there were
no associations between dosage, timing and complications related to vasoactive agent use.
Despite this, this group of authors did not recommend the routine use of vasoactive agents
for microsurgery, but did endorse careful communication within the operating room
environment in situations where vasoactive agents are necessary.
Finally, Harris et al (2012) also retrospectively reviewed a similar population.23 In
their study of 485 patients, there were 496 free tissue transfers for head and neck
reconstruction with a total major complication rate of 5.2%. This included 11 (2.2%)
complete failures. This did not include a partial failure rate of 1.4% or a take back rate of
1.6 %. Of the 485 patients in the study, 320 (66%) received intraoperative vasoactive agents
with over 97% receiving phenylephrine. Like Monroe et al., they found no relationship
between administration of phenylephrine and flap failure. The most common free flap in this
series was also a radial forearm flap. They found 8 flap failures in the vasoactive agent
group and 3 failures in the nonvasoactive agent group. They did look at timing of drug
administration and found no significant relationship between the time of administration and
adverse flap outcome. Dose of phenylephrine given also did not correlate with flap failure.
They did however exclude patients who received multiple doses of vasoactive agents in this
analysis.
40
Despite the retrospective clinical data, which would suggest that drugs like
phenylephrine do not statistically affect free flap failure, few microsurgeons endorse the use
of vasoactive agents during free flap surgery.
2.65 Animal Studies
The effect of vasopressors on free flap perfusion has been studied in animal models,
however, like the clinical studies the results are controversial. These animal studies focused
on the relationship between systemic or locally administered vasoactive agents, MAP and
flap perfusion. Corderio et al. (1997) used a musculocutaneous porcine flap model that
allowed simultaneous monitoring of systemic as well as flap hemodynamic parameters.26
They used a vertically raised rectus abdominus muscle flap to look at the relationship
between systemic and flap effects of 3 common vasoactive drugs. They used a flow probe
around the artery to measure blood flow into the flap and cannulated the pulmonary artery
and aorta to measure cardiac output and aortic root pressures. The model was then used to
measure the effects of varying doses of dopamine (D1, β1 and α1 agonist; dosage
dependant), dobutamine (β1, β2 agonist; preferentially β1) and phenylephrine (α1 agonist)
on systemic and flap measures of blood flow. They found cardiac output was increased with
low and high dose dopamine and dobutamine but decreased with increasing doses of
phenylephrine. Flap flow increased only with dobutamine, remained unchanged with
dopamine and decreased with high dose phenylephrine. Relative to cardiac output both
dopamine and dobutamine also decreased flap flow. They concluded that phenylephrine
clearly affects flap flow adversely and should be avoided while dopamine and dobutamine
should be used with caution. They proposed that despite the flap being denervated, intrinsic
factors that regulated vascular tone could still be in play. They proposed it was this delicate
41
balance of intrinsic and extrinsic factors that ultimately determined the actual blood flow
and perfusion of the flap.
This paper was the first to use a large animal model to measure both systemic and
flap hemodynamic parameters. Criticism of the Cordeiro paper focused on the flap design
as it probably best mimicked a pedicled flap since the superior epigastric artery was left
intact. In this group’s defense they do not call it a free flap, but state that by removing the
adventitia of the vessels, they created a denervation model which mimics the situation in
free flap surgery when the pedicle is cut prior to transport to the recipient bed. A second
criticism is that the doses of phenylephrine used in the study were supratherapeutic with
respect to clinical correlation.23
Banic et al. (1999) was interested in the role of sodium nitroprusside (SNP; an
arterial vasodilator) and phenylephrine on blood flow in a free musculotaneous flap.121
Using a porcine model, latissimus dorsi flaps were raised and used to cover a lower
extremity defect using microsurgical anastamosis of the flap. This represents a true free flap
model. Total blood flow in the flap was measured after re-anatamosis using ultrasound
flowmetry and microcirculatory blood flow was measured using laser doppler flowmetry.
Systemic administration of SNP resulted in a 30% decrease in MAP without changing
cardiac output. Total flow in the flap decreased by 40% with microsurgical flow decreasing
by 23% in the skin and 30% in the muscle. SNP infused directly into the flap via a feeding
artery resulted in an increase in total flap flow by 20%. Systemic administration of
phenylephrine caused a 30% increase in MAP, without any changes in heart rate, cardiac
output or flap blood flow. Local administration of phenylephrine via a feeding artery
directly into the flap caused a decrease in total flap flow by 30% without any effect on skin
or muscle blood flow. The authors concluded that systemic phenylephrine in a dose which
42
increased systemic vascular resistance, had no adverse effects on blood flow in the flap
while SNP, in a dose which resulted in decreased vascular resistance and arterial pressure,
caused a severe reduction in free flap blood flow. Banic et al. concluded that changes in
total blood flow and vascular resistance in the flap by SNP suggested that there was a
pharmacologically reversible vascular tone in the flap vessels despite total sympathectomy.
The nature of this tone (reperfusion injury, circulating catecholamines or other vasoactive
substance) was unknown. Failure of phenylephrine to provide vasoconstriction of the flap
was attributed to its pure α1 agonist properties and its effect on predominantly larger
arterioles (100 um). They concluded that phenylephrine may be safe to use in microsurgery,
but cautioned that their results applied only to phenylephrine and not broadly to other
vasoconstrictors. As well they cautioned that all experiments were done in a setting of
normovolemia and situations of hypovolemia may have different results.
In an attempt to try to reconcile these differences, Massey and Gupta (2007) also
used a porcine vertical rectus flap model to measure pedicle artery blood flow and
microvascular perfusion during systemically administered intravenous phenylephrine or
epinephrine in a dose dependant fashion.27 They found that phenylephrine consistently
decreased pedicle artery blood flow and microvascular perfusion of the flap while
epinephrine increased both flows. The increases seen in cardiac output with administration
of epinephrine also correlated well with increased pedicle blood flow and microvascular
perfusion. They concluded that epinephrine may be the preferential agent for treating
intraoperative hypotension during flap surgery.
The premise that vasoactive drugs have little effect on flap perfusion comes from the
concept that the entire body has flow that is dependent on systemic perfusion pressure. If
you improve systemic perfusion pressure then by extension this would improve flap
43
perfusion. The opposite school of thought is that vasoactive agents like phenylephrine cause
vasoconstriction of the isolated flap pedicle artery and microvasculature due to the isolation
of the detached microcirculation from peripheral support. While Banic et al. and Massey
and Gupta’s work seem to support the first premise, Codeiro et al.’s work would support the
latter. From these three studies, the role of phenylephrine in flap surgery was still not clear.
2.7 The Effect of Temperature on Tissue Metabolism and Perfusion
Temperature is a factor that can affect skeletal muscle perfusion during surgery.
Hyperthermia is generally associated with peripheral vasodilation in attempt to promote heat
loss. In fact, clinicians will place patients who have undergone free muscle flap transfer in
warm rooms after surgery to promote vasodilation for the benefit of flap perfusion.126 In
contrast, hypothermia, although effective at preserving organs, extremities, and free flaps by
reducing metabolic rate, can be detrimental to skeletal muscle and flap tissue in the intact
living body. Cooling is associated with peripheral vasoconstriction, increased vascular tone,
increased blood viscosity and platelet aggregation which can all contribute to jeopardized
muscle and muscle flap perfusion during surgery. 127-131 Hussl et al (1986) examined the
effect of temperature on blood flow and metabolism in neurovascular island skin flaps and
demonstrated that the blood flow and metabolism declined with decreasing temperature.130
After cooling the flap from 35oC to 20oC, the blood flow was 65% of the baseline while the
oxygen consumption was only 25% of baseline . Blood flow was observed to cease when
cooled to 14oC, which the authors attributed to increased blood viscosity. Faber et al (1988)
observed an additional increase in constriction of the large arterioles in rat cremaster skeletal
muscle when the temperature was cooled from 34oC to 26oC in a norepinephrine bath.128
Kinnunen et al (2002) have demonstrated that hypothermia significantly decreases blood
44
flow and postocclusive reactive hyperaemia in the rat epigastric pedicled groin flap which
may increase the risk of ischemic flap complications unless rewarming is performed.127
Binzoni et al (2012) have continuously monitored blood flow on small muscle masses at
different temperatures with laser doppler flowmetry.132 They warmed human skeletal
muscle from 15oC – 40oC with a water bath and found that increased temperature resulted in
increased blood flow speed. Conversely, decreasing muscle temperature would result in
reduced muscle blood flow to the skeletal muscle. Therefore increasing temperature will
lead to increased vasodilation and increased blood flow, whereas cooling will result in
peripheral vasoconstriction and decreased blood flow and metabolism in the skeletal muscle
and muscle flaps. Temperature has been used as an assessment of flap perfusion during
surgery, however Kaufman et al (1987) have suggested that it is an unreliable indicator.133
Similarly, a pink healthy looking flap may not be reflective of an accurate perfusion status
of the flap. Examining indicators of flap perfusion by assessing quantitative muscle tissue
PO2 measurements will be more reliable than temperature and the colour of the flap. It is
generally recommended that warming blankets should be used to maintain patient body
temperature preoperatively, intraoperatively, and postoperatively for two days to prevent
cooling during surgery. In our rat experiments, we have used a heating pad and heating
lamps in an attempt to keep the animal warm.
45
CHAPTER 3 METHODS
3.0 Experimental Design:
This thesis is comprised of both clinical and experimental animal studies in an attempt to
provide a translational approach to understanding mechanisms which impair muscle
perfusion during surgery.
Part 1 Clinical Human Study (A) A prospective clinical study was designed to investigate the potential causes of increased
serum lactate characteristic of inadequate perfusion in patients during neurosurgery.
This prospective clinical study involved perioperative arterial blood gas and urine
sample analysis and postoperative data collection in the intensive care unit. The study
was undertaken to determine the significance of the frequently observed increase in
serum lactate which occurred in patients undergoing craniotomy for brain tumour
resection. The initial hypothesis, that serum lactate was dependent on length of surgery
was revised with the early observation that the early rise in serum lactate has been
correlated to the patient body mass index (BMI) suggesting that patient mass induced
muscle compression during surgery may be responsible for impaired skeletal muscle
perfusion. Evidence of associated muscle break down may be verified by muscle
damage markers (creatine kinase, and myoglobinuria).
Part 2 Experimental Rat Study
(B) An experimental rat model was developed to evaluate skeletal muscle and muscle flap
perfusion during free tissue transfer surgery and the effects of vasopressor mediated
vasoconstriction (phenylephrine) on flap perfusion. These experiments were performed
in a number of progressive protocols in which a number of parameters were assessed in
46
response to a stepwise increase in phenylephrine infusion. Important outcomes included;
femoral vs. carotid artery blood flow; microvascular blood flow and tissue PO2 in
skeletal muscle and muscle free flaps.
3.1 Clinical Study Methods: 3.11 Study Design
Institutional research ethics board (REB) approval was obtained for a single centre
observational study to assess ASA 1-4 adult patients scheduled for brain tumour resection
craniotomy.
3.12 Study Population
The study population includes 18 of 20 patients that were consented in this pilot study. 3.13 Study Protocol:
Inclusion criteria for the study included men and women with an ASA physical
status 1-4 and age greater than 18, undergoing prolonged surgery for complex brain tumour
resection with an estimated surgical time ≥ 5 hrs.
Exclusion criteria included: the presence of clinical co-morbidities that might cause
an increased level of lactate (sepsis, shock, renal or hepatic dysfunction, infusion of
catecholamine, antiretroviral drugs, limb or mesenteric ischemia, severe COPD, severe
anemia), history of myopathy or muscular dystrophy, presence of acquired causes of
methemoglobinemia and a preoperative hemogloblin less than 100g/L
3.14 Data Collection:
Pre-operatively patient gender, age, past medical history (hypertension, asthma,
COPD, diabetes, peripheral vascular disease, myopathy, renal or hepatic disease),
medications and BMI were recorded. During the surgery, no changes to standard care were
47
made. Intraoperative mean arterial blood pressure (MAP), body temperature, blood loss,
intravenous fluid intake, urine output, and vasopressor use were recorded. Anesthesia was
standardized with the use of desflurane and remifentanil infusion and performed by a single
anesthesiologist (Dr. Marco Garavaglia). All patients received 0.9% NaCl intravenously.
Blood samples were obtained for lactate, arterial blood gases, hemoglobin concentration,
creatine kinase (CK) and urine myoglobin collected at baseline, and at 3- 4 hour intervals
during the surgery and until 48h postoperatively. ABGs and serum lactate was assessed
every 3 hours during surgery, while CK and myoglobin was assessed every 4 hours during
surgery from baseline. Patient positioning, operative time, tumour type, and world health
organization tumour grade were recorded.
3.15 Statistical Analysis
Data (mean +/- SD) were assessed by one way repeated measures ANOVA and
linear regression. A p value <0.05 was taken to be significant.
3.2 Rat Experimental Methods 3.21 Animals
All animal procedures were approved by the Animal Care Committee at St.
Michael’s Hospital Li Ka Shing Knowledge Institute (Toronto, Ontario, Canada) and
followed the standards of the Canadian Council on Animal Care. Male sprague dawley rats
(500g) were ordered from Charles River Laboratories (Montreal, Quebec, Canada) and
housed under standard conditions with food and water.
48
3.22 Surgical Procedure
All rats were anesthetized with isoflurane (5.0%, Abbott Laboratories, St. Laurent,
Quebec, Canada) in 50% oxygen for 10 minutes before tracheostomy was performed and the
rat was attached to a ventilator (Kent Scientific). Isoflurane was reduced to 2.0% after
completion of tracheostomy and the rat was ventilated to achieve normcapnia and normoxia.
The tail artery was cannulated to measure mean arterial pressure through a pressure
transducer (Memscap SP884) connected to Power Labs (Power Lab 16/30; AD Instruments,
Colorado Springs, CO, USA) and to assess arterial blood gases and hemoglobin
concentration through cooximetry (Radiometer ALB500 and OSM3; London Scientific,
London, Ontario, Canada) before and after the experiment. Similarly, the rat tail vein was
cannulated for intravenous drug infusion (phenylephrine). Four EKG electrodes were
subcutaneously inserted into the limbs of the rat to record heart rate. Rectal temperature was
recorded by rectal temperature probe (Physitemp) and an associated heating pad kept the
animal warm. Heating lamps were also used when necessary to maintain the temperature of
the rat around 36-37oC. The physiological data was recorded continuously during
experimentation and acquired digitally from Powerlabs.
3.23 Free Flap Reanastomosis Surgery
A midline incision was made between the bilateral rectus abdominus muscles of the
rat after elevation of the skin and subcutaneous tissue. Another incision was made just
lateral to the lateral wall of the left rectus sheath then the muscle origin was cut and the deep
superior epigastric vessel was ligated. The muscle insertion was then partially cut and small
fascia was left to hold the muscle in place. The abdominal viscera were reflected away from
the common iliac artery and the external iliac artery with the deep inferior epigastric artery
49
(DIE) were identified under a microscope. A double arterial clamp was applied to the
external iliac artery proximal to the DIE artery origin then transection of the vessel between
the clamp arms was performed followed by re-anastomosis using 9/0 nylon. The ischemia
time was recorded [average ischemia time was 27.7 ± 6.1 minutes (n = 11).] and the flow
was checked clinically by the microscope after completion of the anastomosis.
3.24 Arterial Blood Gas and Co-Oximetry Analysis
Arterial blood gas and co-oximetry were collected in sterile syringes and assessed
before the 10 minute baseline phase of the experiment immediately after successful
completion of the flap reanastomosis surgery and at the end of the experimentation after the
30 minute recovery phase before euthanizing the animal. Variables such as pH, PaCO2,
PaO2, blood oxygen content, and hemoglobin concentration (Hb) were assessed.
3.25 Ultrasound Doppler and Arterial Blood Flow
Transonic ultrasound doppler flowmetry was used to assess arterial blood flow
velocity (ml/min) in the carotid and femoral arteries in the rat. Transonic probes (Transonic
Systems Inc) were clipped around the isolated conduit arteries and blood velocity was
assessed. A piezoelectric crystal located on the wall of the probes transmitted ultrasound
towards the flowing blood along the carotid/femoral artery. Some of the sound was
reflected by moving red blood cells within the blood resulting in a doppler shifted
ultrasound frequency that travelled back to the crystal. The reflected waves had a lower
frequency because the red blood cells are moving away from the transmitter crystal
characteristic of the doppler effect. The blood flow velocity was determined by the
difference in frequency between the transmitted and reflected sound wave. The diameter of
the vessel clipped by the probe allowed for the assessment of volume (ml). Blood flow is
50
proportional to blood cell velocity (ml/min) when the diameter is constant. Ultrasound
doppler was used in our carotid vs femoral blood flow protocol (Protoco1 1) to assess
conduit artery blood flow.
3.26 Laser Doppler and Microvascular Blood Flow
Laser doppler was used to assess microvascular blood flow determined by red blood
cell velocity in a noninvasive manner. The fiberoptic cable laser doppler flow probe was
carefully positioned over the muscle tissue of interest and held in place with a probe holder.
The laser doppler emits a monochromatic light that enters the muscle and flap tissue. Some
of this emitted light was absorbed and some was reflected. The reflected light was gathered
by the fiberoptic cables to photodetectors and frequency changes in light caused by the
movement of red blood cells were detected. Light being reflected by static structures
retained the same initial frequency, however light reflected by moving red blood cells have a
doppler shifted frequency. This frequency shift was proportional to the velocity of moving
red blood cells. Thus, flow velocity was a measured averaged value of the doppler shift of
light striking many moving blood cells at various angles of incidence over a volume of 1
mm3. This laser doppler output signal represents the flux of red blood cells and is expressed
in perfusion units (PU), which is linearly correlated to microvascular blood flow. Laser
doppler flowmetry was used to assess microvascular blood flow in bilateral rectus
abdominus muscle protocol (Protocol 2) and muscle flap vs contralateral muscle control
protocol (Protocol 4).
