Time-Dependent Reduction in O2 Supply and Utilization in the Diaphragm during Mechanical Ventilation

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Title:
Time-Dependent Reduction in O2 Supply and Utilization in the Diaphragm during Mechanical Ventilation Role of Vascular Dysfunction
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english
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Davis, Robert Thomas, III
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Health and Human Performance, Applied Physiology and Kinesiology
Committee Chair:
Behnke, Bradley Jon
Committee Members:
Adhihetty, Peter
Powers, Scott K
Clanton, Thomas
Muller Delp, Judy Marie

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Subjects / Keywords:
diaphragm -- mv -- oxygenation
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
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Health and Human Performance thesis, Ph.D.
Electronic Thesis or Dissertation
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )

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Abstract:
Mechanical ventilation (MV)engenders several clinical complications associated with the duration ofMV.  One consequence of MV is thedifficulty to successfully wean a large portion of patients from theventilator.  In skeletal muscle, areduced O2 supply results in contractile dysfunction and prematurefatigue when performing external work. However, whether MV induces an O2 supply-usage imbalance inthe diaphragm, which contributes to weaning difficulties, remains unknown.  Here we demonstrate that MV induces atime-dependent reduction in diaphragm blood flow which results in a greatlydiminished microvascular oxygenation. Further, MV after as little as 6 hr severely compromised the ability toincrease blood flow within the diaphragm during contractions.  Consistent with an O2 supplylimitation, MV after 6 hr resulted in a ~80 % reduction in O2 uptakeduring contractions compared to that achieved immediately after the onset ofMV, which would force reliance on non-oxidative energy sources and hasten diaphragmfatigue. We also provide clear evidence for vascular dysfunction (i.e. NitricOxide (NO)-mediated, structural alterations) as one potential mechanism thatcontributes to diminished diaphragm blood flow. These new and importantfindings reveal that prolonged MV results in a time-dependent decrease in theability of the diaphragm to augment blood flow to match O2 demand inresponse to contractile activity. To our knowledge this are the firstexperiments that sought to determine the effects of mechanical ventilation ondiaphragm oxygenation and vasomotor control.
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Behnke, Bradley Jon.
Statement of Responsibility:
by Robert Thomas Davis III.

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1 TIME D EPENDENT REDUCTION IN O2 SUPPLY AND UTILIZATION IN THE DIAPHRAGM DURING MECHANICAL VENTILATION: ROLE OF VASCULAR DYSFUNCTION By ROBERT THOMAS DAVIS III A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSI TY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Robert Thomas Davis III

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3 I would like to dedicate this work to my parents, friends and professors who have play ed a role in my educational growth. If it were not for your c ontinued support and enthusiasm I wouldnt be where I am today. Thank you for your love, understanding, and encouragement throughout my graduate career.

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4 ACKNOWLE DGMENTS I would like to acknowledge those who made the successful completion of this project possible: my mentor, Dr. Brad Behnke; and my committee members, Dr. Scott Powers, Dr. Thomas Clanton, Dr. Judy Delp, and Dr. Peter Adhihetty. Collectively and indi vidually, my committee provided me with invaluable guidance, instruction, and patience. In addition, I would like to acknowledge Dr. Christian Bruells for his invaluable contribution to this project. I would also like to give a special thanks to members of the laboratory: Danielle J. McCullough, John N. Stabley, Payal Gosh and Bei Chen for their enthusiastic participation assured the success of these experiments and also my research achievements.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 14 2 LITERATURE REVIEW .......................................................................................... 18 Brief Histo ry of Oxygen ........................................................................................... 18 Joseph Priestley (1733 1804) ........................................................................... 19 Antoine Laurent Lavoisier (17431794) ............................................................ 19 Carl William Scheele (1742 1786) .................................................................... 20 Oxygen Cascade: Atmosphere to Mitochondria ...................................................... 21 Oxygen Uptake: The Dy namic VO2 Response ....................................................... 22 Oxygen Transport and VO2; Does O2 Delivery Limit VO2? ..................................... 23 Diaphragmatic Microvascular Oxygenation (PO2m) .......................................... 26 Skeletal Muscle Resistance Vasculature ................................................................ 27 Mechanical Ventilation ............................................................................................ 30 History of MV .......................................................................................................... 31 Characteristics of the Diaphragm ............................................................................ 31 Mechanisms of MV Induced Diaphragm Weakness ............................................... 32 Skeletal Muscle Atrophy ................................................................................... 32 Contractile Dysfunction .................................................................................... 34 ROS Production ............................................................................................... 35 Xanthine oxidase ....................................................................................... 35 NO synthase .............................................................................................. 35 NAD(P)H oxidase ....................................................................................... 36 Mitochondrial oxidants ............................................................................... 36 Blood Flow and Oxygen Delivery to the Diaphragm with MV .................................. 37 Summ ary ................................................................................................................ 37 3 METHODS .............................................................................................................. 39 Statistical Analysis .................................................................................................. 40 General Experimental Protocols and Analyses ....................................................... 40 Animals ............................................................................................................. 40

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6 Mechanical Ventilation ..................................................................................... 41 Blood Flow ........................................................................................................ 41 Phosphorescence Quenching .......................................................................... 42 Calculation of diaphragm VO2 .................................................................... 43 Diaphragm contractions ............................................................................. 44 Isolated Microvessel Technique .............................................................................. 44 mRNA Analysis ....................................................................................................... 46 4 RESULTS ............................................................................................................... 48 Animals Weight, Hemodynamic Data, Arterial Blood Gases and pH ...................... 48 Diaphragm Blood Flow and V ascular Conductance are Diminished with Mechanical Ventilation ......................................................................................... 48 Mechanical Ventilation Reduces Resting Diaphragm Microvascular PO2 (PO2m) in a Time Dependent Manner .............................................................................. 51 Resting Diaphragm O2 Uptake (VO2) ...................................................................... 51 Mechanical Ventilation Reduces the Ability to Augment Diaphragm Blood Flow, Match O2 Delivery to Utilization, and Increase VO2 During Muscular Contractions ........................................................................................................ 52 Mechanical Ventilation Reduces NO Mediated Vasodilation in Diaphragm Resistance Arterioles ........................................................................................... 53 Pressure Diameter Relationship Is Altered in Diaphragm Arterioles Following Prolong Mechanical Ventilation ........................................................................... 54 5 DISCUSSION ......................................................................................................... 68 Mechanistic Basis for the Diminished Diaphragm Blood Flow Following Mechanical Ventilation ......................................................................................... 69 Implications from Altered PO2m Dynamics .............................................................. 72 Reduced O2 Delivery, Diaphragm VO2 and Cellular Energetics .............................. 73 Reduced PO2m and Cellular/Molecular Signals for Mitochondrial Dysfunction, Atrophy, and Autophagy ...................................................................................... 74 QO2 and VO2 During Contractions: Ramifications on the Weaning Process .......... 75 Future Directions .................................................................................................... 76 Summary ................................................................................................................ 78 LIST OF REFERENCES ............................................................................................... 80 BIOGRAPHICAL SKETCH ............................................................................................ 92

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7 LIST OF TABLES Tab le page 4 1 Body and diaphragm mass, blood basses and hematocrit. ................................ 55 4 2 Renal, respiratory and select hindlimb skeletal muscle blood flows. .................. 55 4 3 Microvascular PO2 dynamics across the rest to contractions transition after mechanical ventilation. ....................................................................................... 55

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8 LIST OF FIGURES Figure P age 4 1 Resting diaphragm muscle blood flow (A) and vascular conductance (B) measur ed during spontaneous breathing and after 30 min and 6 hr of MV ...... 56 4 2 Resting blood flow (A) and vascular conductance (B) to regionally delineated portions of the diaphragm muscle dur ing spontaneous breathing and after 30 min and 6 hr of MV ............................................................................................ 57 4 3 Resting diaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing and mec hanically ventilated rats after 30 min and 6 hr.. ........................................................................................ 58 4 4 Comparison of blood flow (A) and vas cular conductance (B) measured at rest between the diaphragm, red portion of the gastrocnemius muscle complex, soleus, and intercostal muscles dur ing spontaneous breathing and after 30 min and 6 hr of MV ............................................................................................ 59 4 5 Representative resting diaphragm microvascular PO2 profiles measured over time (A) and the average diaphragm microvascular PO2 (B) measured during spontaneous breathing and a fter 30 min and 6 hr of MV .. .................................. 60 4 6 Mean microvascular PO2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular PO2 profiles (B) in response to electrically stimulated muscle contractions a fter 30 min and 6 hr of MV .. ........... 61 4 7 Blood flow (A) and oxygen consumption (VO2) (B) at rest and during the steady state of electrically stimulated contractions in the diaphragm m uscle after 30 min and 6 hr of MV as compared to spontaneous breathing ................. 62 4 8 O2 delivery (A) and fractional O extraction (B) at rest and during electrically stimulated diaphragm muscle contractions after 30 min and 6 hr of MV as compared to spontaneous breathing ................................................................. 63 4 9 Flow mediated vasodilation in diaphragm arterioles duri ng spontaneous breathing and after 30 min and 6 hr of MV .. ....................................................... 64 4 10 Dose responses to the endothelial dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N G nitro l argine methyl ester ( L NAME) (A), Dose responses to the endothelial dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N G nitro l argine methyl ester (LNAME) + COX inhibitor Indomethacin (INDO) (B), NO dependent (endot helium dependent) dilation (max dilationACh max dilationACh + LNAME) (C), Dose responses to the endothelium independent vasodilator sodium

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9 nitroprusside (SNP) (D) in diaphragm arterioles duri ng spontaneous breathing and after 30 m in and 6 hr of MV ........................................................ 65 4 11 eNOS mRNA expression in diaphragm arterioles during spontaneous breathing and after 30 min and 6 hr of MV .. ....................................................... 66 4 12 Active and passive myogenic responsiveness in arterioles duri ng spontaneous breathing and following 30 min and 6 hr of MV .. ......... 67

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10 L IST OF ABBREVIATION S ADP ADENOSINE DIPHOSPHATE ATP ADENOSINE TRIPHOS PHATE cAMP CYCLIC ADENOSINE MONOPHOSPHATE cGMP CYCLIC GUANOSINE MONOPHOSPHATE COX CYCLOXYGENASE EDRF ENDOTHELIALDERIVED RELAXATION FACTOR EDHF ENDOTHELIALDERIVED HYPERPOLARIZING FACTOR eNOS ENDOTHELIAL NITRIC OXIDE SYNTHASE Gastred RED PORTION OF THE GA S TROCNEMIUS GTP GUANOSINE TRIPHOSPHATE iNOS INDUCIBLE NITRIC OXIDE SYNTHASE L NAME L NG NITROARGININE METHYL ESTER MAP MEAN ARTERIAL PRESSURE MV MECHAN ICAL VENTILATION NAD+ NICOTINAMIDE ADENINE DINUCLEOTIDE NADH REDUCED FORM OF NAD+ NO NITRIC OXIDE NOS NITI RIC OXIDE SYNTHASE ONOOPEROXYNITRITE PCr PHOSPHOCREATINE PGI2 PROSTACYCLIN Pi INORGANIC PHOSPHATE P O2m MICROVASCULAR OXYGENATION

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11 QO2 OXYGEN DELIVERY Q CARDIAC OUTPUT ROS REACTIVE OXYGEN SPECIES SNP Sodium Nitroprusside VO2 OXYGEN UPTAKE VIDD VENTILATORINDUCED DIAPHRAGM DYSFUNCTION VSMC VASCULAR SMOOTH MUSCLE CELL XO XANTHINE OXIDASE

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of D octor of Philosophy TIME DEPENDENT REDUCTION IN O2 SUPPLY AND UTILIZATION IN THE DIAPHRAGM DURING MECHANICAL VENTILATION: ROLE OF VASCULAR DYSFUNCTION By Robert Thomas Davis III December 2012 Chair: Brad Behnke Major: Health and Human Performance Mec hanical ventilation (MV) engenders several clinical complications associated with the duration of MV. One consequence of MV is the difficulty to successfully wean a large portion of patients from the ventilator. In skeletal muscle, a reduced O2 supply re sults in contractile dysfunction and premature fatigue when performing external work. However, whether MV induces an O2 supply usage imbalance in the diaphragm, which contributes to weaning difficulties, remains unknown. Here we demonstrate that MV induc es a timedependent reduction in diaphragm blood flow which results in a greatly diminished microvascular oxygenation. Further more, after as little as 6 hr of MV there is a severely compromised ability to increase blood flow within the diaphragm during co ntractions. Consistent with an O2 supply limitation, MV after 6 hr resulted in a ~80 % reduction in O2 uptake during contractions compared to that achieved immediately after the onset of MV, which would force reliance on nonoxidative energy sources and h asten diaphragm fatigue. We also provide clear evidence for vascular dysfunction (i.e. Nitric Oxide (NO) mediated, structural alterations) as a potentia l mechanism that contributes to the diminished diaphragm blood flow. These new and important findings

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13 re veal that prolonged MV results in a timedependent decrease in the ability of the diaphragm to augment blood flow to match O2 demand in response to contractile a ctivity. To our knowledge these are the first experiments that sought to determine the effects of mechanical ventilation on diaphragm oxygenation and vasomotor control.

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14 CHAPTER 1 INTRODUCTION Mechanical ventilation (MV) is used clinically as a lifesaving intervention to sustain adequate pulmonary gas exchange in patients that are incapable of maintaining sufficient alveolar ventilation (e.g., patients in respiratory failure, coma, and drug overdose). The removal of patients from MV is termed weaning and problems in weaning are extremely common. The inability to wean patients from MV results in in creased risk of morbidity and mortality along with higher health care costs to patients, insurance companies, and hospitals. Furthermore, long term mechanical ventilation results in perturbation in diaphragm function, collectively known as ventilator induc ed diaphragm dysfunction (VIDD; Vassilakopoulos et al., 2004). The physiological mechanisms responsible for VIDD are unclear but likely multifaceted. Previous studies have indicated increased reactive oxygen species (ROS) production, mitochondrial damage, skeletal muscle atrophy, and muscle fiber remodeling as potential contributors to weaning failure (GayanRameriz et al., 2002, Kavazis et al., 2009, Sassoon et al., 2002, Sieck et al., 2008). However, one aspect of VIDD that has not been investigated is the impact of prolonged MV on diaphragmatic blood flow, oxygen delivery and vasomotor control In this regard, it is established that during acute MV (e.g. 30 minutes) blood flow to the diaphragm is decreased (Robertson et al., 1977, Uchiyama et al., 200 6, Virres et al., 1983, Hussain et al., 1985). Recently, our group has expanded these observations and our data reveal that extending MV from 30 minutes to 6 hr results in an addit ional decrease in diaphragmatic blood flow However, at present, there are n o published reports regarding the impact of longer periods of MV (i.e., > 3 hrs) on blood flow, oxygen delivery to the diaphragm, or vasomotor control of the

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15 resistance vasculature (i.e., arterioles). Improving our knowledge of MV induced changes in blood flow and oxygen delivery to the diaphragm will advance our understanding of how these variables contribute to VIDD and weaning difficulties. Therefore, the overall objective of this project was to investigate the temporal pattern of MV induced alterations in diaphragmatic blood flow/oxygen delivery and investigate mechanism(s) responsible for decrements in diaphragmatic blood flow and oxygen delivery as well as putative mechanism(s) responsible for decreme nts in diaphragmatic blood flow Our central hypothesis is that prolonged MV results in a progressive decline in diaphragmatic blood flow and oxygen delivery resulting from impaired vasomotor function in resistance arteries due, in part, to decreased production/bioavailability of nitric oxide (NO). Our cent ral hypothesis was tested in four highly integrated specific aims using a well established animal model of MV. Specific Aim 1: To determine whether diaphragm blood flow decreases as a function of time during MV. Rationale : Previous work has demonstrated a significant reduction in diaphragm blood flow immediately after the onset of MV due to the decreased metabolic activity of the diaphragm (Robertson et al., 1977, Uchiyama et al., 2006, Virres et al., 1983, Hussain et al., 1985). ROS generation in the diaphragm is significantly elevated after 6 hours of MV (i.e. prolonged MV) and an increased superoxide generation within the diaphragm vasculature would likely reduce the bioavailability of NO (Kang et al., 2009). NO has been demonstrated as a key regulator o f resting arterial tone (Hirai et al., 1994, Schrage et al., 2007) and a reduced bioavailability of NO after prolonged MV would likely diminish resting diaphragm blood flow.

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16 Hypothesis : Diaphragm blood flow will be signi ficantly reduced following 6 hr MV versus blood flow measured immediately after the onset of MV. Specific Aim 2: To determine the effect of prolonged MV on the matching of diaphragmatic O2 deliveryto O2 consumption (QO2VO2) at rest and during contractions ; this will be accomplished through the measurement of microvascular P O2 ( P O2m). Rationale : In order to sustain diaphragmatic metabolic and contractile function there must be a tight coupling between the rates of O2 delivery (QO2) to that of O2 utilization (VO2) (Poole et al., 2001). A n imbalance in either of these variables may force an increased reliance on nonoxidative energy sources and, consequently, respiratory muscle failure (for Rev see (Poole et al., 1997)). At present, we are unaware of any other investigations determining w hether the duration of MV affects diaphragm O2 delivery at rest, or induce alterations in the capacity to augment O2 delivery with muscular contractions (e.g., during weaning). Hypothesis : Following 6 hr MV there will be a diminished ability of the diaphragm to augment O2 delivery rapidly during electrically induced muscle contractions resulting in a reduced P O2m. Specific Aim 3: To determine how prolonged MV modifies resistance artery function (i.e. vasomotor control). Rationale : Previous work demonstrates that changes in shear stress through resistance arterioles can alter vasomotor control (e.g., flow induced dilation) in as little as 4 hours (Woodman et al., 2005). Therefore, in tissues that normally sustain a high blood flow, a reduced shear stress asso ciated with inactivity may rapidly alter vascular

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17 control (e.g. blunted endothelial dependent vasodilation). With a prolonged diminished blood flow (and thus shear stress) to the diaphragm during MV (versus that of spontaneous breathing) we would expect a down regulation of vasodilatory pathways similar to that observed in other skeletal muscle models of disuse (McCurdy et al., 2000). In addition, we wanted to test whether prolonged MV results in structural alterations that have been demonstrated in other m odels of inactivity ( i.e. bed rest, Demiot et al., 2007). Hypothesis : In diaphragm resistance arteries (i.e. arterial branch which provides highest vascular resistance to blood flow) endothelium dependent vasodilation, and passive diameter responsiveness will be diminished after prolonged MV. Specific Aim 4: To determine whether eNOS mRNA expression is reduced with time of MV. Rationale : Prolonged MV will result in a diminished blood flow (and thus shear stress) an d stimulate a reduction in mRNA expression of eNOS following 6 hr MV compared to that of spontaneous breathing. Hypothesis : There will be a time dependent reduction in eNOS mRNA and protein expression in diaphragm resistance arterioles.

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18 CHAPTER 2 LITERATURE REVIEW Being the primary muscle of vent ilation, normal diaphragm function is requisite for breathing. In this regard, it is clear that any perturbation to diaphragm muscle function will have a negative impact on the o verall health of the individual Because MV has been associated with deleter ious effects on respiratory muscle function, elucidating the mechanisms of VIDD is crucial. One aspect of VIDD that remains unknown is the effect of MV on diaphragm muscle oxygen supply. It follows that obtaining new insights regarding diaphragm oxygenation during prolong MV is important and will help us develop therapeutic strategies to combat VIDD Hence, this forms the rationale for the experimental design within this proposal. In this literature review, the discovery of oxygen will first be described, followed by our current understanding of O2 transport and the pathways involved therein. To study diaphragm O2 exchange we will measure blood flow, muscle oxygenation, and vascular properties of diaphragm arterioles, all of which will be discussed. After the foundation of these measures is established, the unique characteristic of the diaphragm and the effects of MV will be discussed followed by a short summary. Brief H istory of O xygen To many chemists and physiologists oxygen is regarded as the elixir of life being the essential element for all living things. In Scandinavia, students are taught that Swedish apothecary Carl William Scheele discovered oxygen in 1772, although his publication is dated 1777. In America, it is taught that English Unitarian minister and chemist Joseph Priestly discovered oxygen on August 1st, 1774 and personally informed the French chemist Antoine Lavoisier in Paris in September of the same year.

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19 Lavoisier realized this gas was a new element which he termed oxygen (Roach et al., 2003). Despite the contentious history behind the identification of oxygen, it is regarded as the most important discovery in chemistry (Roach et al., 2003). This section will provide a brief background of each of the three scientists and their cont ribution to the discovery of this vital element. Joseph Priestley (17331804) Joseph Priestly was born near Leeds, England in 1733 and was the oldest of six children. Initially he was a successful cloth dresser (Schofield, 1997) and later became a self tau ght chemist and teacher at Warrington Academy (Severinghaus, 2002). Priestly was known to be very outspoken and a ferocious, freethinking philosopher (Schofield, 1997). On August 1st, 1774, he discovered a gas was liberated when he heated the mineral red mercuric oxide in a sealed glass chamber. In the presence of this gas, he demonstrated that a candle would burn more brightly and a mouse could live longer in this chamber compared to a similarly sealed volume of air. He stated that this gas is much bette r than common air (Comroe, 1977). Interestingly, Priestley also discovered photosynthesis by demonstrating that a mint leaf left in the air in which the mouse had died regenerated the substance needed to keep a mouse alive. In September 1774 Priestly was invited to Paris by Antoine Laurent Lavoisier to present his findings to a group of distinguished French scientists. As a consequence of this meeting, Lavoisier began his own experiments and it is thought that this visit was the critical catalyst of Lavoi siers revolution of modern chemistry (Roach et al., 2003). Antoine Laurent Lavoisier (1743 1794) Antoine Lavoisier was born in Paris, France. His parents had a strong educational background in science, philosophy, literature, and law. Lavoisier was a man

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20 of many professions. He was primarily a chemist but also a statesman, financier, ec onomist manufacturer, and landowner (Poirier, 1993). Following Priestleys visit, Lavoisier repeated Priestleys experiments using more elaborate techniques. During this t ime, it is speculated that Carl William Scheele had also written a letter to Lavoisier asking him to repeat his studies (completed in 1771) using his more elaborate burning lens (Roach et al., 2003). Lavoisier later published his discovery and termed thi s eminently breathable air oxygen in 1775 (West, 1980; West, 1996). Collectively, Lavoisiers work and reinterpretation of previous findings revolutionized the scientific world. Carl William Scheele (17421786) Although Priestley and Lavoisier are us ually credited with the discovery oxygen, Carl William Scheele, a Swedish apothecary, may have generated this element as early as 1771 (Severinghaus, 2002). Scheele was one of eleven children and received very little formal education. In 1770, he became a lab assistant at Uppsala University in Sweden. During his tenure there Scheele discovered oxygen by heating MnO2 with H2SO4 (Oseen, 1942). Scheele wrote a book On Air and Fire describing his findings but unfortunately it was not published until 1777. A couple of reasons for his delay in the publication of his book were: 1) at this time he failed to fully understand how important this discovery was and 2) he wanted to publish a book containing all his discoveries collectively versus publishing independent papers (Severinghaus, 2002). Undoubtedly, the debate regarding who discovered oxygen will always remain. What is important to understand is that each individual scientist contributed in their own way to this indispensable discovery. Whether it was the first person to isolate and collect pure O2, differentiate it from other species of gases, or demonstrate that it is

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21 requisite for combustion; the discovery of oxygen was dependent on all three of these remarkable scientists. Oxygen C ascade: Atmosphere to Mitochondria As mentioned previously, an adequate supply of oxygen is needed to sustain normal cellular functions. The processes involved in the transport of oxygen from the air to the cellular mitochondria are well defined (Weibel, 1984). Briefly, the maj or steps include the convective movement of atmospheric oxygen (via the act of breathing) down the airways to the alveoli. Thereafter, the oxygen undergoes alveolar gas mixing via diffusion and convective forces from the lungs. Following this process, oxy gen diffuses out of the alveolar gas and into the pulmonary capillaries. This step is passive and dependent on the magnitude of the diffusive gradient of O2 and the physicochemical properties of the alveolar membrane. The fourth step in this cascade is the convective transport of oxygen to the peripheral tissues and organs. Finally, oxygen diffuses from the microvasculature into the tissue and ultimately the mitochondria. This oxygen cascade is dependent on a sufficiently high P O2 gradient to sustain diffus ion from the atmosphere to the mitochondria. Currently, the mechanism(s) responsible for skeletal muscle fatigue are complex and not completely understood. There is compelling evidence demonstrating each step in the oxygen cascade can modify oxygen transport and can cause impaired muscle function (Roca et al., 1989; Pugh et al., 1964; Powers et al., 1989; Buick et al., 1980; Saltin, 1985). As a result, the current notion is that all steps act in an integrated fashion, and a disturbance to any of the steps i n the cascade will result in a reduction of total O2 transport and skeletal muscle fatigue (Wagner, 1995). The following section will very

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22 briefly summarize the evidence that supports the notion that skeletal muscle oxygen delivery is a major factor in det e rmining exercise tolerance. Oxygen Uptake: The Dynamic VO2 R esponse In the conscious human, we are rar ely in a resting metabolic steady state ; rather we are continuously cycling between different metabolic demands. Therefore, a greater understanding of the metabolic control and dynamics of oxygen exchange is of great benefit to physiologists and clinicians alike. The speed of VO2 response (i.e. kinetics) provides unique insight into skeletal muscle energetics and substrate utilization. For example, faster VO2 kinetics across the rest to exercise transition limit reliance on short term energy sources (e.g. glycolysis and phosocreatine (PCr) which can ultimately impair skeletal muscle contractile function (e.g excess proton formation from accelerated glycoly sis). On the contrary, slower VO2 kinetics will mandate a greater disturbance of the intracellular milieu (e.g. H+, PCr, ADPfree and inorganic phosphate) and deplete finite glycogen stores causing impaired contractile function and muscle fatigue. Historica lly, oxygen uptake (VO2) has been predominantly measured at the level of the mouth, allowing for the characterization of pulmonary VO2 kinetics. However, in order to investigate how the muscle responds to contractions, mitochondria located within the muscl e must be studied. However in vivo measurements of mitochondria function are not currently technically feasible. At exercise onset, ATP demand increases in a square wave fashion whereas the pulmonary VO2 response displays a finite kinetic response (Barst ow et al., 1994). There are three distinct phases of the pulmonary VO2 response in moderate intensity exercise. Phase I constitutes an increase in cardiac output (Q; Casaburi et al., 1989) and