3.27 Microsensor G4 Oxyphor and Interstitial PO2 Measurements
Interstitial measurements of muscle tissue PO2 were performed using G4 Oxyphor
and oxygen-dependent quenching of phosphorescence. When the phosphorescent probe
51
was excited by pulse of light [λ = 635 um], it emitted phosphorescence [λmaxima = 813um]
over a course of tens-to-hundreds of microseconds. The lifetime (τ) of the phosphorescence
decay is inversely proportional to the partial pressure of oxygen (PO2) in the environment
according to the Stern-Volmer relationship. [1/ τ = 1/ τ0 + Kq[PO2]], where τ0 is the
phosphorescence lifetime (PLT) when PO2 is 0, Kq is the quenching constant, and PO2 is
the partial pressure of oxygen. (Figure 3A) PLT was measured to calculate interstitial tissue
PO2 with the Stern-Volmer equation. In this oxygen dependent quenching of
phosphorescence method, a high PLT corresponds to low muscle/flap tissue PO2, whereas a
low PLT corresponds to high muscle/flap tissue PO2. G4 oxyphor was used in direct tissue
PO2 measurements as a part of an insertable microsensor in muscle and flap tissues. The
signals of the probes have been calibrated under physiological pH and temperature and
shown to provide quantitative, selective and absolute measurements of PO2 in vivo. This
novel methodology was used in the bilateral rectus abdominus muscle tissue PO2 (Protocol
3) and muscle flap vs contralateral muscle control PO2 protocols (Protocol 5).
3.28 Calibration of the Effect of Temperature on the Oxygen Quenching Constant
The oxygen quenching constant is dependent on the temperature, and thus it was
necessary to calibrate the Kq value in respect of the recorded muscle and flap temperature.
The oxygen quenching constant (Kq) and the temperature is linearly correlated by the
equation: Kq = 6.25 (Temperature) + 28.75. At 37oC, the Kq is reported to be 260mmHgg-
1s-1 by Wilson and colleagues. However, our muscle experiments recorded cooler
temperatures in the skeletal muscle (33oC) and muscle flaps (30oC). The Kq for the
respective temperatures were calculated from the linear relationship (Figure 3B). At 33oC,
52
the Kq is 235 mmHg g-1 s-1 and at 30oC the Kq is 216 mmHg g-1 s-1. These Kq values were
used to calculate the tissue PO2 with the Stern Volmer relationship. (Figure 3A) 3.29 Invivo Calibration of T0
T0 is the phosphorescence lifetime recorded when there is no oxygen present. To
determine the T0 value which is used as part of the stern volmer equation to calculate tissue
PO2, we recorded the muscle tissue phosphorescence lifetimes in dead rats after
experimentation (n=4). The averaged T0 value was around 48 usec. We therefore used this
value for T0 in the calculation of experimental PO2 values with the Stern-Volmer
relationship. (Figure 4 A,B)
53
A THE STERN VOLMER EQUATION
1/τ = 1/τ0 + Kq [PO2]
B
Temperature (oC)
20 22 24 26 28 30 32 34 36 38 40
K q (m
mHg‐1 s‐1)
100
120
140
160
180
200
220
240
260
280
300Kq = 6.25 (Temperature) + 28.75
At 37 degrees Kq = 260 At 33 degrees Kq = 235At 30 degrees Kq = 216
Figure 3: Stern Volmer Relationship and Calibration of Temperature Effect on
Quenching Constant in G4 Microsensor Oxyphor (A) The Stern-Volmer Relationship Equation for calculating tissue PO2, where τ is the measured phosphorescence lifetime, τ0 is the phosphorescence lifetime in the absence of oxygen (PO2~zero), Kq is the quenching constant, and PO2 is the partial pressure of oxygen. (B) Calibration for the effect of temperature on the quenching constant (Kq) in G4 microsensor oxyphor. Using this in vitro calibration curve, a specific Kq value is chosen based on the tissue temperature during each experiment to correct for any temperature dependent effect on the relationship between tissue PO2 and G4 oxyphor phosphorescence lifetime.
54
Time (minutes)
0 20 40 60 80
Phosph
orescence Lifetime (usec)
44
46
48
50
Experiment 1Experiment 2Experiment 3Experiment 4
Time (minutes)
0 20 40 60 80 Pho
spho
rescen
ce Life
time (usec)
25
30
35
40
45
50n = 4
Maximum Phosphorescence Lifetime = 47. 60
A
B
Figure 4: Calibration of To in Euthanized Rats (n = 4). (A) Individual measurements of phosphorescence lifetime in acutely euthanized rats under anesthesia in vivo when PO2 is expected to be zero (τ0) (n = 4). (B) The mean value for τ0 in vivo is near 48. A value of 48 µseconds was used for τ0 in all experiments.
55
3.3 Initial Developmental Protocol
In the initial development of the experimental model (Protocol 2 only), three
different doses of phenylephrine were utilized [PE4 = 10ug/kg/min, PE5 = 20 ug/kg/min,
PE6 = 30 ug/kg/min] (Figure 5A). In a refined experimental model, two additional lower
doses were included to determine the threshold for blood pressure response in the rat model
and the highest doses were reduced [PE1 = 1.5 ug/kg/min, PE2 = 3.0ug/kg/min, PE3 =
6.0ug/kg/min, PE4 = 12.0ug/kg/min, PE5 = 18.0 ug/kg/min] (Figure 5B). This dose range
approximates clinically relevant concentration of phenylephrine and allows for the
reproduction of increased blood pressure in a dose dependent manner. In both cases,
phenylephrine caused a dose dependent increase in blood pressure (one way repeated
measures ANOVA, p < 0.001, * post hoc Tukeys test corrected p value p < 0.05) All
experimental protocols except for the developmental model (Protocol 2) follow the 5 dose
scheme of PE infusion (PE1-PE5). The initial developmental model showed that the
bilateral rectus abdominus muscles respond in a similar manner to PE validating it’s use for
a muscle flap vs contralateral muscle control model in subsequent protocols. In our
developed protocols, a consistent elevation in MAP (Figure 6) and a relatively stable heart
rate response to PE infusion (Figure 7) were observed throughout the experiments. This
validates the consistency of the finalized experimental model.
56
Baseline PE4 PE5 PE6 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
120
140
160
180
**PE4: 10ug/kg/min
PE5: 20ug/kg/minPE6: 30ug/kg/min
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
120
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**PE1: 1.5ug/kg/min
PE2: 3.0ug/kg/minPE3: 6.0ug/kg/minPE4: 12.0ug/kg/minPE5 18.0ug/kg/min
A B
*
Figure 5: Measurement of Mean Arterial Blood Pressure after Two Different Infusion Protocols of Phenylephrine. (A) In the initial development of the experimental model, three different doses of Phenylephrine were utilized. (B) In a refined experimental model, two additional lower doses were included to determine the threshold for blood pressure response. This dose range approximates clinically relevant concentration of phenylephrine in that it causes a dose dependent increase in mean arterial pressure (MAP). In both cases, higher levels of phenylephrine infusion (10 to 30 µg/kg/min) were required to increased MAP in a reproducable manner. (1 way repeated measures ANOVA, p < 0.001; * corrected p < 0.05 post hoc Tukeys test).
57
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
120
140
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180
* *PE1: 1.5ug/kg/minPE2: 3.0ug/kg/minPE3: 6.0ug/kg/minPE4: 12.0ug/kg/minPE5 18.0ug/kg/min
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
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* *
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
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ure
(mm
Hg)
0
20
40
60
80
100
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**
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
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(mm
Hg)
0
20
40
60
80
100
120
140
160
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**
BA
C D
Figure 6: A Consistent Mean Arterial Blood Pressure Response to Phenylephrine was Observed in Four Experimental Different Protocols: (A) Carotid vs Femoral blood flow protocol (n = 7), (B) Flap vs Muscle Laser Doppler Protocol (n =6), (C) Bilateral muscle PO2 protocol (n = 6), and (D) Flap vs Muscle PO2 protocol (n = 9). These data demonstrate that the phenylephrine infusion protocol caused a consistent elevation in blood pressure in response to the dose of phenylephrine administered. In all cases the MAP returned towards baseline 30 minutes after discontinuing the PE infusion. (ANOVA p<0.001 for all) [ANOVA = 1 way repeated measures analysis of variance, and *: p adjusted p <0.05, post hoc Tukey test,]
58
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Hea
rt R
ate
(bpm
)
0
100
200
300
400
*
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Hea
rt R
ate
(bpm
)
0
100
200
300
400
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Hea
rt R
ate
(bpm
)
0
100
200
300
400
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Hea
rt R
ate
(bpm
)
0
100
200
300
400
A B
C D
Figure 7: Heart Rate Response to Phenylephrine in Four Different Experimental Protocols: (A) Carotid vs femoral blood flow protocol (n = 7), (B) Flap vs muscle laser doppler protocol (n = 6), (C) Bilateral muscle PO2 protocol ( n = 6), and (D) Flap vs muscle PO2 protocol (n = 8). Heart rate was generally stable throughout the experimental protocols. There was a slight increase in heart rate at the highest dosage of PE during drug infusion at PE5 observed in the femoral and carotid protocol (ANOVA, p<0.001). [A: ANOVA = 1 way repeated measures analysis of variance, and *: adjusted p <0.05, post hoc Tukey test]
59
3.4 Experimental Protocols 3.41 Protocol 1: Femoral vs Carotid Blood Flow Ultrasound Doppler Flowmetry
Sprague Dawley rats (600-700g, n=7, Charles River) were used in this protocol and
followed the initial surgical procedure described in section 3.22. The femoral and carotid
arteries were then isolated and exposed in the rat. Transonic ultrasound flowmetry recorded
the flow in the femoral and carotid artery through transonic flowprobes (Transonic Systems
Inc (0.7PSB298) and (1PAB3696) respectively, NY, USA) after surgical isolation of the
respective vessels. (3.25) Phenylephrine (PE) (10mg/ml) [SANDOZ] was diluted to 50ug/ml
and infused by a Harvard Apparatus PhD2000 infusion/withdrawal pump. A stable baseline
was recorded for 10 minutes before continuous PE infusion began at increasing doses in 5
minute intervals: PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0 ug/kg/min, PE4 =
12.0 ug/kg/min, PE5 = 18.0 ug/kg/min. A recovery period of 30 minutes was allowed
before the final blood gas and euthanasia of the animal by T61 heart injection under
isoflurane anesthesia.
3.42 Protocol 2: Bilateral Rectus Abdominus Muscle Laser Doppler Microvascular Blood Flow (Initial Developmental Model)
Sprague Dawley rats (500-600g, n=10, Charles River) were used in this protocol and
followed the initial surgical procedures described in section 3.22. The skin covering the
bilateral rectus abdominus muscles was surgically removed to expose the bilateral rectus
abdominus muscles. Two Oxyflo laser doppler probes (SNPR90269, Oxford Optronix Ltd,
Oxford, UK) were positioned over each of the two bilateral rectus abdominus muscles to
record microvascular blood flow characterized by red blood cell velocity. Phenylephrine
(PE) (10mg/ml) [SANDOZ] was diluted to 50ug/ml and was intravenously infused into the
60
rat by a Harvard Apparatus PhD2000 infusion/withdrawal pump. A stable baseline was
recorded for 10 minutes before continuous PE infusion began at an infusion rate of 0.10,
0.20, and 0.30 ml/min at 10 minute intervals: PE1 = 10ug/kg/min, PE2 = 20 ug/kg/min, PE3
= 30 ug/kg/min. A recovery period of 30 minutes was recorded before a final blood gas
was sampled and the animal was euthanized by the injection of T61 into the heart under
isoflurane anesthesia.
3.43 Protocol 3: Bilateral Rectus Abdominus Muscle G4 Oxyphor PO2
Sprague Dawley rats (600-700g, n=6, Charles River) were used in this protocol and
followed the initial surgical procedure described in 3.22. The skin and subcutaneous tissue
overlaying the rectus abdominus muscles were surgically removed to expose the bilateral
muscles. In quantitative oxygen assessment experiments, PMOD1000 microsensor oximeter
probes were inserted into the bilateral rectus abdominus muscle. The PMOD1000 G4
oximeter recorded quantitative oxygen levels within the muscle tissues throughout
experimentation (section 3.27). Phenylephrine (PE) (10mg/ml) [SANDOZ] was diluted to
50ug/ml and infused by a Harvard Apparatus PhD2000 infusion/withdrawal pump. A stable
baseline was recorded for 10 minutes before continuous PE infusion at the following doses
at 5 minute intervals: PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4
= 12.0ug/kg/min, PE5 = 18.0ug/kg/min. A recovery period of 30 minutes was allowed
before the final blood gas and euthanasia of the animal by T61 heart injection under
isoflurane anesthesia.
3.44 Protocol 4: Rectus Abdominus Muscle Flap vs Contralateral Control Laser Doppler Microvascular Blood Flow
Sprague Dawley rats (800-900g, n=6, Charles River) were used in this protocol and
followed the initial surgical procedure described in section 3.22. Free flap surgery was
61
performed by a plastic surgeon (Dr. Sami Alissa) described in detail in section 3.23. Laser
doppler flowmetry (section 3.26) was used to record microvascular blood flow in the flap
and the contralateral muscle. Phenylephrine (PE) (10mg/ml) [SANDOZ] was diluted to
50ug/ml and infused by a Harvard Apparatus PhD2000 infusion/withdrawal pump. A stable
baseline was recorded for 10 minutes before continuous PE infusion at the following doses
at 5 minute intervals: PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4
= 12.0ug/kg/min, PE5 = 18.0ug/kg/min. A recovery period of 30 minutes was allowed
before the final blood gas and euthanasia of the animal by T61 heart injection under
isoflurane anesthesia.
3.45 Protocol 5: Rectus Abdominus Muscle Flap vs Contralateral Control G4 Oxyphor PO2
Sprague Dawley rats (800-900g, n=9, Charles River) were used in this protocol and
followed the initial surgical procedure described in section 3.22. Free flap surgery was
performed by a plastic surgeon (Dr. Sami Alissa) described in detail in section 3.23. In
quantitative oxygen assessment experiments, PMOD1000 probes were inserted into rectus
abdominus muscle and muscle flap. The PMOD1000 oximeter recorded quantitative oxygen
levels within the muscle and flap tissue throughout experimentation (section 3.27).
Phenylephrine (PE) (10mg/ml) [SANDOZ] was diluted to 50ug/ml and infused by a
Harvard Apparatus PhD2000 infusion/withdrawal pump. A stable baseline was recorded for
10 minutes before continuous PE infusion at the following doses at 5 minute intervals: PE1
= 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.00ug/kg/min, PE4 = 12.0ug/kg/min, PE5 =
18.0ug/kg/min. A recovery period of 30 minutes was allowed before the final blood gas
and euthanasia of the animal by T61 heart injection under isoflurane anesthesia.
62
3.46 Protocol 6: Rectus Abdominus Muscle and Flap Temperature Analysis
Sprague Dawley rats (700-800g, Charles River) were used in this protocol and
followed the initial surgical procedure described in section 3.22. The skin and subcutaneous
tissues overlaying the rectus abdominus muscles was removed to expose the bilateral
muscles. In the bilateral muscle temperature experiments (n = 6), two temperature probes
were inserted into the left and right rectus abdominus muscles to assess bilateral muscle
temperature. In the muscle versus muscle flap temperature experiments (n = 5), one
temperature probe was inserted into the muscle flap, and the other temperature probe was
inserted into the contralateral muscle to record muscle and flap temperature from the start of
baseline till the end of the experiment. Rectal temperature was recorded via rectal
temperature probe (Physitemp). After successful completion of the free flap reanastomsis
surgery described in section 3.23, an arterial blood gas was taken and assessed (Radiometer
ALB500 and OSM3; London Scientific, London, Ontario, Canada). A stable baseline of 10
minutes was recorded (MAP and Temperature only), followed by the drug infusion phase in
which phenylephrine was continuously infused at increasing elevated doses in 5 minute
intervals (PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4 =
12.0ug/kg/min, PE5 = 18.0ug/kg/min.) for 25 minutes followed by a 30 minute recovery
period. At the end of the recovery period a final arterial blood gas was assessed and the rat
was euthanized with T61 injection into the heart under isoflurane anesthesia.
3.47 Summarized Experimental Timeline
All protocols followed the same identical timeline and drug infusion protocols with
the exception of the initial developmental model (Protocol 2). After surgery was performed
(1-2 hours), and arterial blood gas was taken before the start of the baseline. Physiological
63
data including heart rate, mean arterial pressure, rectal temperature, and muscle blood flow
and PO2 were recorded continuously throughout the experiment which includes the start of
the 10 minute baseline, Phenylephrine infusion at 5 increasing doses at 5 minute intervals
(PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4 = 12.0ug/kg/min, PE5
= 18.0ug/kg/min.), and the 30 minute recovery phase. An arterial blood gas was assessed at
the end of the experiment and muscle and flap tissue samples were collected for future
analysis of HIF-1a with western blot. (Figure 8)
Figure 8 Experimental Timeline of Phenylephrine Infusion Experiments. After surgery was performed (1-2 hours), and arterial blood gas was taken before the start of the baseline. Physiological data including heart rate, mean arterial pressure, rectal temperature, and muscle blood flow and PO2 were recorded continuously throughout the experiment which includes the start of the 10 minute baseline, phenylephrine infusion at 5 increasing doses at 5 minute intervals, and the 30 minute recovery phase. An arterial blood gas was assessed at the end of the experiment and muscle and flap tissue samples were collected.
64
3.5 Statistical Analysis
Sample size calculations are performed for each experimental protocol assuming a
power of 0.8 and an α of 0.05. Data was analyzed using Sigma Plot version 11.0 (Systat
Software Inc, San Jose, CA, USA). Baseline and post PE values in blood gas and
electrolyte tables were assessed by the T test. All physiological data were assessed to be
normally distributed utilizing tests of homogeneity of variance as assessed by Shapiro-Wilk
and Levene tests. Physiological data (mean arterial pressure, heart rate, and rectal
temperature) were assessed parametrically with one way repeated measures analysis of
variance (ANOVA). Bilateral blood flow and temperature data were assessed
parametrically by a two way repeated measures ANOVA performed to assess treatment,
group, and interaction effects. Tukey tests were used to compare the means when an
adequate F ratio was achieved. All data are presented as mean ± SD and significance was
assigned at p<0.05. P values are reported for ANOVA with post hoc values presented as
adjusted p values.
65
CHAPTER 4 RESULTS
4.0 Clinical Study: Assessing Skeletal Muscle Perfusion during Craniotomy for Resection of Brain Tumours 4.01 Patient Blood Pressure and Body Temperature during Surgery.
Eighteen consecutive patients were consented for surgery (age range 19-74 years
old, and BMI range 18.5-34.4 kg/m2). The average duration of surgery was 8.45 ± 2.84
hours. Craniotomy for brain tumor resection was performed on all patients (n = 18) without
intraoperative complication. Average patient systolic and diastolic blood pressure was
stable throughout the operation, systolic and diastolic pressure was maintained around
120 mmHg and 70 mmHg, respectively throughout the surgical procedure (Figure 9A).
Average patient body temperature ranged from 35 – 36 degrees and gradually increased
overtime (Figure 9B). Arterial blood pressure was relatively stable over time while
temperature increased with time (P<0.001). Blood gas and co-oximetry data are presented in
Table 2 (mean ± SD). Estimated blood loss was 610 ± 504 ml and total urine output was
2590 ± 705 ml intraoperatively. None of the patients demonstrated intraoperative
hypotension.
4.02 Elevated Serum Lactate during Surgery
Serum lactate was observed to increase in all eighteen patients. (Figure 10A) Serum
lactate increased within the first 3 hours of the start of the surgery (2.21 ± 1.22 mmol/L),
and peaked near 9 hours into the surgery (3.73±1.60 mmol/L) (for both, p<0.05 relative to
baseline lactate (1.01±0.47mmol/L)) (Figure 11A). The elevated serum lactate declined after
the surgery while the patients were in the intensive care unit.
66
4.03 Elevated Creatine Kinase and Myoglobinuria in Some Patients
Creatine kinase was also elevated in some patients following the early rise in serum
lactate. There was an increase in CK by 12 hours with a mean value of 739±1251 U/L
(Figure 11B, p = 0.009). In eight patients, the CK values rose to greater than 1000U/L and
six patients had myoglobinuria. (Figure 10B, * = myoglobinuria positive).
4.04 Hemoglobin Levels were Stable during OR and ICU
Hemoglobin levels were stable above 100g/L within the first 30 hours, during the surgery
and in the ICU. After 30 hours, hemoglobin was observed to decline to around 84g/L.
(Figure 11C)
4.05 Body Mass Index Correlated with the Early Rise in Serum Lactate
The patients had an average BMI of 26.5 ± 3.75 (Table 1). The initial increase in
lactate (Δ Lactate3hr) correlated with BMI (p=0.010, r=0.587 r2= 0.334) but not with other
parameters including PaCO2, hemoglobin and length of surgery (Figure 12). Assessment of
peak change in lactate did not correlate with any parameter (Figure 12). No relationships
were observed between increased lactate and tumor type or grade (Table 1). In addition,
urine output corresponded to mannitol dose (p <0.05), but no correlation was found between
Δ Lactate3hr and mannitol administered at the standard dose of 0.5 mg/kg.
4.06 Arterial Blood Gas and Cooximetry
ABG and cooximetry data is presented in Table 2. The pH, PaCO2, PaO2, HCO3-,
base excess, and hemogloblin were within normal limits during the surgery in the OR and in
the ICU. The pH decreased significantly at ICU admission (p<0.001, one way ANOVA)
relative to baseline. PaCO2 decreased significantly at OR 3hours (p=0.001, one way
ANOVA) and OR 6 hours (p = 0.010, one way ANOVA) relative to baseline. PaO2
67
significantly decreased at OR 3hours (p = 0.027, one way ANOVA), ICU admission, ICU 8
hours, ICU 16 hours and ICU 24 hours (p<0.001 for all, one way ANOVA) relative to
baseline. Bicarbonate decreased significantly at OR 3hours, OR 6 hours, ICU admission
(p<0.001, for all, one way ANOVA) and ICU 8hrs (p = 0.018, one way ANOVA) relative to
baseline. Base excess decreased significantly at OR 3hours and OR 6 hours (p<0.001, for
both, one way ANOVA) and at ICU admission and ICU 8 hours (p = 0.002, for both, one
way ANOVA) relative to baseline. Hemoglobin declined significantly at ICU 8 hours (p =
0.006, one way ANOVA) and ICU 16 hours and ICU 24 hours (p<0.001, for both, one way
ANOVA) relative to baseline.
68
0 1 2 3 4 5 6 7 8 9 10
Bloo
d Pressure (m
mHg)
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Systolic Blood Pressure Diastolic Blood Pressure
Time (Hours)
0 1 2 3 4 5 6 7 8 9 10
Tempe
rature (oC)
26
28
30
32
34
36
38
40
* * * *
A
B
Figure 9: Patient Blood Pressure and Temperature during surgery. (A) Average systolic and diastolic blood pressures in patients undergoing craniotomy for brain tumor. No significant drop in blood pressure was observed (n = 18). (B) Average pharyngeal temperature in patients during neurosurgery. There was a slight increase in temperature, relative to baseline, after 6 hours of surgery (one way repeated measures ANOVA, *: P<0.05 (n = 18).
69
Time (Hours)0 10 20 30 40 50 60 70
Creatine
Kinase (U/L)
0
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**
** *
*
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*
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0 10 20 30 40 50 60 70
Serum Lactate (m
mol/L)
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8Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient 10Patient 11Patient 12Patient 13Patient 14Patient 15Patient 16Patient 17Patient 18
A
B
Figure 10: Elevated Serum Lactate and Creatine Kinase in Neurosurgical Patients. (A) Increased serum lactate in patients undergoing craniotomy for brain tumour resection (n = 18). An early increase in lactate occurred in all patients (3 hrs) with variability to the peak value near 9 hours. (B) Elevated serum creatine kinase occurred later than lactate and increases in patients with a higher lactate response (n=6). In some patients with a high CK, myoglobinuria was detected (*= myoglobinuria positive)
70
0 10 20 30 40
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oglobin (g/L)
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*
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*A
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Figure 11. Average Serum Lactate, CK, and Hemoglobin during Surgery and in ICU. Average values for serum lactate (A), creatine kinase (B), and hemogloblin (C) in patients undergoing craniotomy for brain tumour resection (n = 18) (*: P<0.05 vs. baseline; ANOVA).
71
Body Mass Index (BMI)15 20 25 30 35 40
ΔLac
tate
3hr (
mm
ol/L
)
0
1
2
3
4
5
6
Body Mass Index (BMI)15 20 25 30 35 40
Δ La
ctat
e Pea
k (m
mol
/L)
012345678
Length of Surgery (Hours)0 2 4 6 8 10 12 14 16 18 20
ΔLa
ctat
e Peak
(mm
ol/L
)
012345678
ΔHemogloblin3hr (g/L)
-60 -50 -40 -30 -20 -10 0 10 20
Δ Lac
tate
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mm
ol/L
)
0
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6ΔPCO23hr (mmHg)
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ΔLa
ctat
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r (m
mol
/L)
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ΔCK 4hr (U/L)
-20 0 20 40 60 80 100
ΔLac
tate
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(mm
ol/L
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0
1
2
3
4
5
6
N = 18R = 0.587Rsqr = 0.344P = 0.010
N = 18R = 0.283Rsqr = 0.080P = 0.256
N = 18R = 0.118Rsqr = 0.0139P = 0.641
N = 18R = 0.0499Rsqr = 0.00249P = 0.844
N = 18R = 0.137Rsqr = 0.0187P = 0.588
N = 18R = 0.278Rsqr = 0.0775P = 0.263
A B
C D
E F
Figure 12: Positive Correlation between Serum Lactate and Body Mass Index (A) Positive correlation between body mass index (BMI) and change in lactate was observed after 3 hours (∆Lactate3hr) (P=0.010). (B to F) No correlation between BMI and peak lactate (∆LactatePeak) or between ∆Lactate3hr and change in arterial carbon dioxide (∆pCO2), length of surgery and ∆LactatePeak , ∆Hemoglobin3hr and ∆CK4hr were observed.
72
Table 1: Demographic data, characterization of tumour pathology and World Health Organization (WHO) grade relative to lactate and body mass index (BMI)
Table 2: Arterial Blood Gas and Co-oximetry data for craniotomy patients in the operating room (OR) and intensive care unit (ICU). * = statistical significance (p<0.05, one way ANOVA)
Time pH PaCO2 PaO2 HCO3‐
Base Excess
Hemoglobin (Hb), g/L
Baseline 7.39 ± 0.03 39.6 ± 4.5 271.2 ± 86.0 24.4 ± 1.7 ‐1.1 ± 1.5 125 ± 18 OR, 3 hr 7.39 ± 0.03 34.5 ± 4.1* 217.8 ± 64.1* 21.2 ± 1.8* ‐3.7 ± 1.7* 117 ± 13
OR, 6 hr 7.37 ± 0.04 33.6 ± 2.5* 233.1 ± 40.9 20.2 ± 1.9* ‐5.0 ± 2.3* 115 ± 28 ICU, Adm. 7.31 ± 0.05* 40.0 ± 5.8 190.1 ± 42.0* 20.5 ± 2.3* ‐6.1 ± 2.4* 116 ± 15 ICU, 8hr 7.36 ± 0.04 37.8 ± 5.3 130.0 ± 39.5* 22.3 ± 2.0* ‐3.6 ± 2.0* 104 ± 13* ICU, 16hr 7.38 ± 0.02 39.0 ± 4.7 106.4 ± 27.5* 24.1 ± 2.0 ‐1.7 ± 1.6 96 ± 9* ICU, 24hr 7.41 ± 0.03 36.2 ± 1.8 88.6 ± 5.0* 24.2 ± 1.3 ‐1.5 ± 1.6 84 ± 17*
73
Animal Study 4.1 Protocol 1: Assessing Femoral vs Carotid Blood Flow with Ultrasound Doppler Flowmetry 4.11 The Effect of Phenylephrine on Mean Arterial Pressure
Mean arterial pressure (MAP) [n = 7 rats] was recorded at an average baseline value
of 84 ± 7 mmHg and increased with continuous phenylephrine infusion (PE1 =
1.5ug/kg/min, PE2 = 3.0 ug/kg/min, and PE3 = 6.0 ug/kg/min, PE4 = 12.0 ug/kg/min, and
PE5 = 18.0ug/kg/min) at 5 minute intervals. Averaged MAP was 85 ± 8 mmHg after initial
infusion of phenylephrine at PE1 (1.5ug/kg/min). Continued infusion of drug at PE2
(3.0ug/kg/min) resulted in an averaged MAP of 87 ± 8 mmHg. The first two lower doses of
PE did not alter MAP. At the higher doses of PE3 (6.0ug/kg/min), PE4 (12.0ug/kg/min), and
PE5 (18.0ug/kg/min), MAP increased to 101± 8 mmHg, 141 ± 17mmHg, and 152 ± 20
mmHg, respectively. Following a 30 minute recovery period, MAP declined to 100 ± 14
mmHg, but remained above initial baseline value. The rise in MAP was statistically
significant at PE4 and PE5 (both, p<0.001, one way repeated measures ANOVA, Tukey
test). Therefore, the infusion of phenylephrine increased MAP in a dose dependent manner.
(Figure 13A)
4.12 The Effect of Phenylephrine on Heart Rate
Heart rate (HR) [n = 7 rats] was recorded to be at an average baseline value of
293±23 bpm and remained relatively stable throughout most of the treatment with
phenylephrine. Upon continuous infusion of phenylephrine at PE1 (1.5 ug/kg/min), PE2
(3.0 ug/kg/min), and PE3 (6.0 ug/kg/min), and PE4 (12.0 ug/kg/min) for 5 minute intervals,
averaged heart rate was 295 ± 18 bpm, 295 ± 16 bpm, 291 ± 14 bpm, 304 ± 19 bpm
74
respectively. At PE5 (18.0ug/kg/min), heart rate was observed to increase slightly (335 ± 27
bpm) [p<0.001, one way repeated measures ANOVA, Tukey test]. Following a 30 minute
recovery period, averaged heart rate was 309 ± 8 bpm. Therefore heart rate was relatively
stable throughout the experimentation, but increased at PE 5 in this protocol only. (Figure
7A)
4.13 The Effect of Phenylephrine on Carotid Blood Flow
Carotid blood flow [n = 7 rats] was measured using ultrasound doppler flowmetry
and was 7.41 ± 3.96 ml/min at baseline. Upon phenylephrine infusion (PE1 = 1.5ug/kg/min,
PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4 = 12.0ug/kg/min, and PE5 = 18.0
ug/kg/min), averaged carotid blood flow was recorded to be 8.26 ± 3.95 ml/min, 8.51 ± 3.88
ml/min, 8.04 ± 3.84 ml/min, 8.07 ± 2.96 ml/min, and 8.97 ± 3.3 ml/min respectively.
Following a 30 minute recovery period, averaged carotid blood flow was 7.84 ± 4.14
ml/min. The carotid blood flow at PE 5 (8.97 ± 3.3 ml/min) was not statistically different
from baseline (7.41 ± 3.96 ml/min) (p<0.344, one way repeated measures ANOVA, Tukeys
test) (Figure 13C). Therefore, phenylephrine had no significant effect on carotid blood
flow.
4.14 The Effect of Phenylephrine on Femoral Blood Flow
Femoral artery blood flow [n = 7 rats] was measured using ultrasound doppler
flowmetry and the observed baseline value was 1.65 ± 0.77 ml/min. Upon infusion of
phenylephrine (PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0ug/kg/min, PE4 =
12.0ug/kg/min, and PE5 = 18.0 ug/kg/min), averaged femoral blood flow was recorded to be
1.69 ± 0.86 ml/min, 1.76 ± 0.89 ml/min, 1.88 ± 0.87 ml/min, 2.69 ± 1.32 ml/min, and 2.98 ±
1.46 ml/min respectively. An increase in femoral blood flow was statistically significant at
75
PE4 and PE5. (p<0.001, one way repeated measures ANOVA, Tukey test) At recovery
phase, femoral blood flow was 2.05 ± 1.09 ml/min. Therefore, phenylephrine infusion
increased femoral blood flow at the highest dose (PE5) by approximately 1.8 times. (Figure
13D)
4.15 Carotid Blood Flow versus Femoral Blood Flow
At baseline, the carotid blood flow was 7.41 ± 3.96 ml/min whereas the femoral
blood flow was 1.65 ± 0.77 ml/min. There was a clear difference between the magnitude of
the carotid and femoral blood flow.
4.16 Stable Rectal Temperature throughout Experimentation
Rectal temperature [n = 7 rats] was observed to be stable throughout experimentation
ranging from 36.2 – 36.7oC. The averaged baseline temperature was 36.3 ± 0.9oC. Upon
phenylephrine infusion (PE1 = 1.5ug/kg/min, PE2 = 3.0ug/kg/min, PE3 = 6.0ug/kg/min,
PE4 = 12.0ug/kg/min, and PE5 = 18.0 ug/kg/min) at 5 minute intervals, the temperature was
36.6 ± 0.8oC, 36.6 ± 0.8oC, 36.5 ± 0.6oC, 36.8 ± 0.6oC, and 36.6 ± 0.7oC respectively.
Following a 30 minute recovery phase, the temperature was 36.7 ± 0.7oC. There was no
statistical difference observed and temperature was stable throughout at relatively
normothermic temperatures. (Figure 13B)
4.17 Arterial Blood Gas and Cooximetry
The ABG and cooximetry analysis at baseline and post PE at the end of the
experiment is presented in Table 3 under the carotid vs femoral blood flow protocol. The
pH, PCO2, PO2, hemoglobin, and SaO2 were assessed to be within normal ranges at the start
and end of the experimentation. No significant changes were observed.
76
4.18 Electrolyte and Metabolic Data
The electrolyte and metabolic data analysis at baseline and post PE at the end of the
experiment is presented in Table 4 under the carotid vs femoral blood flow protocol. The
K+, Na+, Ca+2, Cl-, glucose, lactate, base, and HCO3- were assessed to be within normal
ranges at the start and end of the experimentation. No significant changes were observed.
4.19 Protocol 1 Summary
Mean arterial pressure increased with PE infusion in a dose dependent manner
(p < 0.001, one way repeated measures ANOVA). Rectal temperature was stable
throughout the experiment (p = 0.190, one way repeated measures ANOVA). There was no
significant change in carotid blood flow (p = 0.344, one way repeated measures ANOVA)
and femoral blood flow was statistically significantly increased at PE 4 and PE5 (p <0.001,
one way repeated measures ANOVA).