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23 augmented perfusion through the pulmonary vasculature. This ph ase is dependent upon the cardiopulmonary system (Wasserman et al ., 1973) and is not indicative of the muscle VO2 response (Grassi et al., 1996). Phase II represents the arrival of desaturated venous blood from the exercising muscle, and reflects temporal ly the increase in oxygen consumption at the level of the muscle (Whipp & Mahler, 1980; Barstow et al., 1990, Grassi et al., 1996). The final phase (phase 3) represents the steady state in oxygen consumption. Oxygen Transport and VO2; Does O2 Delivery L im it VO2? The regulation of VO2 kinetics (i.e. the dynamic transition of VO2 upon the initiation of exercise) during muscular contractions has been an area of debate since A. V. Hills work in the early 1900s. The two main hypotheses for the regulation of VO2 are that 1) there is an inherent inertia within the metabolic machinery of the muscle (Whipp and Mahler, 1980., Grassi et al., 1996, Behnke et al., 2001) and 2) O2 delivery regulates the speed at which VO2 can increase (Hughson et al., 1991, 1993). More r ecently, it has become apparent that these two hypotheses are not mutually exclusive and the regulation of VO2 kinetics is highly dependent on the health status of the individual and the paradigm with which VO2 is measured. As our central hypothesis is tha t the diaphragm displays a greatly reduced O2 delivery after MV, the evidence of an O2 delivery limitation to VO2 will be discussed. The notion of an O2 transport limitation to VO2 kinetics has been pioneered by Dr. Richard L. Hughson (Hughson et al., 1 991, Hughson et al., 1993, Hughson et al., 1997) and others (Karlsson et al., 1975, Convertino et al., 1984, Hortsman et al., 1976). Peripheral tissue oxygen delivery can be calculated as the product of cardiac output (Q) and arterial oxygen content (CaO2) and is an indicator of the rate of convective transport

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24 of oxygen to the tissues. In addition, there is a strong linear relationship between VO2 (i.e. oxy gen consumption) and O2 delivery (Wagner, 1995). An example of how an alteration in VO2 kinetics and thus muscle function might be related to a change in O2 transport is provided by examining the effects of supine exercise and heavy intensity priming exercise. In the supine position, the hydrostatic gradient for muscle perfusion due to gravity is reduced and muscle O2 availability may be compromised (Hughson et al., 1993, Hughson et al., 1991, MacDonald et al., 1998). Indeed, it has been demonstrated in the submaximal domain, pulmonary VO2 kinetics at exercise onset are slowed in the supine position (i.e. reduction in the pressure head for muscle O2 delivery during supine exercise in which the gravitational assist to muscle blood flow is absent) (Hughson et al., 1991, Macdonald et al., 1998, Jones et al., 2006) and when performing rhythmic forearm exerc ise with the arm elevated above the heart compared to when the arm is places below the hear (Hughson et al., 1997). Under such conditions priming exercise accelerated blood flow dynamics during a subsequent bout of exercise and removed possible constrai nments of O2 delivery to VO2 and sped VO2 kinetics ( Jones et al., 2006). Similar to priming exercise lower body negative pressure, which increases the perfusion gradient for blood flow from the heart to the contracting muscle returns VO2 kinetics toward that of upright position (Hughson et al., 1993). Furthermore, Jones and colleagues (2006) demonstrated that prior heavy exercise resulted in a speeding of VO2 kinetics in the supine position. It is noteworthy to mention that slower VO2 kinetics (seen with supine exercise) can arise via: 1) slower dynamics of O2 utilization at the level of the myocyte (i.e. inherent inertia in the metabolic contractile machinery), or 2) a reduction in O2 transport.

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25 In this regard, it has been demonstrated that the activity of the sympathetic nervous system (SNS) is attenuated in the supine position (Saul et al., 1991). This diminished SNS may lead to an increased perfusion of nonexercising tissue (i.e. cutaneous circulation, splanchnic regions) and affect the dynamic matching of O2 delivery to consumption in the active tissue. As mentioned above, VO2 kinetics are slower during forearm exercise performed above the heart, as compared to below or at the level of the heart. In this position blood flow may be compromised at exerci se onset, and therefore this exercise paradigm is analogous to supine exercise (Hughson et al. 1993). Oxygen delivery/supply regulates muscle function in numerous conditions such as during strenuous exercise in the healthy individual. All levels of the respiratory system and oxygen cascade present some inherent level of limitation (e.g. convective oxygen transport to muscle, diffusive O2 conductance, and rate of O2 consumption in the myocytes). Therefore, in order to fully understand the mechanism(s) responsible for skeletal muscle fatigue, it is necessary to assess cellular metabolism as close to the site of O2 utilization as possible (i.e. within the microcirculation). Microvascular Oxygenation ( P O2m) As arterial blood transits from the central circulati on to the periphery, oxygen exits through the capillary wall by an oxygen diffusive gradient that extends from the red blood cell all the way to the mitochondria (Tsai et al., 2007). There have been many different techniques utilized to measure skeletal muscle oxygenation (e.g., microelectrodes, near infrared spectroscopy). However, these techniques have had several limitations (e.g. electrodes cause damage to the microvascular environment and m uscle fibers). In the late 1980s, David Wilson and colleagues ( Vanderkooi et al., 1987, Rumsey et al., 1988) developed a technique that allows the indirect measurement of

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26 microvascular P O2m (which is indicative of these driving forces). This noninvasive technique, known as phosphorescence quenching, utilizes palladiu m porphyrin compounds injected into the circulation. Following excitation of these compounds, the only molecule that can quench phosphorescence is oxygen ( Rumsey et al., 1991). Once the lifetime decay has been determined, a simple manipulation of the SternVolmer relationship allows for t he calculation of microvascular P O2 ( Rumsey et al., 1991 Wilson et al., 1991). This technique has since been revised to examine muscle microvascular P O2 in vivo and is detailed in the methods section. Diaphragmatic M icrova scular O xygenation ( P O2m) The diaphragm is a unique skeletal muscle in that it is continuously active and has a higher oxidative capacity than most other skeletal muscle (for exception see red gastrocnemius; Hoppeler et al., 1981, Poole et al., 2000, Poole et al., 1992, Delp and Duan., 1996, Powers et al., 1996). However, similar to other skeletal muscle, the diaphragm can become fatigued during exercise (Johnson et al., 1993) and with chronic diseases (e.g. emphysema) (Poole et al., 2001). This fatigue ultimately leads to respiratory muscle failure and exercise intolerance. David Poole and colleagues (1995) were the first to demonstrate that phosphorescence quenching is a feasible tool to measure P O2m in the diaphragm. Since this seminal study, there have been other investigations that sought to determine the P O2m in the diaphragm in healthy and diseased conditions (e.g. emphysema; Poole et al., 2001). Geer and colleagues (2002) demonstrated that the diaphragm has a higher resting P O2m (i.e. driving pressure for oxygen) than other skeletal muscle (e.g. spinotrapezius). I n addition, at the onset of muscular contractions, the kinetics of P O2 in the diaphragm are more rapid than other skeletal muscle (i.e. faster oxygen exchange dynamics). A higher P O2 in the diaphragm

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27 is beneficial as it would reduce the degree of intracellular perturbations necessary to achieve a given mitochondrial ATP flux (Wilson et al., 1977), and conserve limited glycogen stores. Skeletal Muscle Resistance V asculature As P O2m is dictated by the balance of QO2to VO2, mechanisms which regulate the former, primarily the resistance vasculature, will be discussed. The most mechanistic way to acutely regulate changes in peripheral skeletal muscle blood flow is through changes in the vasomotor tone of the resistance vasculature. Vascular tone is controlled by a balance between cellular signaling pathways (e.g. neural, humoral, and metabolic) that mediate either vasoconstriction or vasodilation and ultimately the internal diameter of the blood v essel. This section will focus on the structural conformation of the resistance arterioles and how each component interacts functionally to control vascular diameter and regulate blood flow. Arterioles can be defined as the primary resistance vessels that enter an organ to distribute blood flow into capillary beds and provide in excess of 80% of the resistance to blood flow in the body (Martinez Lemus, 2011). Consequently, they are vital in the regulation of hemodynamics, regional distribution of blood flow (Chri stensen et al., 2001, Meininger et al., 1984) and the maintenance of arterial pressure. Anatomically, the vessel wall of arterioles consists of three structurally distinct layers, namely the tunica intima, media, and adventitial layer (Rhodin et al. 1967). The intima layer of resistance arterioles is predominantly composed of endothelial cells. These endothelial cells play a role in the control of vascular tone by the production and release of vasoactive factors that exert their action on neighbori ng smoo th muscle cells (Martinez Lemus, 2011). Endothelial cells are arranged

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28 longitudinally and in the direction of flow (Rhodin, 1980), and a large amount of evidence indicates that these cells have the capacity to sense and transduce mechanical forces and pr oduce vasoactive compounds (Lui et al., 2008, Su et al., 2002, Brum et al., 2005, Loufrani et al., 2008). The contribution of endothelium derived products in regulating blood vessel tone is well recognized [e.g. nitric oxide (NO), endothelium derived hyperpolarizing factor (EDHF), prostaglandins (PGI2), endothelinvasoactive compound released from the endothelium is NO. NO is a relatively stable gas, with the ability to easil y diffuse through the cell membrane and interact with various substances in the cell (Nathan et al., 1994). NO stimulates guanylate cyclase in the same cell or in a target cell, which converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) causing the concentration of cGMP to rise; and lead to vascular smooth muscle relaxation (Vane et al., 1990). Another important vasoactive compound that relaxes the vascular smooth muscle is EDHF. However, at present, this factor has not been fully identified. On the contrary, there are at least three vasoconstrictor substances that are released by the endothelium including endothelin, prostanoids and thomboxane (Rubanyi et al., 1985). Endothelin1 is the most potent of these vasoconstrictors and was discovered in 1988 (Yanagisawa et al., 1998). Its release can be initiated by numerous substances such as thrombin, epinephrine, and interleukin1 (Yanagisawa et al., 1988). Endothelin 1 is the most active pressor substance discovered, with potency 10 times that of angiotensin II (Vane et al., 1990).

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29 The tunica media, or medial layer of arterioles, is primarily composed of vascular smooth muscle cells (VSMC) and an internal elastic lamina (Martinez Lemus, 2011). VSMCs activity are primarily regulated by changes in intracellular calcium concentrations, activation of myosin light chain kinase, and increase in the phosphorylation of the regulatory light chains (Kamm and Stull, 1985, Sommerville and Hartshorne, 1986, Hai and Murphy, 1989). In addition, it has been established that an increase in transmural pressure produces membrane depolarization of VSMC and ultim ately vasoconstriction (Harder et al., 1984). As stated previously, intracellular calcium levels play a key role in regulating vascular tone (Mis siaen et al., 1992). Calcium influx occurs predominantly through voltagegated and receptor o perated calcium channels (Hurtw itz et al., 1986), and calcium efflux occurs via the sarcolemmal calcium pump and Na+/Ca2+ exchanger (Missiaen et al., 1992). An inc rease in cAMP or cGMP will induce VSMC relaxation by decreasing intracellular calcium via activation of potassium channels (Nelson et al., 1990) or via the sarcolemmal calcium pump and calcium uptake by the sarcoplasmic reticulum (Missiaen et al., 1992). The adventitial layer consists of fibroblasts embedded in an extracellular matrix made of thick bundles of collagen (Rhodin, 1967, Sangiorgi et al., 2006) and elastic fibers which allow the resistance arterioles to elongate and recoil (Martinez Lemus, 2011). Traditionally, it has been thought that the role of the adventitial layer was to provide structural support only. However, current evidence suggests that adventitial fibroblast are capable of producing ROS that can modulate the activity of smooth muscle cells and initiate vascular remodeling (Haurani et al, 2007). In addition, adventitial fibroblast can produce other growth factors and vasoactive compounds (e.g.