77
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Rec
tal T
empe
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re (o
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**PE1: 1.5ug/kg/min
PE2: 3.0ug/kg/minPE3: 6.0ug/kg/minPE4: 12.0ug/kg/minPE5 18.0ug/kg/min
A B
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Baseline PE1 PE2 PE3 PE4 PE5 Recovery
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5
**
Figure 13: The Effect of Phenylephrine on Carotid and Femoral Blood Flow ( n = 7). (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p < 0.001). (B) Rectal temperature was stable throughout the experiment (ANOVA, p = 0.190). (C) There was no significant change in carotid blood flow. (ANOVA, p = 0.344) (D) Femoral blood flow was statistically significant at PE 4 and PE5. (ANOVA, p <0.001) [ANOVA = one way repeated measures analysis of variance *: adjusted p <0.05, post hoc Tukey test,]
78
4.2 Protocol 2: Assessing Bilateral Rectus Abdominus Muscle Laser Doppler Microvascular Blood Flow (Initial Developmental Model) 4.21 The Effect of Phenylephrine on Mean Arterial Pressure
Mean arterial pressure (MAP) [n = 10 rats] was recorded to be at an average baseline
value of 73 ± 11 mmHg and increased with continuous phenylephrine infusion (PE4 =
10ug/kg/min, PE5 = 20 ug/kg/min, and PE6 = 30 ug/kg/min) at 10 minute intervals.
Averaged MAP elevated to 94 ± 20 mmHg at the first dose of 10 ug/kg/min phenylephrine
infusion (PE3). At PE4 (20 ug/kg/min) the averaged MAP rose to 125 ± 23mmHg and was
statistically significant compared to baseline (p<0.001, one way repeated measures
ANOVA, Tukey test). At the highest dose PE6 (30 ug/kg/min) MAP was statistically
significant and peaked at 140 ± 25 mmHg (p<0.001, one way repeated measures ANOVA,
Tukey test). Following a 30 minute recovery period, the MAP decreased down to 99 ± 24
mmHg, but was still statistically significant and above the baseline value (p = 0.013, one
way repeated measures ANOVA, Tukey Test). Therefore continuous infusion of
phenylephrine significantly elevated MAP in a dose dependent manner. (Figure 14A)
4.22 The Effect of Phenylephrine on Heart Rate
Heart rate (HR) [n = 10 rats] was recorded to be at an average baseline value of 277
± 37 bpm and remained relatively stable throughout the treatment with phenylephrine.
Upon continuous infusion of phenylephrine at PE4 (10ug/kg/min), PE5 (20ug/kg/min), and
PE6 (30ug/kg/min) for 10 minute intervals, averaged heart rate was 268 ± 40 bpm, 272 ± 38
bpm, 274 ± 46 bpm respectively. Following a 30 minute recovery period, averaged heart
rate was 275 ± 46 bpm. Therefore heart rate was relatively stable throughout the
experimentation and there is not a statistically significant difference.
79
4.23 The Effect of Phenylephrine on Bilateral Rectus Abdominus Microvascular Muscle Blood Flow
Bilateral microvascular muscle blood flow response to phenylephrine infusion was
measured by laser doppler flowmetry. (Figure 14C,D) In the left rectus abdominus muscle,
an averaged baseline value of 1391.75 ± 989.384 perfusion units [PU] was recorded. After a
10 minute infusion of phenylephrine at PE3 (10ug/kg/min) the microvascular blood flow
was 1262.71 ± 950.77 PU. Subsequent phenylephrine infusions at PE5 [20ug/kg/min] and
PE6 [30ug/kg/min] further reduced microvascular blood flow to 892.56 ± 396.79 PU and
893.78 ± 528.08 PU respectively. Following a 30 minute recovery period, averaged
microvascular blood flow remained significantly reduced at 898.62 ± 444.47 PU. Analyzing
the data by normalizing the microvascular blood flow data revealed a statistically significant
decline in microvascular blood flow at PE5 (0.77 ± 0.25), PE6 (0.75 ± 0.25), and recovery
phase (0.76+/-0.26) compared with baseline (1.00 ± 0.00).
In the right rectus abdominus muscle, an averaged baseline value of 1050.55 ±
814.69 PU was recorded. The average microvascular blood flow in response to infusion of
phenylephrine: PE4, PE5, and PE6 was 1063.84 ± 834.26 PU, 899.62 ± 903.36 PU, and
782.18 ± 529.07 PU respectively. Following a 30 minute recovery period, average
microvascular blood flow was 806.39 ± 996.26 PU. Analyzing the data by normalizing the
microvascular blood flow data in the right rectus abdominus muscle also revealed a decline
at PE5 (0.84±0.24), PE6 (0.82±0.33), and Recovery (0.72±0.32). There was no difference in
the muscle blood flow response between the right and left side of the muscle. (p = 0.495
(absolute data), p = 0.560 (normalized data); two way repeated measures ANOVA) There
was no interaction effect. There was a significant grouped treatment effect at PE5, PE6, and
recovery phase in both the absolute and normalized data. (p =0.002 (absolute), p <0.001
80
(normalized), two way repeated measures ANOVA) Therefore PE infusion at elevating high
doses significantly reduced microvascular blood flow in the bilateral rectus abdominus
muscles in a dose dependent manner.
4.24 Stable Rectal Temperate during the Experiment
The rectal temperature was stable throughout the experimentation ranging 32.5-
33oC. Throughout the protocol (Baseline, PE4, PE5, PE6, Recovery), the average rectal
temperatures measured were 33.0 ± 2.0oC, 33.0 ± 1.9oC, 32.7 ± 1.9oC, 32.5 ± 1.9oC, and
32.5 ± 2.1oC respectively. Although the temperature was hypothermic, it was relatively
stable throughout the experimentation. (Figure 14B)
4.25 Arterial Blood Gas and Cooximetry
The ABG and cooximetry analysis at baseline and post PE at the end of the
experiment is presented in Table 3 under the bilateral muscle blood flow protocol. No
significant changes in pH, PCO2, PO2, hemoglobin, and SaO2 were observed.
4.26 Electrolyte and Metabolic Data
The electrolyte and metabolic data analysis at baseline and post PE at the end of the
experiment is presented in Table 4 under the bilateral muscle blood flow protocol. No
significant changes in K+, Na+, Ca+2, Cl-, glucose, lactate, base, and HCO3- were observed.
4.26 Protocol 2 Summary
Mean arterial pressure increased with PE infusion in a dose dependent manner
(p<0.001, one way repeated measures ANOVA, Tukeys test). Rectal temperature decreased
slightly by the later stages of the experiment (p = 0.001, one way repeated measures
ANOVA, Tukeys test). Absolute muscle blood flow decreased in a dosage dependent
manner with increased PE infusion and did not recover upon discontinuation of PE infusion
81
(p =0.002, two way repeated measures ANOVA). There was no difference between the left
and right muscle (p = 0.628, two way repeated measures ANOVA). No interaction existed
between the treatment and muscle side (p = 0.495, two way repeated measures ANOVA).
Normalized rectus abdominus muscle blood flow also decreased in a dosage dependent
manner (p<0.001, two way repeated measures ANOVA). There was no difference between
the left and right side of the muscle (p = 0.560, two way repeated measures ANOVA). No
interaction exists between the treatment and muscle side (p = 0.636, two way repeated
measures ANOVA).
82
Baseline PE4 PE5 PE6 Recovery
Mea
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rter
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**
Baseline PE4 PE5 PE6 Recovery
Rec
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empe
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40PE4: 10ug/kg/minPE5: 20ug/kg/minPE6: 30ug/kg/min
Baseline PE4 PE5 PE6 Recovery
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Abd
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Left Rectus Abdominus MuscleRight Rectus Abdominus Muscle
A B
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Baseline PE4 PE5 PE6 Recovery
Mus
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(BPU
)
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500
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Left Rectus Abdominus MuscleRight Rectus Abdominus Muscle
**
** * *
*
Figure 14: The Effects of Phenylephrine on Bilateral Rectus Abdominus Muscle Blood Flow (n = 10). (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p<0.001). (B) Rectal temperature decreased slightly by the later stages of the experiment (ANOVA, p = 0.001). (C) Absolute muscle blood flow decreased in a dose dependent manner with increased PE infusion and did not recover upon discontinuation of PE infusion (ANOVA, p =0.002). There was no difference between the left and right muscle (ANOVA, p = 0.628). No interaction existed between the treatment and muscle side (ANOVA, p = 0.495). (D) Normalized rectus abdominus muscle blood flow also decreased in a dosage dependent manner (ANOVA, p<0.001). There was no difference between the left and right side of the muscle (ANOVA, p = 0.560). No interaction exists between the treatment and muscle side (ANOVA, p = 0.636). [A,B,: ANOVA = 1 way repeated measures analysis of variance, C, D: ANOVA = 2 way repeated measures analysis of variance, *: adjusted p <0.05 post hoc Tukey test,]
83
4.3 Protocol 3: Bilateral Rectus Abdominus Muscle G4 Oxyphor PO2 4.31 The Effect of Phenylephrine on Mean Arterial Pressure
The baseline value of the averaged MAP was 89 ± 15 mmHg and a gradual increase
in MAP [n = 6 rats] was observed with increasing phenylephrine infusion (PE1 = 1.5
ug/kg/min, PE2 = 3.0ug/kg/min, PE3 = 6.0ug/kg/min, PE4 = 12.0ug/kg/min, and PE5 = 18.0
ug/kg/min) at 5 minute intervals. At the low doses, PE1 and PE2, MAP was unchanged at
89 ± 13 mmHg, and 91 ± 12mmHg respectively. A slight rise in MAP was observed at PE3
where MAP increased to 99 ± 10 mmHg. Furthermore, the elevation of MAP was
statistically significant at the high doses of phenylephrine, PE4 and PE5, in which MAP rose
to 124 ± 21 mmHg, and peaked at 137 ± 26 mmHg respectively. (p<0.001, one way
repeated measures ANOVA, Tukey test) Following a 30 minute recovery period, the
averaged MAP decreased back down to 105 ± 16 mmHg, but remained above baseline
value. A clear increasing trend in MAP was evident with the infusion of phenylephrine.
(Figure 15A)
4.32 The Effect of Phenylephrine on Heart Rate
Heart rate (HR) [n = 6 rats] was recorded to be at an average baseline value of 305 ±
38 bpm and remained stable throughout the treatment with phenylephrine. Upon continuous
infusion of phenylephrine at PE1 (1.5 ug/kg/min), PE2 (3.0 ug/kg/min), PE3 (6.0
ug/kg/min), PE4 (12.0 ug/kg/min), and PE5 (18.0ug/kg/min) for 5 minute intervals,
averaged heart rate was 310 ± 42 bpm, 314 ± 45 bpm, 312 ± 49 bpm, 303 ± 36 bpm, and
304 ± 29 bpm respectively. Following a 30 minute recovery period, averaged heart rate was
311 ± 33 bpm. There was not a statistically significant difference (P=0.614, one way
84
repeated measures ANOVA) detected and the heart rate was relatively stable throughout the
experimentation. (Figure 7C)
4.33 The Effect of Phenylephrine on Phosphorescence Lifetime and Muscle PO2
The phosphorescence lifetime (n = 6 rats) was recorded with the PMOD device to
calculate the tissue PO2 levels based on oxygen dependent phosphorescence quenching and
a general decline in phosphorescence lifetime was observed with increasing infusion of
phenylephrine corresponding to an increase in muscle tissue PO2. On the left side of the
muscle, baseline lifetime of phosphorescence was 35.2 ± 4.3 usecs, which decreased at the
highest dose of phenylephrine (PE5) to the lifetime of 32.5 ± 3.0 usecs. As the
phosphorescence lifetime is inversely proportional to the partial pressure of oxygen, left
muscle PO2 increased from a baseline value of 33.8 ± 16.7 torr to a peak value of 43.1 ±
11.4 torr at PE5. Similarly on the right side of the muscle, the baseline phosphorescence
lifetime of 36.1 ± 2.8 usecs decreased to 32.4 ± 1.5 usecs at the highest dose of
phenylephrine (PE5) and corresponded to an increase in muscle baseline PO2 from 29.9 ±
8.9 torr to a peak value of 42.9 ± 5.8 torr. There was no statistical difference observed
between the left and the right sides of the bilateral rectus abdominus muscles (p = 0.630,
two way repeated measures ANOVA), and no statistical interaction effect between the drug
treatment (PE1 – PE5) and muscle side (left or right). There was a statistically significant
treatment effect at PE4, PE5, and during recovery phase on PLT and muscle PO2. (p<0.001
for all, two way repeated measures ANOVA, Tukey test) Therefore phenylephrine infusion
resulted in an increase in muscle tissue PO2 characterized by the decrease in
phosphorescence lifetime observed on both the left and right sides of the muscle. (Figure 15
C,D)
85
4.34 Rectal Temperature was Stable throughout the Experimentation
Rectal temperature (n = 6 rats) was stable throughout the experimentation ranging from
36.1 to 36.3 oC. The average baseline temperature was 36.3 ± 0.6oC. Upon continuous
phenylephrine infusion (PE1, PE2, PE3, PE4, and PE5) the recorded temperatures were 36.3
± 0.6oC, 36.2 ± 0.7oC, 36.2 ± 0.7oC, 36.2 ± 0.7oC, and 36.2 ± 0.7oC respectively. During the
recovery phase the average temperature was 36.2 ± 0.7oC. There was not a statistically
significant difference (p = 0.164, one way repeated measures ANOVA) and rectal
temperature was stable throughout the experimentation. (Figure 15B)
4.35 Arterial Blood Gas and Cooximetry
The ABG and cooximetry analysis at baseline and post PE at the end of the
experiment is presented in Table 3 under the bilateral muscle PO2 protocol. There was a
significant decrease in pH and PO2 (T test, p = 0.006 and p = 0.032 respectively). No
significant change in PCO2, hemoglobin, and SaO2 were observed.
4.36 Electrolyte and Metabolic Data
The electrolyte and metabolic data analysis at baseline and post PE at the end of the
experiment is presented in Table 4 under the bilateral muscle PO2 protocol. There was a
significant increase in Na+ (T test, p =0.011) and Cl- (T test, p = 0.048) and a significant
decrease in glucose (T test, p = 0.002). No significant change in K+, Ca+2, lactate, base, and
HCO3- were observed.
4.37 Protocol 3 Summary
MAP increased with PE infusion in a dose dependent manner (p <0.001, one way
repeated measures ANOVA, Tukey test). Rectal temperature was stable throughout the
experimentation (p = 0.164, one way repeated measures ANOVA). Phosphorescence
86
lifetime declined and muscle PO2 increased with continued infusion of phenylephrine
(p<0.001for both, two way repeated measures ANOVA) There was no significant difference
between the phosphorescence lifetime or muscle PO2 recorded from the left and right
muscle (p = 0.703 (PLT), p = 0.630 (muscle PO2), two way repeated measures ANOVA).
No interaction effect existed between the treatment and the muscle side (p = 0.446 (PLT), p
= 0.400 (muscle PO2), two way repeated measures ANOVA).
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Figure 15: The Effect of Phenylephrine on Bilateral Rectus Abdominus Muscle Tissue PO2. (n = 6) (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p <0.001) (B) Rectal temperature was stable throughout the experimentation. (ANOVA, P = 0.164) (C) Phosphorescence lifetime declined with continued infusion of phenylephrine (ANOVA, p<0.001) There was no difference between the phosphorescence lifetime recorded from the left and right muscle. (ANOVA, p = 0.703). No interaction effect existed between the treatment and the muscle side. (ANOVA, p = 0.446). (D) Muscle tissue PO2 increased with continuous phenylephrine infusion (ANOVA, p <0.001). There was no difference between the muscle PO2 in left and right side. (ANOVA, p = 0.630). No interaction effect existed between the treatment and the muscle side (ANOVA, p = 0.400). [A,B,: ANOVA = one way repeated measures analysis of variance, C, D: ANOVA = two way repeated measures analysis of variance, * post hoc Tukey Test, adjusted p <0.05]
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4.4 Protocol 4: Rectus Abdominus Muscle Flap vs Contralateral Control Laser Doppler Microvascular Blood Flow 4.41 The Effect of Phenylephrine on Mean Arterial Pressure
Mean arterial pressure [n = 6 rats] was recorded at an average baseline value of 86
±14 mmHg and increased with continuous phenylephrine infusion (PE1 = 1.5ug/kg/min,
PE2 = 3.0 ug/kg/min, and PE3 = 6.0 ug/kg/min, PE4 = 12.0 ug/kg/min, and PE5 =
18.0ug/kg/min) at 5 minute intervals. Averaged MAP was 85 ± 14 mmHg after initial
infusion of phenylephrine at 1.5ug/kg/min. Continued infusion of drug at PE2
(3.0ug/kg/min) resulted in an averaged MAP of 86 ± 15 mmHg. The two low doses of PE
did not alter MAP. At the high doses of PE3 (6.0ug/kg/min), PE4 (12.0ug/kg/min), and PE5
(18.0ug/kg/min), MAP increased to 94 ± 19 mmHg, 122 ± 26 mmHg, and 128 ± 27 mmHg
respectively. Following a 30 minute recovery period, MAP declined to 92 ± 21 mmHg, but
remained above initial baseline value. The rise in MAP was statistically significant at PE4
and PE5 (p<0.001, one way repeated measures ANOVA, Tukey test). Therefore, the
infusion of phenylephrine increased MAP in a dose dependent manner. (Figure 16A)
4.42 The Effect of Phenylephrine on Heart Rate
Heart rate (HR) [n = 6 rats] was recorded to be at an average baseline value of 271±
37 bpm and remained stable throughout the treatment with phenylephrine. Upon continuous
infusion of phenylephrine at PE1 (1.5 ug/kg/min), PE2 (3.0 ug/kg/min), and PE3 (6.0
ug/kg/min), PE4 (12.0 ug/kg/min), PE5 (18.0ug/kg/min) for 5 minute intervals, averaged
heart rate was 272 ± 33 bpm, 273 ± 31 bpm, 272 ± 29 bpm, 272 ± 24 bpm, and 280 ± 27
bpm respectively. Following a 30 minute recovery period, averaged heart rate was 281 ±
19 bpm. There was not a statistically significant difference (P=0.616, one way repeated
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measures ANOVA) detected and the heart rate was relatively stable throughout the
experimentation. (Figure 7B)
4.43 The Effects of Surgery and Phenylephrine on Microvascular Blow Flow in Rectus Abdominus Muscle and Muscle Flap
Microvascular blood flow was assessed in the rectus abdominus muscle flap and the
contralateral nonoperated muscle control. (n = 6 rats) At baseline muscle blood flow (989.58
± 313.86 PU) was much greater than muscle flap blood flow (479.31 ± 126.90 PU), flap
blood flow is approximately 50% of muscle control blood flow. The magnitude of muscle
blood flow and flap blood flow was statistically different throughout the experiment
(p<0.050, two way repeated measures ANOVA). Furthermore phenylephrine infusion
resulted in a decline in microvascular blood flow in both the muscle and the flap tissue.
Muscle blood flow declined to 669.06 ± 192.05 PU at the highest dose of drug infusion at
PE5, whereas muscle flap blood flow declined to 381.79 ± 60.28 PU at PE5 and further
declined during the recovery period to 328.00 ± 73.53 PU. Both the muscle blood flow and
flap blood flow was reduced with increasing phenylephrine infusion in a dose dependent
manner. Analyzing the data through data normalization verifies a 30% reduction in blood
flow in the muscle and a 16% reduction in muscle flap blood at the highest infusion of
phenylephrine. There was a grouped treatment effect on reduced microvascular blood flow
observed at PE4, PE5, and recovery phase that was statistically significant. (p<0.05, two
way repeated measures ANOVA, Tukey test) (Figure 16 C,D)
4.44 Rectal Temperature was Stable throughout the Experimentation
Rectal temperature (n = 6 rats) was stable throughout the experimentation ranging
from 34.7 to 35.0 oC. The average baseline temperature was 35.0 ± 2.8oC. Upon
continuous phenylephrine infusion (PE1, PE2, PE3, PE4, and PE5) the recorded
90
temperatures were 34.9 ± 2.8 oC, 34.8 ± 2.8oC, 34.7 ± 2.7oC, 34.7 ± 2.6oC, and 34.8 ± 2.7oC
respectively. During the recovery phase the average temperature was 35.0 ± 2.9oC. There
was not a statistically significant difference (p = 0.666, one way repeated measures
ANOVA) and rectal temperature was stable throughout the experimentation. (Figure 16B)
4.45 Arterial Blood Gas and Cooximetry
The ABG and cooximetry analysis at baseline and post PE at the end of the
experiment is presented in Table 3 under the muscle vs flap blood flow protocol. No
significant changes in pH, PCO2, PO2, hemoglobin, and SaO2 were observed.
4.46 Electrolyte and Metabolic Data
The electrolyte and metabolic data analysis at baseline and post PE at the end of the
experiment is presented in Table 4 under the muscle vs flap blood flow protocol. No
significant change in K+, Na+, Ca+2, glucose, lactate, base, and HCO3- were observed. There
was a significant increase in Cl- (T test, p = 0.014).
4.47 Protocol 4 Summary
Mean arterial pressure increased with PE infusion in a dose dependent manner (p =
<0.001, one way repeated measures ANOVA). Rectal temperature was stable throughout
the experimentation. (p = 0.666, one way repeated measures ANOVA). Muscle flap blood
flow was 50% of the muscle control blood flow during the baseline period and phenyephrine
infusion reduced both muscle and flap blood flow in a dose dependent manner (treatment
p<0.001, muscle type p = 0.009, and interaction effect p = 0.137, two way repeated
measures ANOVA). Normalized muscle and free muscle flap blood flow was reduced by
approximately 30% and 16% respectively from baseline after infusion of phenylephrine and
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during the recovery phase (treatment p<0.001, muscle type p = 0.990, interaction effect p =
0.224, two way repeated measures ANOVA).
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Figure 16: The Effect of Phenylephrine on Muscle and Flap Microvascular Blood Flow (n = 6). (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p = <0.001). (B) Rectal temperature was stable throughout the experimentation. [ANOVA, p = 0.666]. (C) Muscle flap blood flow was 50% of muscle control blood flow during the baseline period and phenyephrine infusion reduced both muscle and flap blood flow in a dose dependent manner. (ANOVA, treatment p<0.001, muscle type p = 0.009, and interaction effect p = 0.137). (D) Normalized muscle and free muscle flap blood flow was reduced by approximately 30% and 16% respectively from baseline after infusion of phenylephrine and during the recovery phase. (ANOVA, treatment p<0.001, muscle type p = 0.990, interaction effect p = 0.224). [A,B,: ANOVA = 1 way repeated measures analysis of variance, C, D: ANOVA = 2 way repeated measures analysis of variance, *: adjusted p <0.05post hoc Tukey test]
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4.5 Protocol 5: Rectus Abdominus Muscle Flap vs Contralateral Control G4 Oxyphor PO2 4.51 The Effect of Phenylephrine on Mean Arterial Pressure
Mean arterial pressure (MAP) [n = 9 rats] was recorded at an average baseline value
of 83 ± 15 mmHg and increased with continuous phenylephrine infusion (PE1 =
1.5ug/kg/min, PE2 = 3.0 ug/kg/min, and PE3 = 6.0 ug/kg/min, PE4 = 12.0 ug/kg/min, and
PE5 = 18.0ug/kg/min) at 5 minute intervals. Averaged MAP was 80 ± 16 mmHg after
initial infusion of phenylephrine at 1.5ug/kg/min. Continued infusion of drug at PE2
(3.0ug/kg/min) resulted in an averaged MAP of 79 ± 17 mmHg. At the high doses of PE3
(6.0ug/kg/min), PE4 (12.0ug/kg/min), and PE5 (18.0ug/kg/min), MAP increased to 83 ± 17
mmHg, 111 ± 19mmHg, and 124 ± 20 mmHg respectively. Following a 30 minute recovery
period, MAP declined to 86 ± 17 mmHg, but remained above initial baseline value. The rise
in MAP was statistically significant at PE4 and PE5 (both, p<0.001, one way repeated
measures ANOVA, Tukey test). Therefore, the infusion of phenylephrine increased MAP in
a dose dependent manner. (Figure 17A)
4.52 The Effect of Phenylephrine on Heart Rate
Heart rate (HR) [n = 8 rats] was recorded to be at an average baseline value of 281 ±
43 bpm and remained relatively stable throughout most of the treatment with phenylephrine.
Upon continuous infusion of phenylephrine at PE1 (1.5 ug/kg/min), PE2 (3.0 ug/kg/min),
PE3 (6.0 ug/kg/min), PE4 (12.0 ug/kg/min), and PE5 (18.0ug/kg/min) for 5 minute
intervals, averaged heart rate was 278 ± 46 bpm, 276 ± 45 bpm, 274 ± 44 bpm, 278 ± 41
bpm, and 295 ± 36 bpm respectively. Following a 30 minute recovery period, averaged
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heart rate was 278 ± 36 bpm. There was no statistically significant change in heart rate.
Therefore heart rate was relatively stable throughout the experimentation. (Figure 7D)
4.53 The Effects of Surgery and Phenylephrine on Phosphorescence Lifetime and Muscle and Flap PO2
Phosphorescence lifetime [n = 9 rats] was measured in the muscle control tissue and
muscle flap tissue to calculate tissue PO2 via the O2 dependent quenching of
phosphorescence method. Phosphorescence lifetime within the muscle control tissue was
relatively stable throughout the experimentation ranging from 36.2 – 37.5 usecs. There was
no difference observed within the phosphorescence lifetime detected within the control
muscle tissue. Similarly, phosphorescence lifetime detected within the muscle flap was
stable throughout the experimentation, however the phosphorescence lifetime was much
greater compared to control muscle ranging from 45.7-45.8 usecs. The phosphorescence
lifetime recorded was inversely proportional to the tissue PO2 values that were calculated.
There was a large difference between the muscle control PO2 and the flap PO2, as the
nonoperated muscle had greater PO2 values compared to the flap that had microvascular
surgery and reanastomosis. Muscle tissue PO2 [n = 9 rats] in the control muscle started at a
baseline value of 26.1 ± 10.1 torr and remained relatively stable throughout the
experimentation. Similarly, the flap tissue PO2 also remained relatively stable throughout
the experimentation starting with a baseline value of 4.53 ± 2.66 torr. Clearly,
phenylephrine infusion did not augment the flap PO2, as the flap PO2 remained low
throughout the experiment. Therefore, the nonoperated control muscle had greater muscle
PO2 compared to the flap tissue, and phenylephrine did not improve flap perfusion. (Figure
17 C,D)
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4.54 Rectal Temperature was Stable throughout the Experimentation
Rectal temperature (n = 9 rats) was stable throughout the experimentation. The
average baseline temperature was 34.4 ± 1.8oC. Upon continuous phenylephrine infusion
(PE1, PE2, PE3, PE4, and PE5) the recorded temperatures were 34.3 ± 1.9 oC, 34.3 ± 2.0oC,
34.2 ± 2.0oC, 34.1 ± 2.1oC, and 34.0 ± 2.1oC respectively. During the recovery phase the
average temperature was 33.9 ± 2.2oC. Therefore, the rectal temperature was relatively
stable throughout the experimentation. (Figure 17B)
4.55 Arterial Blood Gas and Cooximetry
The ABG and cooximetry data at baseline and post PE at the end of the experiment
is presented in Table 3 under the muscle vs flap PO2 protocol. No significant change in
PCO2, PO2, hemoglobin, and SaO2 were observed. There was a significant decrease in pH
(T test, p = 0.002).
4.56 Electrolyte and Metabolic Data
The electrolyte and metabolic data analysis at baseline and post PE at the end of the
experiment is presented in Table 4 under the muscle vs flap PO2 protocol. No significant
changes in Na+, Ca+2, glucose, and lactate were observed. There was a significant increase
in K+ (T test, p = 0.002) and Cl- (T test, p = 0.001). A significant decrease in base (T test, p
= 0.018) and HCO3- (T test, p = 0.003) was also observed.
4.57 Protocol 5 Summary
Mean arterial pressure increased with PE infusion in a dose dependent manner (p =
<0.001, one way repeated measures ANOVA). Rectal temperature was stable during the
experiment. Phosphorescence lifetime in the muscle and the flap was stable throughout
experimentation. There was a higher phosphorescence lifetime recorded in the flap tissue
96
compared with the muscle tissue. There is a significant difference between the muscle and
flap phosphorescence lifetime (p<0.001, two way repeated measures ANOVA), but no
treatment effect (p=0.075, two way repeated measures ANOVA), and no interaction effect
(p = 0.194, two way repeated measures ANOVA). Muscle and flap tissue PO2 was inversely
proportional to phosphorescence lifetime. A significant difference between the muscle and
flap PO2 was evident (p <0.001, two way repeated measures ANOVA), muscle PO2 was
greater than flap PO2 throughout the experiment. There was no treatment effect as the
muscle and flap PO2 were both stable (p =0.057, two way repeated measures ANOVA) and
no interaction effect existed (p = 0.123, two way repeated measures ANOVA).
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Figure 17: The Effect of Phenylephrine on Muscle and Flap Tissue PO2. (n = 9) (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p = <0.001). (B) Rectal temperature was stable during the experiment. (C) Phosphorescence lifetime in the muscle and the flap was stable throughout experimentation. There was a higher phosphorescence lifetime recorded in the flap tissue compared with the muscle tissue. There is a significant difference between the muscle and flap phosphorescence lifetime (ANOVA, p<0.001), but no treatment effect (ANOVA, p=0.075), and no interaction effect (ANOVA, p = 0.194). (D) Muscle and Flap Tissue PO2 was inversely proportional to phosphorescence lifetime. A significant difference between the muscle and flap PO2 was evident (ANOVA, p <0.001), muscle PO2 was greater than flap PO2 throughout the experiment. There was no treatment effect as the muscle and flap PO2 were both stable (ANOVA, p =0.057) and no interaction effect existed (ANOVA, p = 0.123). [A,B,: ANOVA = 1 way repeated measures analysis of variance, C, D: ANOVA = 2 way repeated measures analysis of variance, *: adjusted p <0.05 post hoc Tukey test,]
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4.6 Protocol 6: Rectus Abdominus Muscle Flap and Contralateral Muscle Temperature 4.61 Mean Arterial Pressure Response to Phenylephrine MAP responded in a similar manner to PE as previously described in other protocols. 4.62 Bilateral Muscle Temperature during Experimentation
The temperature of the bilateral rectus abdominus muscles were recorded with
temperature probes which revealed that the temperature of the left muscle at baseline was
(32.9±1.5oC) and the right muscle temperature at baseline was (32.8±0.8oC). Both left and
right muscle temperatures were relatively similar around 33oC and the temperature was
lower than the measured rectal temperature (36.3±0.6oC). Temperature was stable
throughout the experimentation (p=0.177, one way repeated measures ANOVA) (Figure
18A).
4.63 Muscle Control and Muscle Flap Temperature during Experimentation The temperature of the muscle flap and contralateral muscle control was measured with
temperature probes and revealed similar temperatures between the muscle flap and the
muscle control. The flap temperature (30.2±1.0oC) and the muscle control temperature
(31.2±1.8oC) at baseline were very similar. There was a one degree difference between the
muscle and flap temperature. Both the muscle and flap temperature was stable throughout
the experimentation. (p=0.113, one way repeated measures ANOVA) Rectal temperature
(35.4±1.2oC) was also recorded and was stable throughout the experimentation. (p = 0.532,
one way repeated measures ANOVA) Temperature was stable throughout the experiments
and was unlikely to have a large effect on the results (Figure 18B).
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Figure 18 Assessing Temperature in Rectus Abdominus Muscle and Muscle Flaps. (A) Bilateral Muscle temperature protocol. (n= 6) The left muscle at baseline was (32.9±1.5oC) and the right muscle temperature at baseline was (32.8±0.8oC). Both left and right muscle temperatures were relatively similar around 33oC and the temperature was lower than the measured baseline rectal temperature (36.3±0.6oC). Temperature was stable throughout the experimentation (ANOVA, p=0.177) (B) Contralateral muscle and muscle flap temperature protocol (n = 5). The flap temperature (30.2±1.0oC) and the muscle control temperature (31.2±1.8oC) at baseline were very similar. There was a one degree difference between the muscle and flap temperature. Both the muscle and flap temperature was stable throughout the experimentation. (ANOVA, p=0.113) Rectal temperature (35.4±1.2oC) was also recorded and was stable throughout the experimentation. (ANOVA, p = 0.532) [ANOVA = one way repeated measures ANOVA]
100
Table 3: Arterial Blood Gas and Cooximetry Data Analysis: pH, partial pressure of carbon dioxide (PCO2), partial pressure of oxygen (PO2), hemoglobin (Hb), and oxygen saturation (SaO2) were assessed at baseline and post PE (at the end of the experiment) in each protocol. * = statistically significant relative to baseline (p<0.05, T test).
Table 4: Electrolytes and Metabolic Data Analysis: K+, Na+, Cl-, Glucose, Lactate, Base, Bicarbonate (HCO3
-) were assessed at Baseline and Post PE (at the end of the experiment) in each protocol. * = statistically significant relative to baseline (p <0.05, T test)
101
CHAPTER 5 DISCUSSION
A variety of factors may contribute to inadequate skeletal muscle perfusion during
surgery. These factors may eventually lead to muscle breakdown, rhabdomyolysis and free
muscle flap failure. We examined four specific factors that may contribute to impaired
muscle perfusion in this thesis: (1) muscle compression, (2) phenylephrine use, (3) free flap
surgery, and (4) temperature. Skeletal muscle health was evaluated in patients undergoing
craniotomy by measuring serum lactate, creatine kinase and myogloblin levels. In addition,
rodent models of rectus abdominus muscle and muscle flap perfusion were also established
to examine the effects of vasopressor use and muscle flap preparation on muscle perfusion
during surgery. In animal models, we utilized measurements of microvascular blood flow,
and quantitative tissue PO2 to assess muscle perfusion. Both our clinical and experimental
studies reveal different mechanisms that may jeopardize skeletal muscle perfusion during
surgery.