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30 TGF 1) and regulate vascular tone (Di Wang, et al., 2010). Therefore, at present, it appears that the adventitial layer has a higher level of plasticity than previously thought. Mechanical Ventilation The overall objective of the research herein is to elucidate the mechanism(s) of prolonged MV induced alterations in diaphragmatic ox ygen delivery and utilization. We also want to learn how these variables may contribute to diaphragm muscle weakness and the diminished ability to successfully wean patients from the ventilator. We will utilize a highly integrative approach (e.g. blood flow, oxygen transport, microvascular oxygenation, vasomotor control) to investigate events occurring within the diaphragm during prolonged MV. MV is an intervention utilized to sustain adequate alveolar ventilation in patients who are incapable of doing so on their own (e.g. spinal cord injury, drug overdose, surgery, and unconsciousness). The process of removing patients from MV is termed weaning. Unfortunately, there are many problems associated weaning and this can account for more than 40% of the tot al time spent on the ventilator (Esteban et al., 1994). At present, the precise mechanism responsible for weaning difficulties is not known, however, there are numerous studies that suggest MV induced diaphragmatic weakness is due, in part, to muscle atro phy, contractile dysfunction, and increased reactive oxygen species (ROS) production (GayanRameriz et al., 2002, Kavazis et al., 2009, Sassoon et al., 2002, Sieck et al., 2008). In order to focus the scope of this section, the remainder will provide a br ief discussion of the history of MV and also discuss our current knowledge and understanding of MV induced diaphragmatic weakness. Furthermore, it will provide a brief overview of new evidence collected by

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31 our laboratory supporting the notion that a reduc tion in diaphragmatic oxygen delivery is one possible mechanism responsible for respiratory muscle weakness and weaning difficulties. History of MV Claudius Galenus (129 AD circa 200 AD), better known as Galen, was a prominent Roman physician. It is thought that he is one of the first to describe the artificial ventilation of an animal (Galen, 1954). In 1543, Andreas Vesalius, also described the importance of ar tificial ventilation by stating that life may be restored to the animal; an opening must be atte mpted in the trachea, into which a tube or reed can be put; you will then blow into this, so that the lungs may rise again and the animal take in air (Chamberlain, 2003, Vesalius, 1543). Beginning in the 1800s, there was the development and use of negativ e pressure ventilation (as opposed to positivepressure ventilation). This gave rise to the first use of an Iron Lung, which occurred in 1928 (Colice, et al., 1994). This type of ventilation was responsible for saving numerous lives during the poli omyel itis epidemics in the 1940 s (Colice, et al., 1994). Collectively, it is easy to comprehend how the use of MV can have profound importance in the health field and be an indispensable lifesaving tool in certain clinical populations. Unfortunately, the dele terious effects of MV often arise and increase patient morbidity and mortality. Characteristics of the D iaphragm The largest muscle involved in mammalian ventilation is the diaphragm. It accounts for up to 75% of the work of breathing during normal respiration and unlike other skeletal muscle is continuously active throughout life. The diaphragm is a highly plastic tissue, and therefore undergoes distinct adaptations to meet physiologic or

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32 pathophysiologic demands (Poole et al., 1997). The diaphragm is composed of three distinct regions, including the central tendon (noncontractile segment), and the costal and crural regions (muscular portions). The fibers of the peripheral muscular regions radiate towards the central tendon. In addition, the costal and crural segments have different embryologic origins, segmental innervation, and functional attributes (De troyer et al., 1988, Duron et al., 1979). The metabolic characteristics of the diaphragm reflect its high tonic contractile activity. In the rat, the diaphragm is composed of four different major muscle fiber types (i.e. I, IIa, IId/x, and IIb) which are heterogenously distributed (Delp and Duan, 1996, Powers et al., 1996). In addition, the oxidative capacity of the muscle fibers in the diaphragm is higher than most other hindlimb skeletal muscle (Delp and Duan, 1996, Powers et al., 1992, Powers et al., 1990, Sexton et al., 1995, Sieck et al., 1987).The diaphragm also expresses a regional distribution of blood flow, with the medial and dorsal costal regions accounting for most of its blood flow (Sexton et al., 1995). Other unique characteristics of the diaphragm include; differential vasomotor regulation (Aaker and Laughlin, 2002), prevalence of counter current capillary flow (Kindig & Poole, 1998), and higher capillarity compared to other hindlimb skeletal muscle (Hoppeler et al., 1981, Poole et al., 1992, Reid et al. 1992). Mechanisms of MV Induced Diaphragm Weakness Skeletal M uscle A trophy MV is a method of reducing or removing the work of the diaphragm It is one of several experimental models and clinical conditions which result in skeletal muscle atrophy (e.g. hindlimb unloading, immobilization, prolonged bed rest). Atrophy of the diaphragm has been observed in both animal and human experiments follow ing MV

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33 (Anzueto et al., 1997, Bernard et al., 2003, GayanRamirez et al., 2003, Knisely et al., 1988, Le Bourdelles et al., 1994, Levine et al., 2008, Shanley et al., 2002, Yang et al., 2002). MV induced diaphragmatic atrophy is unique in that it occurs more rapidly (i.e. within 12 hrs) when compared to disuse atrophy seen in other hindlimb skeletal musculature (Le Bourdelles et al., 1994, McClung et al., 2007, Yang et al., 2002). The rate of skeletal muscle atrophy is dependent on a decrease in protein sy nthesis (Ku et al., 1995); an increase in prot ein degradation (Bodine et al., 2001); or due to a combination of both of these variables. Work from Dr. Scott Powers laboratory has demonstrated a reduction in protein synthesis in as little as 6 hr MV (Shanl ey et al., 2004). In addition, reductions in insulinlike growth factor and myosin heavy chain (i.e. type I and IIx) are evident after 1224 hrs of MV (GayanRamirez et al., 2003, Shanley et al., 2004). However, at present, it is believed that that rapid onset of diaphragmatic atrophy is primarily due to an increase in proteolysis (DeRuisseau et al., 2005, McClung et al., 2007, Shanley et al., 2002). The three primary pathways involved in skeletal muscle proteolysis are: 1) lysosomal proteases (i.e. cathespins), 2) calcium actives proteases (i.e. calpains), 3) and the protesome pathway. In regards to protease activation in the diaphragm following MV; it appears lysosomal proteases do not play a dominant role in the MV induced skeletal muscle degeneration. Co ntrarily, calpains/caspases and the proteasome pathways have been shown to contribute to the muscle protein breakdown seen with MV ( Shanely et al., 2002, DeRuissea et al., 2005). Specifically, elevated levels of intracellular calcium (seen with inactivity) can activate both calpain and caspases, which are responsible for sarcom eric protein release (Communal et al., 2002,

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34 Koh et al., 2000, Purintrapiban et al., 2003). This is thought to be the initial step in muscle protein loss during MV induced diaphragmat ic atrophy. The ubiquitin proteasome pathway is the major proteolytic pathway responsible for skeletal muscle protein breakdown and muscle atrophy following release for myofibrallar proteins. The total proteasome complex (26S) consists of a core subunit (2 0S) coupled with a regulatory complex (19S) at each end of the core subunit (Grune et al., 2003, Hasselgren et al., 1997). The coordinated action of this threeenzyme system is requisite for skeletal muscl e protein breakdown (DeRuissea et al., 2005). Furth ermore, evidence indicates there is an increase in 20S proteasome activity following MV (Shanley, et al., 2002, DeRuissea, et al., 2005). Contractile D ysfunction Utilizing a variety of animals models (i.e. rats, rabbits pigs, baboons), it has been demonstr ated that MV results in contractile dysfunctio n (Anzueto et al 1997, Sassoon et al., 2002, Zhu et al., 2005, Shanely et al., 2003, Powers et al., 2002, Criswell et al., 2003) which occurs in a time dependent manner (Powers et al., 2002). For example, it has been demonstrated that there is a significant reduction (~20%) in diaphragmatic specific force in as little as 12 hrs of MV (Powers et al., 2002, Criswell et al., 2003). It is noteworthy that decrements in force cannot be attributed to atrophy alone because the force of the diaphragm was normalized to the cross sectional area. In addition, it appears the effects of MV are confined to the diaphragm, as there were no changes in sole us skeletal muscle mass (Powers et al., 2002). Interestingly, GayanRamirez and colleagues (2002) demonstrated that intermittent spontaneous breathing during MV can retard contractile dysfunction. These results provide supporting evidence that diaphragm inactivity plays a fundamental role in promoting weaning failure.

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35 ROS P roducti on It is well established that ROS are produced in inactive skeletal muscle thanks to the seminal studies by Kondo et al. (1991, 1992, and 1993). Given the diaphragm is inactive during controlled MV (Powers et al., 2002, Sassoon et al., 2004), the potenti al for ROS production clearly exists. When cellular oxidant production exceeds the capacity of intracellular antioxidants to scavenge these oxidants, oxidativestressinduced cellular injury can occur. In this regard, it has been previously demonstrated th at there is oxidative injury in the diaphragm during MV (within as little as 6 hr after the onset of MV) (Falk et al., 2006, Shanely et al., 2002, Zergeroglu et al., 2003). At present, numerous ROS producing pathways exist in the cell and may contribute i ndependently or cooperatively to myocyte damage including the following: 1) xanthine oxidase; 2) production of NO via nitric oxide synthase (NOS); 3) NADPH oxidase; and 4) mitochondrial production of oxygen radicals. Xanthine oxidase Xanthine oxidase (XO) is produced via sulfhydrydryl oxidation or proteolysis of xanthine dehydrogenase by calcium activated proteases (i.e. calpain) (Halliwell, et al 1999). XO catalyzes the formation of superoxide radicals and uric acid in the presence of oxygen and purine substrates (i.e. hypoxanthine, xanthine). Superoxide radicals can then lead to the formation of other damaging reactive species (e.g. peroxynitrite (ONOO)) (Halliwell et al., 1999). Nonetheless, it is unclear whether if XO induced production of oxidants in t he diaphragm contributes to muscle dysfunction. NO synthase The free radical NO is produced v ia the enzyme NO synthase (NOS), and can lead to the formation of other damaging reactive nitrogen species (e.g. ONOO ). There

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36 are three isoforms of NOS that exist s (Kaminski et al., 2001, Stamler et al., 2001) which include: 1) type 1 or neuronal (i.e. nNOS), which is calcium activated, 2) type II or inducible (i.e. iNOS), which is calcium independent, and 3) type III or endothelial (i.e. eNOS), which is also calci um activated. Both nNOS and eNOS are expressed in the diaphragm (Stamler et al., 2001, Van Gammeren et al., 2007). The formation of nitrogen species is associated with cellular injury including mitochondrial dysfunction, lipid peroxidation, and nitrosylati on of proteins (Barreiro et al., 2002, Nin, et al, 2004, Stamler et al., 2001, Supinski et al 1999). At present it appears that NO may not play a role in MV induced diaphrag matic dysfunction (Van Gammeren et al., 2007), however, further studies are warranted. NAD(P)H oxidase NAD(P)H oxidase is a membraneassociated enzyme that catalyzes the oneelectron reduction of molecular oxygen into superoxide using NADPH or NADH as the electron donor (Javesghani et al., 2002). It has been shown that numerous factors such as the calcium sensitive PKC ERK1/2 pathway can increase NAD(P)H oxidase activity in cells (Hazan et al.,1997). Given that skeletal muscle inactivity (e.g. MV) causes an increase in calcium, one could assume that NAD(P)H activity would increase and b e a possible source of oxidant production. Mitochondrial oxidants The primary function of the mitochondria is to produce ATP; however, its also well known that electron leakage from the electron transport chain (via complex I and III) can result in format ion of superoxide and subsequently hydrogen peroxide (Andreyev et al., 2005; Cadenas et al., 2000; Powers et al., 2005). Clearly, the mitochondria have to ability to alter cellular redox balance. Previously, it was thought that there was minimal

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37 mitochondr ial damage following prolonged MV (Fredriksson et al., 2005), however, recently Powers and colleagues (2011) demonstrated the mitochondrial target antioxidants help protect the diaphragm from VIDD. Blood Flow and Oxygen D elivery to the D iaphragm with MV In order to maintain contractile function and prevent muscle fatigue, the diaphragm must be able to tightly regulate the rates of O2 delivery to those of O2 utilization (Poole et al., 2001, Poole et al., 2012). In this regard, it has been demonstrated at the onset of MV there is a reduction in diaphragm blood flow (Uchiyama et al., 2006) due to the reduced recruitment (and thus metabolic load) which allows for redistribution of cardiac output to other vital organs (Viires et al., 1983; Hussain et al., 1985). However, it is unknown whether prolonged MV results in a reduction in diaphragmatic blood flow or has a negative impact on the diaphragms ability to increase O2 delivery during muscular contractions (e.g. weaning). Clearly, a substantial fall in diaphragm blood flow following prolonged MV could result in impairment in the ability of the diaphragm muscle to contract (e.g. weaning) and contribute to VIDD. In this regard our laboratory has demonstrated MV induces a time dependent reduction in diaphragm oxygenation (Davis et al., 2012). These data are the first to demonstrate there is a reduced oxygen supply following prolonged MV, which may also contribute mechanism to weaning difficulties. Summary Failure to wean patients from MV is an important clinical problem and respiratory muscle weakness is a major contributor to weaning difficulties. Improving our knowledge of how changes in blood flow and O2 delivery to the diaphragm (induced by prolonged MV) will help us design modalities to successfully wean patients off MV.