5.0 The Significance of Hyperlactatemia during Craniotomy for Brain Tumour Resection
5.01 Clinical Significance of Increased Serum Lactate
Hyperlactatemia has been reported in patients during neurosurgery cases for brain
tumour resection, however the clinical significance of the increased lactate has not been
established. Serum lactate is an end product of anaerobic glycolysis and is a clinical marker
for inadequate tissue perfusion during surgery. Our data revealed that lactate increased
within the first three hours of surgery (2.21 ± 1.22 mmol/L), and peaked near 9 hours into
the surgery (3.73±1.60 mmol/L). (p<0.05 relative to baseline (1.01±0.47mmol/L)). An
increased serum lactate can be representative of increased production or reduced metabolism
of lactate. The early increase in lactate correlated with BMI causing us to focus on one of
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two possible explanations: 1) high patient BMI contributed in muscle compression and
inadequate muscle perfusion leading to increased lactate production; or 2) high patient BMI
was associated with metabolic dysfunction in the liver resulting in decreased lactate
metabolism. As postoperative assessment of liver enzymes (ALP, AST, ALT, LD, and Bili)
in our patients was reported to be within normal limits, we therefore speculate that the
increased serum lactate was a result of excessive production due to inadequate perfusion,
and not inadequate lactate metabolism in the liver. This demonstrated that the patients were
at risk of hypoperfusion during brain tumour resection surgery. In the eighteen patients,
lactate was observed to increase. We further examined markers of muscle injury such as
creatine kinase and myoglobin which are released during muscle damage. In eight patients,
the CK values rose to greater than 1000U/L and six patients had myoglobinuria
postoperatively. These data supported our hypothesis that the lactate may be indicative of
inadequate muscle perfusion during surgery. Serum lactate may be an early indicator that
skeletal muscle perfusion is at risk, followed by muscle damage and the release of muscle
enzymes into the bloodstream. Indeed, the elevated lactate, creatine kinase and presence of
myoglobinuria suggested that muscle perfusion is jeopardized during brain tumour resection
craniotomy. Taken together, serum lactate, creatine kinase, and myoglobin are a cascade of
clinical markers that indicate muscle hypoperfusion and damage during craniotomy.
The frequent increase in lactate has been reported in neurosurgery and may occur in
other forms of surgery. Craniotomy may be predisposed the patient to increased lactate. The
rise in lactate may be due to increased sympathetic activity and high levels of
catecholamines associated with open brain surgery, which can also contribute to impaired
perfusion via increased vascular tone. In addition, the use of diuretics may decrease
intravascular volume and decrease tissue perfusion. Mannitol is a common diuretic that is
103
used during neurosurgery to prevent edema and reduce intracranial pressure which may
deplete intravascular volume and was considered as a potential cause of impaired muscle
perfusion. A positive correlation between urine output and mannitol was evident, however
no significant correlation was found between increased serum lactate and mannitol dose.
Therefore we excluded mannitol use during surgery as a factor that may be influencing
muscle perfusion as there was no significant relationship. Finally the frequent use of
steroids (dexamethasone) to reduce peritumoral edema during neurosurgery may alter
glucose metabolism and increase lactate.134 We found no evidence that these factors
influenced lactate production.
5.02 The Potential Source of Increased Serum Lactate
All tissues have the potential to produce lactate, especially during anaerobic
conditions, but only tissues with active glycolysis produce excess lactate from glucose under
normal conditions and release it into the bloodstream. At rest lactate is produced from
skeletal muscle (25%), skin (25%), brain (20%), red cell (20%), and gut (10%). The liver is
the primary site of lactate clearance (60%) and the kidneys metabolize approximately 20 to
30% of daily lactate. The balance between release into the bloodstream and hepatorenal
uptake maintains plasma lactate at about 1 mmol/l. 41, 87, 88 During craniotomy for brain
tumour resection in eighteen patients, we observed that serum lactate increased to a peak
value of 3.73±1.60 mmol/L at 9 hours into the surgery. We hypothesized that the skeletal
muscle was the ultimate source of excessive lactate production as skeletal muscle covers
majority of the body and can produce lactate under anaerobic conditions. Other vital organs
such as the heart, brain, and liver may also contribute to lactate production, however
perfusion to these organs were well monitored and maintained by the anesthesiologist
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during surgery and no cases of intraoperative hypotension occurred. All patients survived
craniotomy for brain tumour resection and did not suffer from any significant brain or
cardiovascular complications resulting from malperfusion during surgery. Assessment of
liver enzymes in patients postoperatively did not detect any abnormalities or dysfunction in
the liver. Thus it was unlikely that the heart, brain, or liver were engaged in anaerobic
production of serum lactate during craniotomy. The kidney may also contribute to lactate
production during hypoxia, however no signs of kidney injury resulting from malperfusion
during surgery were observed in our patients during postoperative care.
Interestingly, the brain tumour was also examined as a potential source of lactate,
and the tumour size, tumour grade and pathology were assessed, however the brain tumour
is very small in size compared to the overall mass of skeletal muscle tissues and was
unlikely to be the ultimate source of lactate production. Indeed metastatic cancerous
tumours are known to produce lactate by aerobic glycolysis through the Warburg effect,135-
137 however a large majority of the pathologies in our patient population were benign
tumours such as meningiomas that had a low tumour grade and were unlikely to be the
source of lactate production. No significant relationship between tumour grade and lactate
production was found in our 18 patients. If the tumour was the source of the lactate
production, we would expect our baseline values of lactate to start at a high value and
remain high until the tumour was removed, however, our baseline lactate values in the
eighteen patients started very low and gradually increase overtime within the first 3 hours of
surgery. Furthermore, elevated creatine kinase (CK) and positive myogloblinuria was
observed in some patients (8/18 CK, 6/18 myoglobinuria) suggestive of downstream muscle
damage and rhabdomyolysis resulting from inadequate muscle perfusion. This supported
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our hypothesis which investigated skeletal muscle as the possible predominant source of
increased serum lactate over other vital organs and the tumour.
5.03 Body Mass Index as a Risk Factor for Increased Serum Lactate during Craniotomy
Although an elevation serum lactate, creatine kinase and myogloblin suggested that
the most likely source of serum lactate was indeed the ischemic muscle tissues, the precise
mechanism and cause leading to ischemia and muscle damage remained unclear. The
positive correlation between the body mass index and the early rise in serum lactate
supported our hypothesis that the skeletal muscle was the predominant source of lactate.
Indeed a high body mass index characteristic of obese patients is associated with co-
mordities such as cardiovascular disease, and diabetes and metabolic syndrome.138 Our data
lead us to derive a plausible mechanism involving BMI. While immobile heavy patients lay
on the operating table under the same pressure points impaired muscle perfusion can result.
It was hypothesized that muscle compression by the patient’s body mass may be involved
with the impairment of muscle perfusion resulting in rhabdomyolysis. Surprisingly, there
was no correlation between serum lactate and the length of surgery, which further supports
the role of body mass index in the mechanisms leading to increased lactate and muscle
damage. This mechanism supports the notion that impaired muscle perfusion resulting from
muscle ischemia leads to increased lactate production.
5.04 Mechanism 1: Muscle Compression leading to Muscle Ischemia and Rhabdomyolysis
Rhabdomyolysis (RM) can range from an asymptomatic condition involving an
increase in serum creatine kinase to a dangerous life threatening stage characterized by CK
levels > 5000 U/L myoglobinuria, electrolyte imbalances, and acute renal failure.5, 9, 11, 85
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We propose that the mechanism behind the muscle hypoperfusion and rhabdomyolysis
observed in our patients was through muscle compression induced tissue ischemia due to
patient’s own heavy body mass. Muscle compression by the patients’ own body mass during
prolonged surgery will compress the microvasculature, resulting in muscle ischemia,
thereby impairing muscle perfusion. The early rise in serum lactate characteristic of
inadequate tissue perfusion, and downstream elevation of creatine kinase and myoglobinuria
was indicative of muscle damage in neurosurgical patients. Indeed, recent case studies
regarding RM in obese patients have been published and support our findings.5, 7 The term
positional rhabdomyolysis has been used by Poli et al to describe a phenomenon in which
muscle damage results when unconscious patients lie on the operating table under the same
pressure points during hours of prolonged surgery.7 Additionly, Alterman et al published a
case report of an overweight patient (22 years old, BMI = 29) in which muscle injury was
suspected to have occurred in the patients left thigh as a result of pressure of the thigh
against the table from prolonged lateral position.5 De Tommasi, and Cusimano also
reported three cases of RM in obese patients who were positioned in the lateral position that
had significant increases in CK levels followed by muscle damage.9 One of our own patients
with the most prolonged surgery (18 hours) did have myoglobinuria and evidence of muscle
breakdown suggesting that prolonged surgery can be a cause of RM. It is proposed that
physical compression of the muscle tissue and blood vessels lead to ischemia induced
necrosis accompanied by the release of muscle enzymes into the bloodstream. Therefore
muscle compression is a factor that may jeopardize skeletal muscle perfusion during surgery
in patients with high body mass index. (Figure 19)
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Figure 19. Muscle Compression during Surgery leads to Muscle Ischemia Followed by Elevated Serum Lactate, Creatine Kinase and Myoglobinuria. Body mass index is a risk factor for muscle hypoperfusion in patients during craniotomy. The heavy body mass of patients may be compressing the muscle and crushing the microvasculature resulting in muscle ischemia and inadequate tissue perfusion characterized by increased serum lactate. Downstream muscle damage is evident as verified by increased creatine kinase and myoglobinuria.
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5.1 Development of the Rat Model of Muscle Perfusion
Our clinical study that assessed of skeletal muscle perfusion in patients during
surgery lead us to establish an experimental rodent model with our collaborators in the
department of plastic surgery. We proposed to study another important question in the
anesthesia and plastic surgery literature regarding the appropriate use of vasopressor during
reconstructive surgery. The hypothesis is that phenylephrine will increase MAP by severe
resistance artery constriction and actually limit perfusion in certain vascular beds (ie.
skeletal muscle). The initial goal of this animal model was to assess the impact of
vasopressor use (phenylephrine) on skeletal muscle perfusion during surgery. Vasopressors
are commonly administered during surgery to treat hypotension and may be a factor that can
influence muscle perfusion. Based on the rectus abdominus muscle model established by
Zhang et al,111 we decided that the bilateral rectus abdominus muscle model was the ideal
muscle perfusion system for our experimental study. The anatomy of the rectus abdominus
muscle in rat was reported to be the very similar to humans with a consistent double blood
supply and multiple musculocutaneous perforators.111 Furthermore, the rectus abdominus
muscle flap is widely used in breast reconstructive surgery which corresponds to the
specialty and research interests of Dr. Melinda Musgrave, one of the main principal
investigators of the project. Thus, we started our experiments by establishing a bilateral
rectus abdominus muscle blood flow model and verified that both the left and right sides of
the rectus abdominus muscle responded in a similar manner to PE treatment. The details
regarding the dosing of PE in our protocols is described in the next section (5.11). We also
measured bilateral rectus abdominus muscle tissue PO2 to study tissue perfusion. The
overall impact of PE increased MAP in a dose dependent manner and increased femoral
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blood flow, but decreased microvascular blood flow; the significance of which will be
discussed in subsequent sections. The bilateral rectus abdominus muscle model allowed us
to study the perfusion of a muscle flap on one side, while using the nonoperated
contralateral muscle as a control. The main focus of our research project was then focused
on muscle flap perfusion. Identification of poor muscle flap perfusion prior to PE treatment
suggested that the muscle flap perfusion may be severely compromised by the flap surgery
procedure and treatments to improve flap perfusion will be the future emphasis.
5.11 Establishing the Dose of Phenylephrine for Increased Mean Arterial Pressure
We established a dose response for PE in the rat model. It was important to find a
dose response which caused minimal to no increase in MAP and higher doses that did
increase MAP. Our initial dose range (PE4 = 10ug/kg/min, PE5 = 20 ug/kg/min, and PE6 =
30ug/kg/min) was too high as all doses caused an increase in MAP. We adjusted the dose to
a lower level in 5 doses. (PE1 = 1.5ug/kg/min, PE2 = 3.0 ug/kg/min, PE3 = 6.0 ug/kg/min,
PE4 = 12.0 ug/kg/min, and PE5 = 18.0 ug/kg/min) The first two doses did not cause an
increase in MAP, but the subsequent 3 doses increased MAP in a dose dependent manner.
Discontinuation of the drug resulted in MAP returning to the baseline suggesting that the
duration of the drug effect was short (<30 minutes).
5.12 The Effect of Phenylephrine on Mean Arterial Pressure
Phenylephrine has been used for the last 60 years to treat intraoperative hypotension
and is a highly selective α1 agonist known to increase MAP by acting at the α1 receptors on
the resistance arteries. 21, 112 Differential distribution of α1 receptors on resistance arteries
occurs such that a larger proportion of α1 receptors in skeletal muscle cause specific
vasoconstriction while fewer α1 receptors in the brain result in less severe constriction.21, 113
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The overall effect of increased MAP is thought to be the centralization of blood flow to the
brain.115 PE will act at the level of the α1 adrenergic receptors of the blood vessels, and
induce vasoconstriction, thereby increasing systemic vascular resistance and mean arterial
pressure, therefore it was expected that mean arterial pressure would increase in a dose
dependent manner with the highest elevation in blood pressure at the highest doses of drug
infusion. The dose dependent rise in mean arterial pressure in response to phenylephrine
infusion was consistent in all protocols. (Figure 6) During the 30 minute recovery phase, the
MAP decreased back down towards baseline value. This may be due to a wear off effect of
phenylephrine as the drug only has a lifetime of 15 minutes in the body.20, 21 With the drug
no longer in effect, the mean arterial pressure will decrease back towards baseline value.
5.13 The Effect of Phenylephrine on Heart Rate
Phenylephrine has minimal effects on the heart because it has very weak β
adrenergic properties and is predominantly an α1 adrenergic agonist.20, 21 In humans an
increase in MAP by PE will result in a decrease in heart rate through the baroreceptor
response to regulate blood pressure. However, in the rat the heart rate is approximately 300
bpm and is different from humans. Indeed in most of our protocols the heart rate was stable
throughout the experimentation and was not influenced by phenylephrine. (Figure 7)
Consistent with our findings, Banic et al also noted no change in heart rate in response to a
30% increase in MAP resulting from PE infusion in their pig model.121 However, in our
carotid and femoral blood flow protocol, heart rate was observed to increase at the highest
dosage of phenylephrine (PE5). This was the only case and it was unexpected as we would
have expected heart rate to decrease as phenylephrine increases mean arterial pressure and
stimulates the baroreceptors to decrease heart rate and control pressure. The elevation in
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heart rate may in fact be a rat specific phenomenon as some other experimental data (Gengo
et al) have reported a 5% elevation in heart rate in sprague dawley rats with the infusion of
phenylephrine.139 Elevated catecholamines may have also contributed to the elevation in
heart rate during the surgery in our one protocol, but the overall pattern was a stable HR
throughout our protocols.
5.2 The Effects of Phenylephrine on Muscle Perfusion and Metabolism 5.21 Mechanism 2: The Effect of Phenylephrine on Muscle Perfusion
We assessed of the effects of phenylephrine (PE), a specific α1 agonist, on skeletal
muscle perfusion in a rodent model. Phenylephrine elevated MAP in the expected dose
dependent manner. Increasing doses of phenylephrine lead to subsequent progressive
increases in MAP which returned to baseline after discontinuing phenylephrine. Since
phenylephrine is used to maintain blood pressure in order to maintain perfusion, we assessed
blood flow in the conduit femoral artery. As might be expected, the higher doses of
phenylephrine almost doubled femoral artery flow as measured by ultrasound doppler. This
finding would support the use of phenylephrine to promote skeletal muscle perfusion.
However, when the effect of phenylephrine was assessed at its site of action in the
microcirculation using laser doppler flowmetry, we observed an early reduction in
microvascular blood flow at low doses of phenylephrine which did not influence blood
pressure. This effect progressed to be maximal at the highest doses of phenylephrine and
persisted even after phenylephrine infusion was discontinued. This data demonstrates that
there is no relationship between MAP and microvascular blood flow in skeletal muscle in
our model. More interestingly, the discrepancy between the increase in femoral blood flow
(conduit artery) and the decrease in microvascular blood flow(skeletal muscle tissue) must
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be explained. As outlined in our summary diagram, we hypothesize that severe increase in
resistance artery constriction lead to the decrease in microvascular flow, and the increased
femoral conduit artery flow may signify the presence of an increased physiological shunting
of blood from the conduit artery to the vein in our model (Figure 20). If such shunting
occurred in the systemic circulation, it should be associated with the bypassing of the
microcirculation, reduction in tissue oxygen delivery and a reduction in tissue PO2.
However, by our measurements, tissue PO2 in the skeletal muscle consistently increased by
about 10% under the same experimental conditions (PE infusion). One plausible means by
which microvascular tissue blood flow can decrease (~20%) while PO2 increases would be
that PE has a direct effect on reducing skeletal muscle oxygen metabolic requirements. The
presence of α1 receptors on skeletal muscle provide plausibility for this explanation.
In summary, PE increased MAP and conduit artery blood flow, however consistently
reduced microvascular perfusion in the skeletal muscle, suggesting that a physiological
shunt had occurred. Furthermore, muscle PO2 was observed to paradoxically increase
suggesting that muscle oxygen consumption was reduced by phenylephrine. To our
knowledge, this is the first description of such an effect of phenylephrine on muscle oxygen
consumption. Therefore, phenylephrine infusion is another mechanism that may jeopardize
skeletal muscle perfusion during surgery.