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38 Data from this project supports the notion that a time dependent reduction in oxygen transport is one mechanism contributing, in part to VIDD. Our long term goal is to develop methods for the prevention of MV induced diaphragmatic weakness and weaning difficulties.

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39 CHAPTER 3 METHODS This section will be divided into two segments. The first segment will contain the experimental design used to test each of our specific aims (14), which were intended to determine the effects of MV on diaphragmati c oxygenation and resistance artery function. The subsequent section will provide detailed methods associated with each experimental technique. Experiment 1: In this experiment we measured diaphragm blood flow during spontaneous brea thing and after 30 min and 6 hr of ventilation using radioactive microspheres in female SpragueDawley (SD) rats. Arterial O2 concentration and mean arterial pressure were measured at these time points to calculate O2 delivery and vascular conductance, respectively. Experiment 2: Utilizing the phosphorescence quenching techniques (Geer et al., 2002, Wilson et al., 1994) we determined if microvascular P O2m is reduced from the spontaneous breathing condition to 30 min of MV, and if there will be a further reduction following six hours of MV. In addition, we determined whether the duration of MV affects the matching of QO2to VO2 across the rest to contraction transition after both acute and prolonged MV. Experiment 3: The purpose this experiment was to determine whether, and thro ugh which signaling pathways (e.g. endothelium dependent or independent, structural/mechanical alter ations) prolonged MV alter diaphragm arteriolar function. Experiment 4: This experiment investigated whether there is a downregulation of eNOS mRNA in diap hragmatic arterioles following prolonged MV. This experiment

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40 provides further mechanistic insight into potential signaling pathways responsible for a reduction in vasomotor control with MV. Statistical Analysis Group and sample sizes were calculated using a power analysis of preliminary data from our laboratory. Comparisons between blood flow, vascular conductance, O2 delivery, and VO2, percent maximal dilation, mRNA were analyzed with oneway ANOVA. Resting, Steady State contracting P O2m, blood pressure, a rterial blood gases, and pH across time were analyzed with repeated measure ANOVA. Individual differences will be examined post hoc using a Tukeys test. All data are presented as means SE. Sig 0.05. General E xperimental Protocols and A nalyses Animals Adult (46 month old, ~250 g) female SpragueDawley obtained from Charles River Laboratories (Boston, MA, USA) were used for this investigation (Spontaneous Breathing : n= 45; 30 min MV: n=41; 6 hr MV: n= 42) The SpragueDawley rat was chosen due to the similar properties (e.g. anatomical and physiological) with the human diaphragm (Metzger et al., 1985, Mizuno et al., 1991, Poole et al., 1997, Powers et al., 1997). Upon arrival, all rats were housed at the University of Florida Animal Care Services Center All procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Animals were maintained in a temperaturecontrolled (23 2 C) room with a 12:12h light dark cycle. Water and rat chow were provided ad libitum through the experimental period.

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41 Mechanical Ventilation All surgical procedures performed used aseptic techniques. Animals in the MV groups were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), tracheostomized and connected to a volumecycled ventilator (Harvard Apparatus; Cambridge, MA). A catheter was implanted in the carotid artery for measurement of blood pressure and peri odic blood sampling (every 3 hr ) for an alyses of blood gases (GemPremier 3000, Instrumentation Laboratory, Bedford, MA). Arterial O2 saturation was monitored continuously by using a Mouse OX (Asbury Park, PA) placed around the rats foot. Expired P CO2 was measured continuously using a microcapnograph (Model Columbia Instruments, Columbus, Ohio). Arterial P O2, P CO2, and pH were maintained within the normal range (80 to 110 mm Hg, 30 to 40 mm Hg, and 7.35 to 7.45, respectively) by minor adjustments to minute volume. A catheter was placed in the jugular vein for infusion of sodium pentobarbital (10 mg/kg/hr) and fluids, when necessary. Body temperature was monitored and maintained at 37 1C (via rectal thermometer) by use of a recirculating heating blanket. Body fluid homeostasis was maintaine d by administrating electrolyte solution (i.v., 2 mL /kg/hr). Operative care during the MV period included expressing the bladder, removal of airway mucus, lubricating the eyes and rotating the animal. The ventilator was maintained at an average breathing frequency of 70 10 breaths/min and tidal volume of 2.2 0.5 mL/breath. Blood Flow Tissue blood flow was measured using radiolabeled microspheres (15 m diameter; 46Scandium, 85Strontium, 57Cobalt; random order) according to the methods of Flaim et al., 1984). Microspheres (i.e. 2.5 X 105 of each label) were injected at three time points; 1) during spontaneous breathing, 2) after 30 min (i.e. acute) and 3)

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42 following 6 hr (i.e. prolonged) MV. After the final microsphere infusion the animal was euthanized and the following muscles were harvested for blood flow determination: diaphragm (dissected into appropriate anatomical sections, i.e., crural, central tendon, dorsal costal, mid costal, and ventr al costal), soleus, red portion of the gastrocnemius, internal and external intercostal, sternocleidomastoid, and scalene. The kidneys were also be harvested to determine adequate distribution of the microspheres (i.e., <15% difference in blood flow between the left and right kidney). Blood flow was expres sed as mL /min/100 g of tissue and vascular conductance was expressed as mL/min/100 g/mmHg to normalize for any possible changes in arterial pressure. Phosphorescence Quenching As stated previously, phos phorescence quenching was utilized to measure diaphragm muscle P O2m during spontaneous breathing and after 30 min and 6 hr of MV. Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg i.p., supplemented as needed) and the right carotid arter y was isolated and cannulated with a fluidfilled catheter (PE 50) to monitor arterial blood pressure (Digi Med BPA Model 400a; Micro Med, Inc.; Louisville, KY). This catheter was also be used for the infusion of the phosphorescent probe R2. The diaphrag m was exposed via laparotomy and the liver reflected gently downward, permitting access to the inferior surface of the medial and ventral regions of the diaphragm. The phosphorimeter light guide was positioned 25 mm from the surface of the medial costal diaphragm, and exposed surfaces were covered with Saran W rap to prevent moisture and heat loss. During the experimental procedure the abdominal cavity was kept moist via superfusion of a Krebs Henseleit bicarbonatebuffered solution equilibrated with 5% CO 2/95% N2 at 37C. A temperature

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43 probe was placed between the liver and the diaphragm to monitor the temperature of the abdominal cavity. The phosphor, palladium mesotetra (4 carboxyphenyl) porphyrin dendrimer (R2; Oxygen Enterprises Ltd., Philadelphia, PA), was infused ~10 min before each experiment at a dose of 15 mg/kg and P O2m measurements were recorded every 2 s for 60 s to provide an average steady state P O2m during spontaneous breathing and after 30 min and 6 hr MV. All P O2m measurements were perf ormed in a dark room to avoid contamination of the signal with ambient light. Upon completion of the experiment each rat was euthanized with an overdose of anesthesia (pentobarbital sodium, >100 mg/kg, i.a.) and a thoracotomy was performed to visually veri fy cardiac arrest Calculation of d iaphragm VO2 Muscle oxygen uptake (VO2) of the medial costal diaph ragm (i.e., region of P O2m measures) was calculated from blood flow and P O2m measurements in the diaphragm during the res ting and contracting conditions as described by Behnke et al., (2002). Specifically, VO2 is calculated using the Fick equation using P O2m as an analogue for venous P O2 (Behnke et al., 2002, Wagner, 1991) and from the O2 dissociation curve and microvascular blood O2 content (CmO2). As no discernible change in blood pH or muscle temperature was observed, we expect there will be no significant shift in O2 dissociation curve. Therefore, diaphragm VO2 was calculated from data collected from arterial blood samples, P O2m and muscle blood flow (Q m) utilizing the following equation: VO2 2CmO2), where VO2 is the oxygen uptake of the diaphragm muscle, Qm is diaphragm muscle blood flow, and CaO2 and CmO2 are the oxygen contents of the arterial and

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44 microvascular blood, respectively. Muscle VO2 was expressed as mL/min/per 100 g tissue. Diaphragm c ontractions The diaphragm was exposed as described above and stainless steel electrodes were sutured (60 silk; Ethicon, Somerville, NJ) to the right ventral costal (cathode) and the right dorsal costal (anode) diaphragm according to the methods of Geer et al. (2002). E lectrically stimulated twitch contractions (1 Hz, 36 V, 2 ms pulse duration) were induced with a Grass S88 stimulator (Quincy, MA) for 3 min. This contraction protocol elicits contr actions at a similar frequency as with spontaneous breathing. Following 30 min and 6 hr MV the stimulation protocol and P O2m was measured at 2 s intervals across the rest to contractions transition. Blood flow determination was made after 180 s of stimul ation to assess the contracting steady state muscle hyperemia. VO2 was then calculated as described above for resting and steady state contracting conditions. Isolated Microvessel Technique diameter) were isolated from the diaphragm and studied in vitro to remove potentially confounding metabolic, humoral, and neural influences. Resistance arterioles will be harvested from three groups of animals which include; 1) spontaneously breathing animals, 2) following acute, and 3) following prolonged MV. With the aid of a dissecting order (1A) arterioles from the diaphragm muscle were isolated a nd removed from the surrounding muscle tissue as previously described (Aaker & Laughlign, 2002, Muller Delp et al., 2002, Spier et al., 2004). The arterioles (length, 0.5 1.0 mm; inner diameter, 90

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45 containing P SS equilibrated with room air. Each end of the arteriole will be cannulated with micropipettes and secured with nylon suture. Following cannulation, the microvessel chamber will then be transferred to the stage of an inverted microscope (Olympus IX70) equi pped with a video camera (Panasonic BP310), video caliper ( Colorado Video 307A ) and dataacq uisition system (PowerLab) for on line recording of intraluminal diameter. Arterioles were initially pressurized to 75 cmH2O with two independent hydrostatic press ure reservoirs. Leaks were detected by pressurizing the vessel, and then closing the valves to the reservoirs and verifying that intraluminal diameter remained constant. Arterioles that exhibit leaks were discarded. Arterioles that were fr ee from leaks wer e warmed to 37C and allowed to develop initial spontaneous tone during a 30 60 min equilibration period. Upon displaying a steady level of spontaneous tone we evaluated vasodilator and pressure responses in these resistance arterioles. To determine endothelial function of diaphragm resistance arterioles, dose responses to acetylcholine (Ach), which mediates smooth muscle relaxation indirectly by binding to endothelial cell M2receptors and stimulates the release of endothelium derived relaxing factor(s) (EDRF), will be tested. EDRFs typically released from vascular endothelial cells are nitric oxide (NO) and prostacyclin (PGI2), and to a lesser extent, endothelium derived hyperpolarizing factor (EDHF). Therefore, the contribution of the nitric oxide synthase (NOS) and cyclooxygenase (COX) signaling pathways to ACh induced vasodilation in the diaphragm resistance arterioles will be determined using the NOS inhibitor AP nitro L arginine methyl ester (LNAME) and the COX inhibitor indomethacin. Flow induced vasodilation (endothelium dependent), endothelium independent (via sodium nitroprusside, SNP), active (myogenic) and

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46 passive diameter responses were assessed in the isolated vessels. The latter was accomplished by opposite directions so that a pressure difference is created across the vessel without altering mean intraluminal pressure. Diameter measurements were then determined in response to incre mental pressure differences of 4, 10, 20, 40 and 60 cmH2O. Kuo et al. 1990; Muller Delp et al. 2002): rbc/1.6)(d/2)2 where (d) is inner diameter and (Vrbc) repres ents mean red blood cell velocity, which will be determined in a subset of arterioles at each of the pressure gradients mentioned and 75 cmH2O for 60 minutes. After equilibration, intraluminal pressure will be increased in 20cmH2O increments from 0 to 140 cmH2O. Diameter was recorded for 3 min at each pressure step and these pressure changes will occur in the absence of flow. mRNA Analysis Diaphragm resistance arterioles were snap frozen and stored at 80C. Arterioles were then pulverized in lysis buffer, and total RNA extracted using a RNAqueous isolation kit (Ambion). cDNA was made using the HighCapacity cDNA Archive kit (Applied Biosystems). Real Time PCR was perform ed as previously described (Spier et al., 2004). time PCR was performed in triplicate, with two notemplate control samples and two reverse transcriptase negative samples (GeneAmp 96well optical reaction plates). eNOS mRNA expression was quan TaqMan primers (Applied Biosystems) using the ABI Prism 7900HT fast real time PCR

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47 was performed using the comparative threshold cycle method.

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48 CHAPTER 4 RESULTS Animals W eight, H emod ynamic D ata, A rterial B lood G ases and pH Body and diaphragm weight were not different between groups (Table 4 1). Mean arterial pressure (MAP), blood gases and pH are summarized in Table 1 for spontaneous breathing and during MV. There was a reduction in MAP after 6 hours of MV, however, it remained above levels that affect P O2m dynamics (Behnke et al., 2006). Indeed, we did not observe any relation between MAP and P O2m dynamics (i.e., mean response time; r2=0.007, P=0.72). Contrary to that reported by Poole et al. (1995), we did not observe a relation between MAP and P O2m (r2=0.11, P=0.29), which may be due to circulatory volumetric changes induced to manipulate MAP in the previous study. Arterial P O2 decreased slightly over time but no relations were observed between arterial PaO2 and VO2 (r2=0.09, P=0.66) or P O2m (r2=0.07, P=0.17). Based on the rightward shifted O2 dissociation curve of the rat (P50= 38 mmHg) small changes in P aO2 above 80 mmHg would have very little impact on O2 content as these P O2 values would fall on the flat portion of the dissociation curve. Diaphragm B lood F low and V ascular C onductance are D iminished with M echanical V entilation To address the changes in diaphragm blood flow that may be a contributing mechanism to diaphragm mus cle weakness with MV we measured diaphragm blood flow as a function of time on the ventilator. During spontaneous breathing, diaphragm blood flow averaged 36 5 mL/min/100 g, which is consistent with what has been reported for the rat diaphragm under anesthesia (Boegehold et al., 1991). Mechanical ventilation was then initiated and set at a rate sufficient to suppress contractions within the diaphragm (i.e., passive ventilation) and blood flow was measured again after 30

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49 min of MV. Passive MV elicited a reduction of ~25% in diaphragm b lood flow after 30 min to ~26 mL/min/100 g (Figure 4 1A). To assess whether diaphragm blood flow decreased in a timedependent manner with passive MV, diaphragm blood flow was again measured after 6 hr MV. There was an ad ditional ~75% reduction in diaphragm blood flow versus that measured at 30 min MV, resulting in an average flow of 7 mL /min/100 g (Figure 4 1A). For comparison, the blood flow to the diaphragm measured herein after 6 hr MV (Figure 4 1A) is considerably less than that measured in the highly glycolytic white portion of the gastrocnemius of ~ 1015 mL/min/100 g (Hirai et al., 2011, Behnke et al., 2011). To normalize for changes in blood flow that may be due to alterations in blood pressure we calculated vasc ular conductance (i.e., blood flow MAP) at the different time points. As demonstrated in Figure 4 1B, reductions in vascular conductance paralleled changes in blood flow with the duration MV, resulting in a significantly lower vascular conductance after 6 hr versus 30 min of MV. Consistent with what others have found (Brancatisano et al., 1991, Sexton et al., 1995) there was a marked heterogeneity in regional distribution of blood flow within the diaphragm (Figure 4 2). During spontaneous breathing bl ood flow to the medial costal portion of the diaphragm was the highest and showed the greatest reduction after the onset of MV. After 6 hr of MV all regions of the diaphragm demonstrated significant reductions in blood flow and vascular conductance versus both spontaneous breathing and 30 min of MV (Figure 4 2A & B). In order to rule out the effects of anesthesia causing the reduction in diaphragm blood flow w e measured blood following 6 hr of anesthesia alone (Figure 4 3). Blood flow and vascular conduct ance to the intercostal

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50 muscles, which are subjected to similar alterations in intrathoracic pressures as the diaphragm, did not change across the measurement period (Figure 4 4A & 4 4 B). The net reduction in blood flow to the diaphragm between the consci ous standing (Sexton et al., 1995, Poole et al., 2000) and the inactive condition (i.e., during MV) is similar to that observed in the soleus muscle between the standing and anesthetized condition (Behnke et al., 2011). Furthermore, the diaphragm has a hi gh daily duty cycle (i.e., ratio of active to inactive times) and the soleus muscle has the highest daily duty cycle of the hindlimb locomotory muscles (Hensbergen et al., 1997). Therefore, we wanted to measure blood flow with inactivity in the soleus mus cle (which is inactive during the entire protocol after the onset of anesthesia) to determine if similar reductions in blood flow as observed in the diaphragm also occur in this muscle over time (Table 4 2). Unlike the diaphragm, blood flow and vascular c onductance remained unchanged in the soleus muscle at every time point measured herein (i.e, up to 6 hr; Figure 44 A & B). Given the high oxidative capacity of the diaphragm, we also wanted to compare blood flow over time in skeletal muscle which display s a similar oxidative capacity and fiber type composition. Therefore, we measured blood flow to the red portion of the gastrocnemius muscle complex (GastRed) as it displays a similar oxidative capacity (citrate synthase activity; diaphragm ~ 39 vs. GastRe d ~ 36 mol/min/g) and oxidative fiber type profile (type I fibers: diaphragm ~44% vs. GastRed ~ 51%; type IIa fibers, diaphragm ~23% vs. GastRed ~ 35%; (Delp and Duan, 1996) as the diaphragm. Similar to that of the soleus, we did not observe any change i n blood flow (Figure 44 A) or vascular conductance (Figure 44 B) in the GastRed across the 6 hr experimental protocol.

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51 Mechanical V entilation R educes R esting D iaphragm M icrovascular P O2 ( P O2m) in a T ime D ependent M anner Figure 4 5A shows representative resting diaphragm P O2m responses from an individual animal during the measurement periods. During spontaneous breathing (SB) diaphragm P O2m was ~ 53 mmHg and was significantly reduced to ~37 mmHg after 30 min of MV (Figure 4 5B). P O2m decreased by an addit ional ~50% after 6 hr of MV versus that after 30 min of MV to 18 5 mmHg (Figure 4 5B). As P O2m reflects the QO2/VO2 ratio in the diaphragm (Poole et al., 1995), the lower P O2m observed after 6 hr could result from the reduced blood flow, an increased VO2, or a reduction in both variables but with a great proportional decrease in O2 delivery. Therefore, we also calculated diaphragm VO2. Resting D iaphragm O2 U ptake (VO2) Relative to spontaneous breathing, 30 minutes of MV significantly decreased diaphr agm VO2 (spontaneous breathing 1.44 0.10; 30 min MV, 1.19 0.08 mL /min/100 g; P<0.05). As the MV rate utilized was sufficient to suppress diaphragm activity, the reduction in VO2 after the onset of MV is due to the quiescence of the diaphragm motor uni ts during passive ventilation. Specifically, as ~1220% of maximal diaphragm force is generated during normal, quiet breathing (Sieck et al., 1989, Mantilla et al., 2010), a fall of ~17% in diaphragm VO2 can be expected with inactivity induced by passive MV. However, after 6 hr of MV there was a trend (P=0.056) for a further reduction in diaphragm VO2 (1.02 0.06 mL/min/100 g) compared to that calculated after 30 min of MV.

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52 Mechanical V entilation R educes the A bility to A ugment D iaphragm B lood F low, M atch O2 D elivery to U tilization, and I ncrease VO2 D uring M uscular C ontractions To determine whether the capacity to increase blood flow and VO2 during a metabolic challenge in the diaphragm is negatively affected after 6 hr of MV, we elicited twitch contractio ns and quantified the resultant blood flow, P O2m and VO2 responses in the diaphragm. Average and representative diaphragm P O2m profiles across the rest to contractions transition are demonstrated in Figure 4 6A and 6B, respectively, after 30 min and 6 hr of MV. Upon initiation of contractions, the diaphragm P O2m after 30 min of MV decreased by ~16 mmHg from the precontracting value to a low P O2m value of ~21 mmHg (Table 4 3), which is similar to that reported by Geer et al. during contractions after acut e MV (Geer et al., 2002). After 6 hr of MV the decrease in P O2m with contractions was significantly less (i.e., ~9 mmHg or roughly 55% of that observed after 30 min of MV; Table 4 3) resulting in a low P O2m value of 8 1 mmHg. When looking at P O2m dynam ics the time delay before a statistically significant fall in P O2m (versus pre contracting values) was shorter in the 6 hr MV group versus that after 30 min of MV (Table 4 3). The shorter time delay suggests that the relative increase O2 delivery was slow er than that of O2 uptake (Behnke et al., 2002) during contractions, resulting in a more rapid exponential decrease (i.e., faster time constant, tau) in PO2m after 6 hr versus 30 min of MV (Table 4 3). In comparison to 30 min of MV, 6 hr of MV elicited si gnificantly faster overall kinetics, with a mean response time of (i.e., TD + Tau) of ~15 s versus ~22 s after 30 min of MV (Table 4 3). When quantifying the hyperemic response elicited by muscular contractions, there was a 2.8 fold increase in diaphragm blood flow compared to the resting value after 30 min of MV versus a change of only 1.3 fold after 6 hr of MV (Figure 4 7A). The

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53 resultant change in diaphragm blood flow from rest to contractions (i.e., steady state contracting value minus precontracting baseline value) was ~90% great er after 30 min of MV (40 6 mL /min/10 0 g) versus 6 hr of MV (4 1 mL /min/100 g). In fact, after 6 hr of MV diaphragm blood flow during contractions was only ~ 80% of that measured in the resting condition after 30 min MV. We also wanted to explore the possibility that the severely reduced ability to augment flow after 6 hr of MV could not be offset by an increase in fractional O2 extraction such that diaphragm VO2 would be reduced during contractions. After 30 min of MV, contractions elicited a marked increase in diaphragm VO2 from 1 .4 0.1 at rest to 7.0 0.4 mL/min/100 g during the contracting steady state (Figure 4 7B). The increase in VO2 was significantly blunted after 6 hr versus 30 min of MV with a change in VO2 from a resting value of 1.0 0.1 to 1.6 0.1 mL/min/100 g during the steady state of contractions (Figure 4 7B). The increase in VO2 during contractions after 6 hr of MV was only ~ 22% of that observed after 30 min of MV during contractions. In additi on, there was a reduced diaphragm O2 delivery (i.e., arterial O2 flow; Figure 8A) and increased O2 extraction (i.e., O2 delivery VO2; Figure 8B) both at rest and during contractions after 6 hr versus 30 min of MV. Mechanical V entilation R educes NO M ediated V asodilation in D iaph ragm R esistance A rterioles In order to investigate the role of MV on resistance vascular function both endothelium dependent and independent mechanisms in diaphragm resistance arterioles were assessed. The developm ent of spontaneous tone did not differ between arterioles from spontaneous breathing (42 4%), 30 min (40 6%), or 6 hr MV (38 5% ) rats (P > 0.05). In addition, the maximum arteriolar diameter was not different

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54 between groups (Spontaneous Breathing, 20 MV, 195 6 of MV, there was a 40% reduction in flow induced vasodilation in diaphragm arterioles (Figure 4 9A). These arterioles also exhibited a significantly lower endothelium dependent vasodilation to A ch (Figure 4 10 C ). There was also a significant reduction in arteriolar vasodilator responsiveness to the exogenous NO donor SNP (Figure 4 10D ) and eNOS mRNA expression (Figure 4 11A ) in diap hragm arterioles following 6 hr of MV. Thes e data demonstrate following prolonged MV vascular dysfunction occurs through endothelium dependent/endot helium independent mechanism s. To our knowledge, this is the first study that provides evidence for a reduction in NO bioavai lability and/or NO mis handling in diaphragm arterioles following prolonged MV. Pressure D iameter R elationship I s A ltered in D iaphragm A rterioles F ollowing P rolong M echanical V entilation Figure 4 12 illustrates the active and passive pressurediameter relationship as intralumin al pressure was increased stepwise from 10 to 130 cm H2O. There were no changes in the active (myogenic) response, however, the passivepressure response was reduced following 6 hr of MV indicating structural alterations within the resistance vasculature.

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55 Table 41. Body and diaphragm mass, blood basses and hematocrit. Spontaneous breathing 30 min MV 6 hr MV Body mass (g) 274 3 268 3 271 2 Diaphragm wt (mg) 873 11 843 30 855 13 MAP (mmHg) 100 4 105 4 81 4 pH 7.4 0.1 7.4 0.1 7.4 1 Arterial PO 2 (mmHg) 96 0.4 90 6 80 5 Arterial PCO 2 (mmHg) 47 1 38 4 30 3 Hematocrit (%) 44 0.3 40 1 37 1 MAP, mean arterial pressure. [Hb], arterial hemoglobin concentration. *P<0.05 versus both spontaneous breathing and 30 min MB. Values are means SE. Table 42 Renal, respiratory and select hindlimb skeletal muscle blood flows. Spontaneous breathing 30 min MV 6 hr MV Kidney 346 43 319 50 284 39 Intercostal 13 3 12 2 8 1 Rectus a bdominus 5 1 4 1 4 1 Soleus 33 10 29 8 28 6 Red g astrocnemius 13 1 15 2 16 1 ml/min/100g tissue. Values are means SE. Table 43 Microvascular PO2 dynamics across the rest to contractions transition after mechanical ventilation. 30 min MV 6 hr MV Baseline PO 2 (mmHg) 37.2 2.4 18.8 1.5* Low PO 2 (mmHg) 20.5 1.9 7.7 0.7 Time d elay (s) 10.8 1.1 7.8 1.4 Time c onstant (s) 11.3 2.1 6.8 1.2 Delta PO 2 (mmHg) 15.9 3.0 8.7 1.0 mmhg. Values are means SE.