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(C) Tissue PO2 Equation
Figure 20. The Effects of Phenylephrine on Rectus Abdominus Muscle Perfusion. (A) Baseline conditions: blood flow was optimal in the large conduit arteries and in the microvasculature. It is assumed that there was minimal physiological shunting of blood. (B). Phenylephrine treatment: femoral blood flow (surrogate of inferior epigastric artery) increased while microvascular blood flow was reduced, suggesting that there was an increase in the shunt fraction as indicated by the larger red arrow. The decrease in microvacscular muscle blood flow occurred in association with a paradoxical increase in muscle tissue PO2. One possible explanation is that phenylephrine directly decreased muscle oxygen metabolism to a greater degree than reduced flow. (C) Tissue PO2 is defined by the oxygen supply over the oxygen demand. An increase in muscle tissue PO2 was observed when microvascular blood flow was reduced suggesting that there was a reduction in muscle oxygen consumption. [Red arrow = blood flow]
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5.22 The Effect of Phenylephrine (α1 Agonist) on Muscle Metabolism
The effects of vasoconstrictor stimuli on hindlimb skeletal muscle metabolism have
been extensively reviewed by Clark et al. Clark et al classify two groups of vasoconstrictors
based on their effects on muscle oxygen metabolism (Type A and Type B).140 Type A
vasoconstrictors are vasoconstrictors that stimulate oxygen consumption in the muscle
tissues. This group of vasoconstrictors include: norepinephrine (at low doses), epinephrine,
phenylephrine, methoxamine, amidephrine, ephedrine, norephedrine, angiotensin II, and
vasopressin, capsaicin, dihydrocapsaicin, [6] –gingerol, [6]-shogaol, and low frequency
sympathetic nerve stimulation.140 In contrast, Type B vasoconstrictors lead to a decrease in
muscle oxygen consumption with increased vascular resistance.140 Some examples of Type
B vasoconstrictors include norepinephrine at high doses (>1uM), serotonin, capsaicin
(>1uM), dihydrocapsaicin (>1uM), [6]-gingerol (>20uM), and high frequency sympathetic
nerve stimulation > 4Hz.140
In experimental models of hindlimb skeletal muscle in rats, oxygen consumption
was assessed by measuring the arteriovenous difference in oxygen content. Richter et al
were to first to show that alpha adrenergic effects of catecholamines increased glucose
uptake and oxygen consumption in the perfused rat hindlimb skeletal muscle.141 Increases in
oxygen consumption stimulated by epinephrine were prevented by α adrenergic blockade
(phentolamine mesylate) but not β adrenergic blockade (propranolol) suggesting that it was
predominantly the α adrenergic effect of the catecholamine that increased oxygen
consumption in skeletal muscle. Further studies revealed that epinephrine, norepinephrine
and phenylephrine elicit an increase in oxygen consumption and lactate efflux by the
skeletal muscle.142-144 Although the literature suggest that phenylephrine, an α1 agonist, is
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associated with an increase in muscle oxygen consumption in relatively low doses, our own
experiments demonstrated that high doses of phenylephrine (18ug/kg/min) reduced
microvascular blood flow and increased skeletal muscle PO2 suggestive of decreased
oxygen metabolism in the nonoperated bilateral rectus abdominus muscles. One possible
explanation for this observation could be that in our model phenylephrine behaves similar to
norepinephrine which has been reported to increase oxygen consumption in low doses (Type
A vasocontrictor) and decreases oxygen consumption at high doses (Type B
vasocontrictor).140, 142 The exact cellular mechanisms that lead to the changes in muscle
metabolism are unknown, however a significant abundance of α1 adrenoceptors have been
reported to be expressed on skeletal muscle cells,140, 145-148 and it may be possible that
phenylephrine is acting through these receptors directly on the skeletal muscle to impact
muscle metabolism.140 Further experimental studies to investigate muscle metabolism and
molecular pathways are necessary.
In summary, phenylephrine is an α1 agonist that has been reported to increase
muscle metabolism and oxygen consumption in rat hindlimb skeletal muscle, however our
study which used high doses of phenylephrine showed a reduction in oxygen metabolism in
nonoperated bilateral skeletal muscle similar to a reduction in oxygen metabolism observed
with high doses of norepinephrine. These differences in the oxygen metabolism trends
observed require further investigation including metabolic studies to investigate actual
oxygen consumption in our model.
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5.3 The Effects of Surgery and Phenylephrine on Muscle Flap Perfusion 5.31 Mechanism 3: The Effects of Muscle Flap Preparation and Microvascular Surgery on Muscle Flap Perfusion
The effects of muscle flap preparation on microvascular flap perfusion was very
surprising as flap perfusion was severely reduced compared to control after the free flap
preparation. Muscle flap blood flow was reduced by about 50% of the muscle control blood
flow, and muscle flap PO2 was a small fraction (~20%) of the muscle control PO2 at
baseline before the treatment of phenylephrine. The flap tissue PO2 was assessed to be
extremely low at around 5 torr. These findings suggest that surgical manipulation and
preparation of the muscle flap has a profound effect in diminishing muscle flap perfusion.
We have not identified any literature that has identified this severe basal reduction in flap
perfusion and interstitial PO2 associated with the creation of a free flap. However, Kamolz
et al have monitored flap tissue PO2 with Licox for over three years and have identified that
flap tissue PO2 was lower than 10 torr in all of their failing flaps.63 Additionally, PO2 values
of 8 – 12 torr is suggestive of ischemia in the brain and values at 2 torr or lower is indicative
of cell death. Our low flap tissue PO2 value of 5 torr at baseline prior to drug infusion
suggests that our muscle flaps which appear healthy and pink visually may indeed be at risk
of flap complications or failure. The effect of surgery on muscle flap perfusion was more
severe than the subsequent effects of phenylephrine infusion. After phenylephrine infusion,
the flap microvascular blood flow was further reduced by 16%. Thus phenylephrine may
have an added detrimental effect in addition to the poor perfusion of the flap at baseline.
The main finding was that flap perfusion was poor from the start of the experiment
immediately after surgery before drug treatment. The low basal perfusion suggests that
resistance arteries were already vasoconstricted with high vascular tone. Further infusion of
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phenylephrine reduced blood flow but did not change muscle flap PO2. Therefore, the
creation of a free flap is another mechanism that can impair muscle perfusion during
surgery. (Figure 21)
5.32 Muscle Flap Oxygen Metabolism after Flap Preparation
The extremely low muscle flap PO2 was suggestive of high oxygen consumption
within the muscle flap. According to the tissue PO2 equation: Tissue PO2 = oxygen supply/
oxygen demand (metabolism), we observed a severe reduction in oxygen supply in the
muscle flap and also extremely low levels of flap tissue PO2 at 5 torr, which is one of the
lowest tissue PO2 value our lab has ever measured in a living tissue. This low PO2 occurred
while 50% of the blood flow was maintained. Thus, this suggests that the oxygen
metabolism within the flap is very high, and that skeletal muscle flap oxygen consumption
is increased as a result of free flap preparation and associated ischemia and reperfusion.
This increase in oxygen consumption occurred during reperfusion of the muscle flap.
Nugent et al have performed in vivo measurements of rat skeletal muscle oxygen
consumption following brief periods of ischemia and reperfusion using phosphorescence
quenching microscopy and demonstrated an increase in oxygen consumption following
ischemia and reperfusion.149 Following 10 minutes of ischemia and reperfusion, oxygen
consumption was observed to be 254% of baseline oxygen consumption five seconds after
reperfusion.149 Forty five seconds later, oxygen consumption remained elevated at 175%
over baseline values.149 In another study, Harris et al used a canine gracilus muscle model to
study the metabolic response of skeletal muscle to ischemia, upon reperfusion of the skeletal
muscles after 7 hours of ischemia, the oxygen consumption was observed to increase greatly
compared to control muscle oxygen consumption.150 Thus, ischemia and reperfusion is
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generally accompanied by an increase in oxygen consumption. These findings support our
findings of high oxygen metabolism within the muscle flap, which was subjected to
approximately 30 minutes of ischemia and reperfusion when the vessels were transected and
reanastomsed during flap surgery. Furthermore, flap tissue may have higher metabolism
compared to control tissue. Im et al studied oxygen consumption and metabolism in skin
flaps. The rate of oxygen consumption in skin flaps was reported to be higher than control
skin.151 Their data suggested that skin flaps may be more metabolically active compared to
normal skin and that demand for energy in flap tissues may be met by increased metabolic
rate.151, 152 Similarly, in our skeletal muscle flap model, skeletal muscle flap tissue may be
more metabolically active than control nonoperated muscle which may explain why oxygen
metabolism in the muscle flap is very high, while the nonoperated control muscle exhibits
lower oxidative metabolism characterized by higher levels of tissue PO2. Thus, the
reperfusion of any tissue is reflected by increased metabolism and blood flow. In the event
that skeletal muscle blood flow is reduced, oxygen extraction and consumption will increase
until there is no oxygen available in which anaerobic metabolism occurs. Therefore, free
flap preparation resulted in a large reduction in skeletal muscle perfusion and the muscle
flap was suggestive of high oxygen consumption as tissue PO2 levels were very low. This
may be in part due to the fact that the microenvironment of the flap has been altered by flap
surgery and a period of associated ischemia and reperfusion which may be attributed to the
differences observed in muscle metabolism between the muscle flap (increased metabolism)
and the nonoperated skeletal muscle (decreased metabolism). Therefore, muscle oxygen
consumption in the muscle flap was observed to be high and may be attributed to surgical
manipulation and ischemia and reperfusion associated with flap preparation.
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5.33The Effect of Phenylephrine on Muscle Flap Perfusion
Phenylephrine also reduced microvascular muscle and muscle flap blood flow.
There was a 30% reduction in blood flow in the control muscle and a 16% reduction in
muscle flap blood flow at the highest infusion of phenylephrine. This reduction in blood
flow was a result of the α1 adrenergic vasoconstriction at the level of the resistance arteries
that was impairing muscle and flap blood flow. Furthermore, phenylephrine had no
significant effect on muscle and flap tissue PO2. The muscle PO2 remained stable, although
there was a slight increasing trend similar to the muscle PO2 response described in the
bilateral muscle protocol, while the flap tissue PO2 was far less than the muscle tissue PO2.
Oxygen consumption within the flap tissue may be very high. Thus, phenylephrine induced
elevation in mean arterial pressure did not improve muscle or muscle flap perfusion.
Furthermore, surgical preparation of the muscle flap had a more profound effect than
phenylephrine on the reduction of muscle flap perfusion. Alternative drug treatments may
be necessary to improve perfusion to the poorly perfused muscle flap.
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(C) Tissue PO2 Equation
Figure 21. The Effects of Free Flap Surgery and Phenylephrine on Muscle Flap Perfusion. (A) Free flap surgery was accompanied with an ischemia time of 27.7±6.1 minutes (B) Surgical manipulation and preparation of the muscle flap resulted in a severe reduction in microvascular blood flow and flap PO2 after reanastomosis. Phenylephrine further reduced microvascular blood flow by about 16% from baseline in the muscle flap without any further decrease in tissue PO2. This may have been because the tissue PO2 was at very low level and will not go down further. (C) In response to a reduction in microvascular blood flow to the flap, the tissue PO2 was extremely low in the muscle flap. This suggests that oxygen metabolism in the muscle flap is very high.
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5.4 The Potential Benefits and Harms of Phenylephrine use during Reconstructive Surgery
Vasopressors such as phenylephrine are commonly administered during surgery to
treat hypotension, however microsurgeons warn against their use as peripheral
vasoconstriction may compromise flap perfusion and lead to flap complications or failure.
Several animal studies, including our own data support this view as microvascular blood
flow to the muscle flaps is compromised.26, 27 One animal study suggests that
phenylephrine has no effect on microvascular blood flow.121 This finding complements
some recent clinical retrospective and prospective studies that investigated the safety of
vasopressor use during reconstructive surgery in breast reconstruction and head and neck
surgery. Indeed many clinical studies have demonstrated that there is no correlation between
vasopressor use and flap complications and failure.22-25 These data suggest that vasopressor
use does not negatively affect flap perfusion. However these studies are limited due to
small sample size, variable flap type, different doses of drug, and variable clinical
conditions. Thus no clear conclusions can be made about use of vasopressor and flap
viability in these studies. Whether or not vasopresor use is safe during reconstructive
surgery is still under clinical debate. We created our rectus abdominus muscle flap perfusion
model in sprague dawley rats with the initial intent on studying the effect of vasopressors on
muscle flap perfusion, and had similar observations with other animal studies performed in
pig models.26, 27 Mean arterial pressure was elevated at the expense of skeletal muscle tissue
perfusion. Phenylephrine reduced muscle and muscle flap microvascular blood flow. Our
animal data suggests that phenylephrine may be detrimental to muscle perfusion, however
our data suggests that the surgical preparation of the free muscle flap has a more profound
effect on muscle flap perfusion before the infusion of the drug. Further investigation is
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required to study the health of the muscle over a longer period of time to assess flap hypoxic
status, flap survival, flap tissue necrosis and cell death to confirm whether or not the
observed reduction in microvascular perfusion will lead to downstream skeletal muscle
damage, flap complications, and flap failure.
5.5 The Effect of Temperature on Muscle Perfusion
Temperature is known to have a profound effect on tissue oxygen metabolism.
Increasing or decreasing temperature causes a proportional change in oxygen consumption.
It is important to assess the impact of temperature on all of our clinical and experimental
models. In our clinical study, patient temperature remained stable throughout the early
phase of surgery (6 hours), and therefore was unlikely to influence the observed increase in
lactate which correlated with BMI at 3 hours. Similarly measurements of rectal, muscle,
and muscle flap temperature remain stable throughout all experimental protocols. By the
same logic we suggest that changes in temperature could not have strongly influenced the
observed changes in blood flow and tissue PO2 associated with phenylephrine infusion or
muscle flap preparation. We did observed that both muscle and muscle flap were colder
than rectal temperature by about 3-6oC, likely due to the superficial position of muscle and
its exposure to the environment during surgery. The stable nature of the muscle and muscle
flap temperature during our experimental protocols suggest that they could not have caused
acute changes in muscle metabolism. Of interest, the muscle flap and contralateral muscle
(internal control) were of comparable temperatures throughout the experiments. Therefore
temperature could not explain the profound reduction in muscle flap PO2 that we measured
in our study. The very low tissue PO2 in muscle flap despite clinical parameters of adequate
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perfusion suggests an important opportunity for improving muscle flap perfusion and
reducing flap failure in the clinic
5.6 The Effect of Isoflurane on Muscle Perfusion
Isoflurane, a vasodilator, is a general anesthetic used in our experiments to
anesthetize the rat in the experimental protocol. Although isoflurane can be a factor that can
influence skeletal muscle perfusion, we kept the isoflurane constant at 2% throughout the
experiment, and thus isoflurane was stable and unlikely to have a large impact on our
results. Of course, muscle perfusion may be higher in awake rats compared to anesthetized
rats as recently demonstrated by Wilson et al48, however, isoflurane was a necessary part of
the experiment. The effect of isoflurane on skeletal muscle blood flow has been reported in
the literature. Hartmen et al have shown that isoflurane resulted in dose related decreases in
skeletal muscle blood flow in dogs due to systemic hypotension.153 Similarly, other studies
showed a sharp significant decrease in skeletal muscle blood flow during isoflurane
anesthesia.154, 155 Wilson et al have examined muscle tissue PO2 in awake and anesthetized
rats under isoflurane. Rats under isoflurane had lower tissue PO2 values compared to awake
rats. Therefore, high dose isoflurane can reduce skeletal muscle perfusion as a result of
decreased perfusion pressure, however isoflurane was unlikely to influence our results as it
was constant at 2.0% in our experiments and we monitored mean arterial pressure and did
not observe hypotension in our rats. The minor effects of the isoflurane would have already
been accounted in the baseline measurements, and the decreases in skeletal muscle blood
flow observed in our protocols was predominantly a result of the infusion of phenylephrine
and not isoflurane anesthesia.
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5.7 Limitations of the Study 5.71 Clinical Study Limitations
One limitation of our clinical study was the limited frequency of sampling for our
clinical parameters (serum lacate, CK, and myoglobin). We only assessed serum lactate
every 3 hours and CK and myoglobin in urine every 4 hours. Our data is discontinuous and
missing data points for the times in between the 3-4 hour intervals where significant events
may have occurred. For instance, serum lactate may have peaked at 2 hours and returned to
lower baseline lactate values at 3 hours. Our 3 hour sampling method would have missed
this peak lactate value. The frequency of sampling for lactate, CK, and myoglobin was
limited by financial cost, however taking too many blood samples may be detrimental to the
patient. Nevertheless, the 3-4 hour sampling interval should be able to capture any
significant trends that occurred over time. For instance, whether or not serum lactate, and
CK increased, decreased, or remained the same during surgery. A small sample size (n = 18
patients) was a major limitation of the study and established trends may become more
reinforced with a larger sample size (n = 1000 patients) or even change. In fact, the positive
correlation between BMI and early rise in serum lactate at 3 hours was only a relatively
moderate correlation (r = 0.587), nevertheless the correlation between lactate and BMI was
established (p = 0.010). Thus, the main finding is based on a low number of patients with a
moderate correlation.
This clinical study provided some insight into a potential mechanism that may
jeopardize skeletal muscle perfusion during surgery, however was unable to definitely
determine the origin of the lactate. Although, it is proposed that the skeletal muscle is the
source of serum lactate during neurosurgery, no clear evidence firmly concludes that the
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lactate production is occurring only from the skeletal muscles, and that other tissues such as
the brain, heart, and tumour are not contributing to elevated lactate levels as well. Thus the
inability to specifically determine the specific source of lactate and the proportion produced
by organ(s) involved was a limitation. Further studies are necessary to specifically identify
the source of serum lactate and the contribution of all organs on a global scale.
5.72 Rat Study Limitations
One major limitation of our experimental study was that we did not create a
hypotension model which is clinically relevant. Instead we focused on assessing the effect
of PE on muscle flap independent of hypotension. It was necessary to first study the effect
of the drug in isolation. Future studies will incorporate a hypotension model for greater
clinical relevance. Another limitation of our experimental study include the inability to
simultaneously measure microvascular blood flow with laser doppler and tissue PO2 with
microsensor G4 oxyphor within the same animal due to wavelength interference between
the two different probes. This means that the blood flow patterns and tissue PO2 patterns
were from different animals, however we performed several experiments with consistent
results. Microvascular blood flow consistently decreased and tissue PO2 consistently
increased with phenylephrine infusion in rats. We concluded that these results indicate a
reduction in muscle metabolism derived from the tissue PO2 formula. However no
experimental procedures were carried out to specifically assess muscle metabolism directly.
Future studies may assess oxygen consumption by measuring arteriovenous PO2 difference.
Additionally, the hypoxic status of the muscle flap was unverified, and can be assessed via
western blot by assessing HIF-1α levels. A larger sample size (n=30 rats) may provide
more statistical power in our experiments, however our results are consistent with the
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smaller sample size used (n=6-10 rats). Temperature was stable throughout our experiments
and unlikely to influence our results, but was not perfectly controlled for at physiological
temperature (37oC). Temperature in our rats ranged from 30oC – 37oC depending on the
protocol and tissue source (ie. rectal (36-37 oC), muscle (33 oC) and flap (30 oC)). The age
group of rats differed between protocols and may contribute to a minor age effect. In our
developmental model we used 500g rats and in our carotid and femoral blood flow model
and bilateral muscle PO2 model we used 600-700g rats. As we progressed with the flap
model, we decided to use larger rats for larger rectus abdominus muscle flaps (800-900g
rats) as discussed with the plastic surgeon (Dr. Sami Alissa). More importantly, the
observed responses in microvascular blood flow and muscle PO2 to phenylephrine between
different age groups were identical. Therefore, rat age did not play a significant role in our
results. Furthermore, the study assessed muscle flap perfusion via blood flow and tissue PO2
in response to phenylephrine infusion, but does not clearly assess the long term outcomes
such as flap necrosis and survival. No clear conclusions can be made regarding the safety of
vasopressor use on flap perfusion without long term monitoring of the muscle flap for flap
survival or failure. Finally, our rectus abdominus muscle flap model may not reflect a true
free flap model, because the rectus abdominus muscle flap was reanastomosed back onto its
original location instead of to another distant region of the body (Ie. groin).
5.8 Future Directions 5.81 Future Directions for the Clinical Study Future directions for our serum lactate clinical study includes a retrospective
analysis of serum lactate in large population of patients. This larger clinical study will
verify the increased serum lactate commonly observed in patients during craniotomy and
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reexamine the correlation between patient BMI and early serum lactate rise in a larger
population of patients (n = 500 patients). A PSI grant has also been submitted to request
funding for another larger and more detailed prospective study. In this new study, we will
be assessing serum lactate, CK and myoglobin in a population of neurosurgical patients and
bariatric patients to compare for differences between surgery type. We will also increase the
sample size to n = 32 patients in each group of surgery for a total of n = 64 patients in the
study. Furthermore, near infrared spectroscopy (NIRS) will be used during the surgery to
monitor patient muscle PO2. Collaboration with radiologists will also enable us to assess for
muscle damage and rhabdomyolysis with MRI. Our two new future clinical studies will
further verify and expand our knowledge in this research field.
5.82 Future Directions for the Experimental Study Although we have shown that microvascular blood flow to the skeletal muscle and
flap tissue is reduced with PE infusion, tissue PO2 was observed to have increased in native
skeletal muscle and remained low in the muscle flap. Whether or not these conditions are
beneficial or detrimental to the muscle remains unknown. This could be suggestive of either
a state of hibernation in the muscle tissue, or inadequate perfusion leading to tissue hypoxia.
Thus, future assessment of the hypoxic status within the muscle tissue and flap tissue via
western blot analysis of HIF-1α will be performed. It is expected that the muscle flap will
express high levels of HIF-1α and the muscle control tissue will express relatively low
levels of HIF-1α. This additional set of experiments will help determine whether or not the
muscle flap is at hypoxic risk due to the effects of phenylephrine and provide some evidence
for the clinical debate regarding vasopressor use during reconstructive surgery. However,
long term experiments are also required to assess whether or not flap necrosis or flap failure
128
occurs after surgery. Flap survival rate over a period of days after the PE infusion protocol
should be examined in order to provide evidence for whether or not PE infusion will
compromise the flap. Thus, one of the directions of our research will be to assess long term
downstream effects of PE on overall flap survival. We will also perform a set of
experiments in which PE will be administered to treat hypotension in a rat model to address
the effects of hypotension.
Another future direction will be examining novel drug therapies that may improve
flap perfusion during reconstructive surgery as we have identified severe impairment of
perfusion to the flap as a result of free flap surgery. The exact cause and mechanism of the
impairment of microvascular blood flow to the muscle flaps is unknown, however we
hypothesize that flap surgery may result in resistance artery dysfunction leading to increased
microvascular tone. A potential mechanism that we will investigate in the future is the
effect of inflammation associated with surgery on the muscle flap perfusion. TNF-1α is an
inflammatory cytokine secreted by immune cells associated with inflammation. Recent
research have shown that skeletal muscle fibers can also synthesize TNF-1α and also
express both type 1 and type II TNF-1α receptors.156 TNF-1α has a significant role in
muscle metabolism as it promotes early myogenesis in undifferentiated myocytes, yet
stimulates the breakdown of muscle fibers in mature myotubes and is associated with
contractile dysfunction in skeletal muscle.156 Thus, high levels of TNF-1α may contribute to
skeletal muscle damage. TNF1α has also been shown to have proconstrictive effects on
microvasculature through sphingosine 1 phosphate signaling.157-159 We hypothesize that the
proconstrictive effects of TNF-1α may be responsible for increasing the microvascular tone
during reconstructive surgery leading to the severe impairment in microvascular skeletal
muscle flap perfusion. Therefore we proposed that the potential cause of the reduction in
129
microvascular perfusion may be due to increased microvascular tone associated with
inflammation and the proconstrictive effects of TNF-1α. Etanercept is a TNF-1α antagonist
that will bind to TNF-1α and prevent it from interacting with it’s receptors and is a potential
treatment that may reduce the microvascular tone in the skeletal muscle flaps. Dantrolene is
another drug that we plan on investigating. Dantrolene is a ryanodine receptor antagonist,
and prevents the release of calcium from the sarcoplasmic reticulum. Calcium is required
for smooth muscle contraction and vasoconstriction of blood vessels. The inhibition of
calcium release will lead to vasodilation of the blood vessels thereby improving perfusion to
the muscle flap. Numerous of vasodilators have been studied in the literature and have been
reported to generally improve local flap perfusion in animal models.160-162
130
CHAPTER 6 SUMMARY 6.0 Summary
Factors that impair tissue perfusion during surgery are associated with significant
adverse events. Identification of reversible causes of inadequate perfusion may help to improve
patient outcome. We have focused on the assessment of factors that may impair skeletal muscle
perfusion during surgery by testing the hypothesis that modifiable factors may reversibly impair
muscle perfusion during surgery including, physical compression, vasopressor administration,
free flap surgery and temperature. To achieve this goal, we assessed muscle tissue perfusion in
translational human and animal models. Muscle perfusion was assessed in patients undergoing
craniotomy for brain tumor resection by measuring systemic lactate, creatine kinase (CK) and
urinary myoglobin levels. A rat model of skeletal muscle and free muscle flap perfusion was
also developed to assess the impact of: 1) phenylephrine (α1-adrenergic agonist); 2) surgical
preparation of a free muscle flap and 3) temperature on skeletal muscle perfusion as assessed by
measurements of tissue blood flow (ultrasound and laser Doppler) and quantitative tissue PO2
(G4 oxyphor microsensors). Our results demonstrated an early increase in serum lactate in
craniotomy patients, which correlated with body mass index, but not length of surgery.
Increased lactate was associated with elevated CK and urine myoglobin, suggesting that muscle
compression contributed to inadequate muscle perfusion and anaerobic production of lactate.
Assessment of rectus abdominus muscle perfusion in rats demonstrated that administration of
phenylephrine, consistently reduced microvascular skeletal muscle blood flow (~20%, laser
doppler). Paradoxically, this reduction in tissue blood flow was accompanied by an increase in
tissue PO2 (~10%), suggesting that the α1-adrenergic agonist also reduced the muscle tissues
metabolic requirement for oxygen by a greater degree than it had reduced tissue blood flow.
131
Preparation of a free skeletal muscle flaps, by cutting and re-anastamosing the feeding artery,
resulted in severe attenuation of both tissue blood flow and PO2 suggesting that a severe degree
of microvascular constriction had attenuated free flap muscle perfusion. Administration of
phenylephrine further reduced blood flow (~20%), but not PO2 possibly because the muscle
tissue PO2 values were very low. The proportionally greater reduction in basal PO2, relative to
flow suggests that muscle ischemia and reperfusion resulted in an increase in muscle
metabolism and PO2 consumption during reperfusion. Finally, in both human and animal
models, muscle temperature did not appear to influence the observed changes in muscle
perfusion as the temperature was stable throughout the experiments. In conclusion, assessment
of muscle perfusion during surgery has identified specific conditions and treatments, which
jeopardize muscle perfusion. These included elevated body mass index of patients undergoing
craniotomy, phenylephrine infusion and surgical preparation of skeletal muscle flaps. In each
case these findings have identified modifiable risk factors for inadequate muscle perfusion
during surgery thereby providing a means to optimize muscle perfusion and minimize adverse
outcomes associated with skeletal muscle damage during surgery.
132
6.1 Key Experimental Findings 6.11 Assessing Skeletal Muscle Perfusion and Health during Neurosurgery
1) Hyperlactatemia occurs during neurosurgery and the skeletal muscle is the most likely source of lactate (Figure 22A)
2) Elevated muscle enzymes (Creatine kinase) and myoglobinuria indicated muscle damage in some patients (Figure 22C)
3) Body mass index is a risk factor for elevated serum lactate and muscle damage markers in patients undergoing craniotomy (Figure 22B)
4) There was no correlation between the peak serum lactate and the length of surgery. (Figure 22D)
5) Muscle compression induced ischemia may lead to rhabdomyolysis in patients 6.12 The Effects of Phenylephrine use on Muscle and Muscle Flap Perfusion
1) Phenylephrine elevated MAP in a dose dependent manner (Figure 23A, 24A), but did not improve muscle or muscle flap perfusion
2) Phenylephrine had minimal effects on heart rate 3) Phenylephrine did not have any effect on the carotid artery, however increased blood flow
in the femoral artery, nevertheless it is at the level of the resistance arteries that perfusion is regulated
4) Phenylephrine reduced microvascular muscle blood flow, but a paradoxical increase in muscle tissue PO2 was observed suggesting reduced muscle O2 metabolism (Figure 23 C,D)
5) Phenylephrine reduced microvascular muscle flap blood flow in a dose dependent manner, flap PO2 was low and remained stable throughout the experiment (Figure 24C,D) 6.13 The Effects of Surgical Free Flap Preparation on Muscle Flap Perfusion during Reconstructive Surgery
1) Surgical manipulation and preparation of the free flap resulted in a reduction in muscle flap perfusion as both microvascular flap blood flow, and flap PO2 was severely diminished compared to the contralateral muscle control perfusion. (Figure 24C,D)
2) Increased microvascular tone associated with inflammation and resistance artery dysfunction may be the cause for the observed impairment in muscle flap perfusion 6.14 The Effects of Temperature on Muscle and Muscle Flap Perfusion
1) Temperature was stable throughout the experiments and was unlikely to have a significant effect on muscle and muscle flap perfusion (Figure 23B, 24B)
133
Time (Hours)0 10 20 30 40 50 60 70
Seru
m L
acta
te (m
mol
/L)
0
1
2
3
4
5
6
7
8Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient 10Patient 11Patient 12Patient 13Patient 14Patient 15Patient 16Patient 17Patient 18
Time (Hours)
0 10 20 30 40 50 60 70
Cre
atin
e K
inas
e (U
/L)
0
2000
4000
6000
8000
10000
A B
C DBody Mass Index (BMI)
15 20 25 30 35 40
ΔLac
tate
3hr (m
mol
/L)
0
1
2
3
4
5
6N = 18R = 0.587Rsqr = 0.344P = 0.010
Length of Surgery (Hours)
0 2 4 6 8 10 12 14 16 18 20
Δ Lac
tate
Peak
(mm
ol/L
)
0
1
2
3
4
5
6
7
8N = 18R = 0.0499Rsqr = 0.00249P = 0.844
*
**
* *
** * *
*
*
Figure 22. Clinical Study Summary: Elevated Serum Lactate, CK and Myoglobinuria Characteristic of Muscle Ischemia Induced Muscle Damage Associated with Patient BMI (A) Increased serum lactate in patients undergoing craniotomy for brain tumour resection (n = 18). (B) Positive correlation between body mass index (BMI) and change in lactate was observed after 3 hours (∆Lactate3hr) (p=0.010). (C) Elevated serum creatine kinase occurred later than lactate increases in patients with a higher lacate response (n=6). In some patients with a high CK, myoglobinuria was detected (*= myoglobinuria positive). (D) No correlation between peak serum lactate and length of surgery (p = 0.844).
134
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
120
140
160
180
*PE1:1.5ug/kg/minPE2: 3.0ug/kg/minPE3: 6.0ug/kg/minPE4: 12.0ug/kg/minPE5: 18.0ug/kg/min
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Tem
pera
ture
(oC
)
26
28
30
32
34
36
38
40
42
Muscle TemperatureRectal Temperature
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mus
cle
PO2
(torr
)
0
10
20
30
40
50
60
70
* * *
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Nor
mal
ized
Mus
cle
Blo
od F
low
0.0
0.2
0.4
0.6
0.8
1.0
1.2
* **
n = 6 muscles n = 12 muscles
A B
C D
Figure 23. Bilateral Rectus Abdominus Muscle Perfusion Model Summary (n = 6). (A) Mean arterial pressure increased with PE infusion in a dose dependent manner (ANOVA, p <0.001), (B) Rectal and muscle temperature was stable throughout the experiment. There did not appear to be an effect of temperature on muscle blood flow. (C) Normalized muscle blood flow decreased with infusion of phenylephrine (ANOVA, p<0.001), and (D) Muscle PO2 increased with phenylephrine infusion (n = 6 rats, 12 muscles). (ANOVA, p < 0.001). [A,B,D,: ANOVA = 1 way repeated measures analysis of variance, *: adjusted p <0.05 post hoc Tukey Test,]
135
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
0
20
40
60
80
100
120
140
160
**
PE1: 1.5ug/kg/minPE2: 3.0ug/kg/minPE3: 6.0ug/kg/minPE4: 12.0ug/kg/minPE5:18.0ug/kg/min
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Tem
pera
ture
(oC
)
0
10
20
30
40 Muscle TemperatureFlap Temperature
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mus
cle/
Flap
Blo
od F
low
(BPU
)
0
200
400
600
800
1000
1200
1400
1600Muscle Blood FlowMuscle Flap Blood Flow
* **
A B
C D
Baseline PE1 PE2 PE3 PE4 PE5 Recovery
Mus
cle
PO2
(torr
)
0
10
20
30
40
50Muscle PO2 Flap PO2
Figure 24. Muscle Flap vs Contralateral Control Muscle Perfusion Model Summary (A) Mean Arterial Pressure increased with PE infusion in a dose dependent manner (ANOVA, p <0.001) (n = 5). (B) Flap and muscle temperature were stable throughout the experiment (n = 5), (C) Absolute muscle blood flow was greater than flap blood flow by 50% during the baseline period and phenyephrine infusion reduced both muscle and flap blood flow in a dosage dependent manner. (ANOVA, treatment p<0.001, muscle type p = 0.009, and interaction effect p = 0.137). (n = 6), and (D) There was a significant difference between muscle and flap tissue PO2 (ANOVA, p <0.001). Muscle PO2 was greater than flap PO2 throughout the experiment. There was no treatment effect of PE on muscle and flap PO2 which were both stable throughout the experiment (ANOVA, p =0.057) and no interaction effect existed (ANOVA, p = 0.123) (n = 9). [A: ANOVA = 1 way repeated measures analysis of variance, B, C, D: ANOVA = 2 way repeated measures analysis of variance, * post hoc Tukey Test, adjusted p <0.05]
136
CHAPTER 7 CONCLUSION 7.0 Conclusion
In our clinical study we characterized an early increase in serum lactate in patients
undergoing craniotomy for brain tumour resection which was characteristic of inadequate
tissue perfusion. Subsequent muscle damage markers such as creatine kinase and
myoglobin also were elevated and suggested rhabdomyolysis. Suprisingly, the early rise in
lactate was correlated to BMI and not length of surgery. This suggests that heavy body
mass may be inducing muscle compression leading to ischemia and muscle damage.
Therefore, muscle compression is a valid mechanism that can impair skeletal muscle
perfusion during surgery. In our experimental rodent model, PE infusion increased MAP but
did not have a large effect on blood flow in the carotid artery, and increased femoral artery
blood flow. At the level of skeletal muscle microvasculature, PE decreased tissue blood
flow but resulted in a paradoxical increase in tissue PO2. These data suggest that PE may
have reduced muscle tissue perfusion and concomitantly decreased the metabolic rate of O2
consumption. Prolonged surgery resulted in a reduction in muscle PO2, possibly due to
impact of inflammatory mediators on microvascular tone. Muscle free flap tissue blood flow
and PO2 (perfusion) were dramatically lower than contralateral control muscle suggesting
that surgical manipulation had severely altered the flap microvascular tone. Creation of a
free muscle flap dramatically impairs muscle perfusion (blood flow and PO2) possibly due
to severe microvascular dysfunction or resistance artery constriction.
In conclusion, assessment of muscle perfusion during surgery has identified specific
conditions and treatments, which jeopardize muscle perfusion. These included elevated
body mass index of patients undergoing craniotomy, phenylephrine infusion and surgical
137
preparation of skeletal muscle flaps. In each case these findings have identified modifiable
risk factors for inadequate muscle perfusion during surgery thereby providing a means to
optimize muscle perfusion and minimize adverse outcomes associated with skeletal muscle
damage during surgery.
138
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