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56 0.0 0.1 0.2 0.3 0.4 0.5 0 10 20 30 40 50 Diaphragm Blood Flow ( mL/min/100 g) Diaphragm Vascular Conductance ( mL/min/100 g/mmHg)Spontaneous Breathing 30 min MV 6 hr MV Spontaneous Breathing 30 min MV 6 hr MV* A B Figure 41 Resting diaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n =5). (mean SE) *P < 0.05 versus spontaneous breathing; P<0.05 versus 30 min MV

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57 0.00.10.20.30.40.50.6 Medial Costal Ventral Dorsal Crural 0 10 20 30 40 50 60 Medial Costal Ventral Dorsal Crural Blood Flow ( mL/min/100 g)Spontaneous Breathing 30 min MV 6 hrs MV* A BVascular Conductance ( mL/min/100 g/mmHg)Spontaneous Breathing 30 min MV 6 hrs MV* Figure 42 Resting blood flow (A) and vascular conductance (B) to regionally delineated portions of the diaphragm muscle during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing; P<0.05 versus 30 min MV.

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58 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 0 10 20 30 40 50 Diaphragm Blood Flow ( mL/min/100 g) Diaphragm Vascular Conductance ( mL/min/100 g/mmHg)30 Min* A B Spontaneous Breathing Mechanical Ventilation Spontaneous Breathing Mechanical Ventilation 6 Hrs*30 Min 6 Hrs Figure 43 Resting d iaphragm muscle blood flow (A) and vascular conductance (B) measured during spontaneous breathing (n=8) and mechanically ventilated r ats (n=7) after 30 min and 6 hr (mean SE) *P<0.05 versus spontaneous breathing; P<0.05 versus 30 min MV.

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59 0.00.10.20.30.40.5 Diaphragm Red Gastrocnemius Soleus Intercostal 0 10 20 30 40 50 Diaphragm Red Gastrocnemius Soleus Intercostal Blood Flow ( mL/min/100 g)Spontaneous Breathing 30 min MV 6 hrs MV*n.s* n.sVascular Conductance ( mL/min/100 g/mmHg)Spontaneous Breathing 30 min MV 6 hrs MV n.s* n.s* A Bn.s n.s Figure 4 4 Comparison of blood flow (A) and vascular conductance (B) measured at rest between the diaphragm, red portion of the gastrocnemius muscle complex, soleus, and intercostal muscles during spontaneous breathing (n=7) and after 30 min (n=8) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing; P<0.05 versus 30 min MV.

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60 0102030405060 0 5 10 15 20 25 30 0 15 30 45 60 Spontaneous Breathing 30 min MV 6 hr MV Microvascular PO2(mmHg)Time (s)Microvascular PO2(mmHg)Spontaneous Breathing 30 min MV 6 hr MV* A B Figure 45 Representative resting diaphragm microvascular P O2 profiles measured over time (A) and the average diaphragm microvascular P O2 (B) measured during spontaneous breathing and after 30 min and 6 hr of MV(n=11). *P<0.05 versus spontaneous breathing; P<0.05 versus 30 min MV.

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61 Figure 46. Mean microvascular P O2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular P O2 profiles (B) in response to electrically stimulated muscle contractions after 30 min and 6 hr of MV (n=5). Contractions were initiated at time zero. Figure 46 Mean microvascular PO2m profiles including 95% CI (dashed lines) (A) and representative diaphragm microvascular PO2 profiles (B) in response to electrically stimulated muscle contractions after 30 min and 6 hr of MV (n=5). Contractions were initiated at time zero.

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62 0 20 40 60 80 6 hr Spontaneous Breathing 30 min MV 6 hr MV 0 2 4 6 8 30 min MV 6 hr MV Rest Contractions* *Blood Flow ( mL/min/100 g)Rest Contractions Spontaneous BreathingVO2(ml/min/100 g)* *#**A 6 hrs Spontaneous Breathing 30 min MV 6 hrs MV Figure 47 Blood flow (A) and oxygen consumption (VO2) (B) at rest and during the steady state of electrically stimulated contractions in the diaphragm muscle after 30 min (n=7) and 6 hr of MV (n=8) as compared to spontaneous breathing (n=7). *P<0.05 versus spontaneous breathing; P<0.05 between 30 min and 6 hr of MV; P<0.05 versus rest in the same condition; #P=0.056 between 30 min and 6 hr of MV.

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63 0.0 0.2 0.4 0.6 0.8 1.0 30 min MV 6 hr MV 024681012 30 min MV 6 hr MV Rest Contractions Spontaneous BreathingO2Delivery (mL O2/min/100 g)* B *Rest Contractions Spontaneous BreathingFractional O2Extraction* *C Figure 48 O2 delivery (A) and fractional O extraction (B) at rest and during electrically stimulated diaphragm muscle contractions after 30 min (n=7) and 6 hr of MV (n=8) as compare d to spontaneous breathing (n=7). *P<0.05 versus spontaneous breathing; P<0.05 between 30 min and 6 hr of MV; P<0.05 versus rest in the same condition.

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64 Flow (nl/sec) -100102030405060Relaxation (%) 01020304050 Spontaneous Breathing 30 min MV 6 hr MV asdasd asdasd Figure 49 Flow mediated vasodilation (A) in diaphragm arterioles during spontaneous breathing ( n=7) and after 30 min (n=8) and 6 hr of MV ( n=7 ) .* A

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65 sds Spontaneous Breathing 30 min MV 6 hr MV DSDSD DSDSDS A B C D Figure 410. Dose responses to the endothelial dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N G nitro l argine methyl ester (LNAME) (A), Dose responses to the endothelial dependent vasodilation Acetylcholine (Ach) in the absence and presence of endothelial NO synthase inhibitor N G nitro l argine methyl ester (LNAME) + COX inhib itor Indomethacin (INDO) (B), NO dependent (endothelium dependent) dilation (max dilationACh max dilationACh + LNAME) (C ), Dose responses to the e ndothel ium independent vasodilator sodium nitroprusside (SNP) (D) in diaphragm arterioles during spontaneous breathing (n=19 ) and after 30 min (n= 21 ) and 6 hr of MV (n= 23). *P<0.05 versus spontaneous breathing. P<0.05 versus 30 min MV.

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66 Spontaneous Breathing 30 min MV 6 hr MV* Figure 411. eNOS mRNA expression in diaphragm arterioles during spontaneous breathing ( n=15) and after 30 min ( n=12) and 6 hr of MV (n=13). *P<0.056 versus spontaneous breathing

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67 Figure 412. Active and passive myogenic responsiveness in arterioles during spontaneous breathing (n=11) and following 30 min (n=10) and 6 hr of MV (n=8). *P<0.05 versus spontaneous breathing P<0.05 versus 30 min MV. Pressure (cmH20) 020406080100120140160Normalized Diameter 0.40.50.60.70.80.91.01.1 Spontaneous Breathing Passive 30 min Passive 6 hr Passive Spontaneous Breathing Active 30 min Active 6 hr Active

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68 CHAPTER 5 DISCUSSION These experiments provide several new and clinically relevant findings regarding the regulation of O2 supply demand and diaphragm contractile function with mechanical ventilation (MV). Specifically, this i s the first investigation to demonstrate: 1) there is a time dependent reduction in diaphragm blood flow and O2 delivery (QO2) with MV that is not apparent in other skeletal muscle (during the time frame used herein), 2) diaphragm microvascular P O2 ( P O2m), which represents the sole driving force for capillary to myocyte O2 diffusion, is reduced as a function of time of MV, 3) the ability to increase blood flow in the diaphragm with muscular contractions is severely reduced after 6 hr of MV, and 4) diaphrag m O2 uptake (VO2) during muscular contractions is reduced by ~80% after 6 hr versus the onset of MV, 5) NO mediated vasodilation is diminished following prolonged MV, 5) there are also structural alterations to the diaphragm resistance vasculature following prolonged MV. Overall, our findings demonstrate an O2 supply demand imbalance in the diaphragm after mechanical ventilation that occurs withi n hours after the onset of MV The functional consequence of this is profound in that the O2 supply demand imbalance is exacerbated during muscular contractions, resulting in a reduced aerobic metabolism in the diaphragm. The time dependent reduction in microvascular O2 supply (blood flow and ultimately O2 pressure) in the diaphragm with MV present several potential consequences, including 1) local areas of hypoxia and/or anoxia that may promote ROS generation within diaphragm mitochondria and activate apoptotic pathways, and 2) an O2 delivery limitation and exacerbated fall in intramyocyte PO2 during elevated

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69 respira tory muscle O2 demand (e.g., during the weaning process), hastening the onset of respiratory muscle fatigue. The precise role of a reduced QO2to VO2 ratio (i.e., diminished P O2m measured herein) in concert with the mitochondrial dysfunction apparent after prolonged MV (Kavasiz et al., 2009), and their relative contributions to VIDD, require additional investigations. Mechanistic B asis for the D iminished D iaphragm B lood F low F ollowing M echanical V entilation To our knowledge this is the first investigation examining the effect of MV over time on diaphragm vasomotor function. Given the large vasodilator reserve evident in the diaphragm even at maximal exercise (Poole et al., 200 0 ), the finding of an relative inability to increase blood flow during contractions following 6 hr of MV (Figure 4 6A) was quite surprising and suggests a reduced vasodilation (possible due to increased oxidative stress/antioxidant imbalances and NO bioavailability (Kang et al., 2009)) and/or structural modifications (e.g., short term remodeling) associated with an increased tonic vasoconstriction (Martinez Lemus et al., 2011, Martinez Lemus et al., 2009) when the diaphragm is inactive. The following section will discuss the evidence for endothelial dysfunction, vascular smooth muscle dysfunction and structural modifications in the resistance vasculature following prolonged MV Endothelial Dysfunction We have demonstrate d prolonged MV results in a reduction in flowmediated (Figure 4 9A) vasodilation and, therefore, a reduction in shear stress along the apical surface of the endothelial cell. Furthermore, it is known that a r eduction in shear stress (steady and oscillatory ) alter s the production of vasoregulating agents such as NO ( Chang et al., 2000; Kuchan et al., 1994; Rubanyi et al., 1986). The structure that

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70 resides on the luminal surface (i.e. interface between blood flow and the endothelial cell) and is in direct contact with the flowing blood is known as the endothelial glycocalyx These membranebound macromolecules are responsible for sensing fluid mechanical shear stress. The physical displacement of these proteoglycans can be transmitted to the cellular surface and provoke an intracellular response, such as production of NO (Davies et al., 1995). Ach induced vasodilation was also blunted following prolonged MV (Figure 4 10 A) and was further reduced following treatment with L NAME or combined blockade with indomethacin and LNAME. Th e inhibitory effect of L NAME on Ach induced vasodilation and reduction in flow mediated v asodilati on indicate a significant role for NO in mediating vascular dysfunction with MV Furthermore, given the drastic decline in diaphragm blood flow, it is also likely that there is a reduced shear stimulus or sensitivity to shear following prolonged MV (evidenced by an reduction in eNOS mRNA expression) following prolonged MV. Although further studies are warranted we believe endothelial glycocalyx disruption (and ultimately reduced NO production) may be one key instigator of the endothelial dysfunction observed in this study In this regard it has also been demonstrated that hypoxia (i.e. alterations in PO2, Ischemia/Reperfusion Injury) can severely damage the glycocalyx (Ward et al., 1993; Czarnowska et al., 1995, RubioGayosso et al., 2006 ) Mo reover, the ese authors also concluded that the damage of the endothelial glycocalyx is associated with the appearance/production of ROS, which i s also elevated during MV (Kavasis et al., 2009) VSMC Responsiveness Endothelial independent vasodilation (i.e. smooth muscle vasodilation) was also diminished following prolonged MV. T he finding of an emaciated vasodilation to

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71 exogenous NO in the prolonged MV group suggests there is NO mis handling (as compared to a reduction NO bioavailabilty) and a reduction sm ooth muscle responsiveness to NO and cGMP mediated relaxation following prolonged MV V ascular smooth muscle dysfunction has been reported by others (Behnke et al., 2010, Delp et al., 1995, Miyata et al., 1992) and it is thought that this is possibly due to reductions in the net intracellular accumulation of cGMP (due to increased cGMP phosphodiesterase activity) in smooth muscle (Moritoki et al., 1988), and/or a reduced scavenging, or increased production of superoxide ( Csiszar et al., 2002, Sindler et al., 2009), causing a reduction in the bioavailabilty of NO ( Gryglewski et al., 1986 Kang et al., 2009) Structural Modifications Another importa nt finding from this study was the reduction in the passive pressurediameter relationship. This indicates the re maybe structural alterations in the resistance vasculature with MV. Pistea and collegues (2008) have demonstrated there maybe inward eutrophic remodeling of arterioles when exposed to chronic vasoconstriction or inhibition of NO mediated vasodilation (f or review see Martinez lemus et al. 2009) In this regard, it has been demonstrated that there is an increased in angiotensin II during prolonged MV ( unpublished observations ) which could be a key contributor to the inward eutrophic remodeling. In additi on, Li Fu and colleges (2011) recently demonstrated that there is an increase in collagen deposition in the diaphragm following prolonged MV. This would likely cause vascular stiffness in diaphragm arterioles and lead the reduction in passivepressure resp onse demonstrated herein (Figure 4 12). These data yet represent another potential vascular mechanism for the reduction in the hyperemic response following prolonged MV. It is important to note that

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72 structural alterations can potentially affect vasodilation, however, the vessel data presented herein was normalized to its maximal diameter, and therefore, we are sure the changes found are not due to changes in the structure of the vessel. It is unclear why the diaphragm displays a timedependent reduction in blood flow which was not apparent in other skeletal muscles that were also inactive (i.e., soleus and gastrocnemius; Figure 4 3). There are several unique properties of the diaphragm in relation to other skeletal muscle (for review see (Mantilla et al. 2003)) which, when coupled with its remarkable activation history, may expedite the timecourse of vasomotor dysfunction with disuse in this muscle. Given the vasomotor dysfunction observed after inactivity (e.g., after bed rest; (Demiot et al., 2007)), we cannot preclude that similar reductions in blood flow to other skeletal muscle do not occur over longer time periods than utilized in our protocol. In fact, reductions in blood flow during disuse are thought to be a key signaling mechanism for structural and functional adaptations within the resistance vasculature of some skeletal muscle (Delp et al., 2009). Nonetheless, the reduced blood flow and O2 delivery to the diaphragm at rest and during contractions following MV (Figures 4 6A and 4 7A, respect ively) can have profound ramifications on intracellular processes that contribute to ATP production and molecular signaling mechanisms for mitochondrial dysfunction, atrophy, and autophagy as discussed below. Implications from A ltered P O2m D ynamics To inve stigate whether the rapidity with which QO2 and VO2 can increase is affected with MV we quantified the P O2m profile (which is representative of the dynamic QO2to VO2 ratio; (McDonough et al., 2011) across the rest to contractions transition. Under healthy conditions, the evidence in vivo (Radedran et al., 1998, Bangsbo et al.,

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73 2000) suggests that that QO2 dynamics are faster relative to VO2, such that the mean venous P O2 (Grassi et al., 1996) or P O2m (Behnke et al., 2001) is maintained or increased during the initial onset of contractions. This is advantageous as it maintains a large capillary to intramyocyte P O2 gradient to facilitate bloodtissue O2 exchange. However, with aging (Behnke et al., 2005) and pathological diseases (e.g., CHF (Behnke et al., 2007,Diederch et al., 2002) and type II diabetes (Padilla et al., 2007)), there is a shorter delay before P O2m decreases, as well as a more precipitous fall in P O2m, after the onset of contractions in skeletal muscle. The faster P O2m kinetics in these co nditions are attributed, in part, to a slower increase in muscle blood flow (Copp et al., 2009) and blunted arteriolar vasodilation dynamics (Behnke et al., 2010). In the present study we observed faster P O2m dynamics (i.e., shorter time delay, faster MRT ; Table 3) during contractions after 6 hr relative to 30 min of MV in the diaphragm. Whereas we did not measure QO2 or VO2 dynamics in separatum the faster P O2m dynamics after 6 hr of mechanical ventilation are consistent with a sluggish increase in QO2 relative to VO2, which would force a greater reliance on nonoxidative energy sources during the critical transition to muscular work. Reduced O2 D elivery, D iaphragm VO2 and C ellular E nergetics Based upon Ficks law of diffusion [VO2=DmO2 (PO2 capillary PO2 intramyocyte)], at a given VO2 and DO2 a significantly higher PO2 in the microvasculature would raise the intracellular PO2. The latter of which would be advantageous in mitigating the degree of intracellular perturbations (e.g. H+, lactate, phosphocreatine (PCr) and inorganic phosphate Pi,) required to sustain a g iven mitochondrial ATP flux However, even a small reduction in O2 supply during the steady state of submaximal exercise can manifest large changes in cellular homeostasis (i.e., PCr degradat ion) (Haseler et al.,

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74 1988, Hogan et al., 1999). This notion can be further conceptualized by considering the equation for oxidative phosphorylation: 5 ADP + 5 Pi + O2 2O Under conditions of altered O2 supply, compensatory changes in the regulatory parameters of mitochondrial respiration (i.e. cytosolic [ATP]/ [ADP][Pi] and mitochondrial [NAD+]/[NADH]) occur to maintain a given rate of ATP production (and thus VO2) (Hogan et al., 1992 ,Wilson et al., 1985). In the current study, 6 hr of MV lowered contracting PO2m (Figure 5), thereby reducing intracellular PO2, and the maintenance of a given rate of oxidative ATP production would mandate lowered intracellular energy levels (i.e., reduced [ATP]/[ADP][Pi], [NAD+]/[NADH], and [PCr]) (Wilson et al., 1977). Reduced P O2m and C ellular/M olecular S ignals for M itochondrial D ysfunction, A trophy, and A utophagy Whereas atrophy in locomotory skeletal muscle requires prolonged disuse (Powers et al., 2007, Jackman et al., 2004, Lawler et al. 2003), the diaphragm displays a rapid atrophic response with MV (Powers et al., 2005, Powers et al., 2007). Furthermore, oxidative stress associated with diaphragm inactivity can occur within 36 hr after the onset of MV (Zergeroglu et al., 2003) and represents a molecular mechanism for diaphragm atrophy (McClung et al., 2007, Betters et al., 2004) and mitochondrial dysfunction (Kavasiz et al., 2009). However, the signaling mechanisms for the enhance ROS production evident with MV remains unclear. The P O2m measurements made in the current investigation reflect the composite P O2 of all the plasma within the sampled region. It is likely that some areas of the diaphragm vasculature would have considerably lower P O2s then the aggregate value reported herein. Therefore, given the large reduction in P O2m following 6 hr of MV in the current

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75 study, we believe there may be hypoxic and/or anoxic loci within many diaphragm myocytes which could promote mitochondrial ROS generation, although this has yet to be determined. It has been demonstrated that antioxidant administration attenuates MV induced muscle atrophy (McClung et al., 2007) and mitochondrial dysfunction (Powers et al., 2011). In addition, in aged animals that have an elevated ROS production, antioxidant administration has been demonstrated to elevate skeletal muscle P O2m (Herspring et al., 2004). Therefore, the beneficial effects of antioxidant administration on mitigating diaphragm contractile dysfunction with prolonged MV may be due, in part, to elevating the QO2to VO2 ratio within the diaphragm. QO2 and VO2 D uring C ont ractions: Ramifications on the W eaning P rocess During muscular work under healthy conditions there is a tight coupling between the increase in VO2 and QO2 (i.e., metabolic demands are precisely met by O2 delivery). However, after 6 hr of ventilation there is an uncoupling between these two variables, resulting in an increased fractional O2 extra ction at rest and during contractions (Figure 7A). In human patients who failed spontaneous breathing trials, there was an increased fractional O2 extraction until failure, which was attributed to a reduced O2 supply (Jubran et al., 1998). Further, it has been demonstrated that impairments in the mechanical chemical coupling in the diaphragm can result, in part, from a decreased O2 availability (Pierce et al., 2001). Accordingly, both an impaired contractile function and limited O2 supply have the potent ial to reduce aerobic metabolism within the diaphragm after MV. In the current study, we observed a ~80% reduction in VO2 during contractions after 6 hr of MV compared to that calculated after 30 min of MV. This large reduction in oxidative metabolism would hasten the onset of diaphragm fatigue, and if this same

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76 reduction in VO2 occurs in the human diaphragm after MV, would predispose the patient to weaning failure. However, from the current study it is not possible to delineate the contributions of a reduced O2 supply from mitochondrial and contractile dysfunction to this impaired VO2 response after MV. Future Directions MV is associated with respiratory muscle dysfunction in animal and human models, however, little is known about the pathophysiology and specific molecular mechanisms behind VIDD. Recently, more investigators are developing and utilizing murine experimental model s of MV. The murine model is very beneficial due to the wide variety of molecular techniques and assays available, as well as the ability to genetically modified animals, allowing for proof of concept experiments and dissection of specific molecular pathways It is important that future investigations attempt to utilize a highly integrative approach (e.g. examining human diaphragm samples as well as utilizing genetically modified mice) in order to obtain the greatest amount of knowledge about this phenomenon. The results of the present study raise the questi on whether diaphragm oxygen supply demand imbalance play s a role in VIDD and weaning difficulties. Future studies are needed to investigate the role of the endothelial glycocalyx in diaphragm arterioles following prolonged MV. The diminished flow mediated and Achinduced vasodilation is indicative of endothelial dysfunction. It w ould beneficial to determine if there is a reduction in NO production following prolonged MV due to the disruption of the glycolcalyx. Additionally new therapeutic strategies ( cationic copolymers) have been developed to target and repair the endothelial glycocalyx (Giantsos et al., 2009). Therefore, further investigations are needed to determine whether these biomimetic

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77 polymers can be a form of infusible therapy to restore endothelial function and ultimately diaphragm oxygenation during prolonged MV Moreover, given the large body of evidence suggesting oxidative stress as a central component of VIDD, interventions aimed to reduce ROS and oxidative damage ( e.g anti oxidant supplementation, exercise training) will also be valuable in further dissecting th e molecular pathways involved in VIDD. Furthermore other potential vascular molecular pathways (e.g. Angiotensin pathway TGF ) need to be examined to gain better insight of diaphragm vascular function following prolonged MV. Another valuable t ool that can significantly improve respiratory muscle function in patients exper iencing weaning difficulties is diaphragm pacing. One of the primary triggers of ROS production in the diaphragm is due to the reduction in contractile activity by the unloadi ng of the diaphragm. The diaphragm, in contrast to other skeletal muscles, is activated rhythmi cally on a continuous period. MV imposes a unique form of muscle disuse as the diaphragm is simultaneously mechanically unloaded, intermittently shortened, and electrically suppressed by the ventilator (Petrof et al., 2010). Intermittent or continuous phrenic nerve stim ulation (and therefore diaphragm muscle contraction) m ay be beneficial in maintenance of the intracellular processes and molecular signaling pathways responsible for ATP production. Moreover, it i s not currently known whether intermittent or continuous pacing will improve the ability of the diaphragm to regulate the matching of O2 delivery to O2 uptake (in response to contractions), or alter diaphrag m vasomotor function following prolonged MV. Conversely, given the data presented in the current investigation, it will also be very beneficial to further examine the effect of flow to the diaphragm following prolonged MV. By the development and imple men tation of a

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78 diaphragm perfusion system, one could easily gain further insight into the role of blood flow to the diaphragm during prolonged MV. Additionally, one could observe whether the maintenance of blood flow during prolonged MV will prevent the characteristics of VIDD (e.g, contractile dysfunction, ROS production, Mitochondrial Dysfunction, Calpain/Protease Activation). Finally, it is imperative that we obtained detailed understanding of the current ventilation protocols and physiological parameters accessed by clinicians and doctors. It is very plausible that these variables (e.g. amount of saline infusion, type and amount of inhalant analgesics used) could have an impact impact on the ability to be weaned from a ventilator. Moreover, mult iple factors other than MV affect diaphragm function ( e.g. sepsis, corticosteroids, neuromuscular blockade, antibiotics, nutritional deficiency ; Laghi et al., 2003) The potential impact of these mediators also needs to be considered when investigating the pathophy siology behind the development of VIDD. Summary In summary, we have demonstrated that mechanical ventilation elicits a significant reduction in the microvascular PO2 in the diaphragm in as little as 6 hr, and this lowered P O2m is associated with a diminished diaphragm blood flow. Further, upon initiation of muscular contractions, there is an inability to augment diaphragm blood flow and O2 delivery which results in an inadequate matching of O2 delivery to O2 uptake. Taken together, the diminished O2 deliver y results in a ~80% reduction in the aerobic metabolism of the diaphragm during contractions. So what is causing this mismatching of O2 delivery to O2 uptake? Although the precise mechanisms are likely multifaceted, we provide clear evidence in favor of v ascular dysfunction being a key instigator in the

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79 oxygen supply/demand mismatch with prolonged mechanical ventilation and believe this is one potential mechanism contributing, in part, to weaning difficulties. Collectively, these data provide strong support that a diminished O2 supply (in addition to mitochondrial dysfunction) contributes to mechanical ventilationinduced diaphragm dysfunction. Moreover, the results from these experiments provide insight into the functional and structural mechanisms responsible for MV induced diaphragm atrophy in the diaphragm, and also for broader topics such as skeletal muscle wasting due to prolonged bed rest, immobilization, and disease states. Important questions to address in future work is the role of the diminished O2 supply on mitochondrial function, and what vasomotor pathways contribute to the reduce blood flow response after mechanical ventilation.

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92 BIOGRAPHICAL SKETCH Robert Thomas Davis III was born in Detroit, MI and spe nt most of his childhood between Winfield, KS and Aurora, IL. He graduated from Kansas State University (KSU) with a bachelors degree in Kinesiology in 2006. He decided continue on his graduate work at KSU and received his masters degree. In 2009 Robert moved to Gainesville, FL to attend the University of Florida and begin work on his Doctor of Philosophy in exercise physiology. Upon completion of his PhD, Robert plans to begin post doctoral training at University of Illinois at Chicago in the area of car diac and skeletal muscle contraction under the direction of Dr. John Solaro.