The Impact of Vasoactive and Inflammatory Reagents on Arteriolar Vasomotion in the Gluteus Maximus of Mice Travis Allen B.Sc, University of Northern British Columbia, 2006 Thesis Submitted in Partial Fulfillment of The Requirements for the Degree of Master of Science In Interdisciplinary Studies The University of Northern British Columbia April 2009 © Travis Allen, 2009 1*1 Library and Archives Canada Bibliotheque et Archives Canada Published Heritage Branch Direction du Patrimoine de I'edition 395 Wellington Street Ottawa ON K1A0N4 Canada 395, rue Wellington Ottawa ON K1A0N4 Canada Your file Votre reference ISBN: 978-0-494-48745-7 Our file Notre reference ISBN: 978-0-494-48745-7 NOTICE: The author has granted a nonexclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distribute and sell theses worldwide, for commercial or noncommercial purposes, in microform, paper, electronic and/or any other formats. AVIS: L'auteur a accorde une licence non exclusive permettant a la Bibliotheque et Archives Canada de reproduire, publier, archiver, sauvegarder, conserver, transmettre au public par telecommunication ou par Plntemet, prefer, distribuer et vendre des theses partout dans le monde, a des fins commerciales ou autres, sur support microforme, papier, electronique et/ou autres formats. The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriete du droit d'auteur et des droits moraux qui protege cette these. Ni la these ni des extraits substantiels de celle-ci ne doivent etre imprimes ou autrement reproduits sans son autorisation. In compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis. Conformement a la loi canadienne sur la protection de la vie privee, quelques formulaires secondaires ont ete enleves de cette these. While these forms may be included in the document page count, their removal does not represent any loss of content from the thesis. Bien que ces formulaires aient inclus dans la pagination, il n'y aura aucun contenu manquant. Canada Abstract Skeletal muscle depends on its arteriole network to meet metabolic demands during physical activity. However, inflammatory mediators can disrupt intercellular communication along arterioles, impairing blood flow regulation and thus negatively impact muscle function. This project evaluated arteriole responsiveness in the gluteus maximus (a locomotory muscle) of C57/BL6 mice in vivo using intravital microscopic techniques. Arterioles were demonstrated as functional using specific endothelial and smooth muscle cell specific reagents. Inducing inflammation with platelet activating factor (PAF) gave rise to an increased degree of arteriolar vasoconstriction as its concentration increased. In contrast, vasodilation transitioned to vasoconstriction as histamine concentration increased. However, the arteriolar effects of histamine were attenuated during NO inhibition with N^-nitro-Larginine methyl ester (L-NAME), illustrating a NO- related mechanism. Therefore, our study established the gluteus maximus as a viable candidate for inflammatory studies related to locomotion, so that the links between physical activity, inflammation, and microvascular health may be investigated. 11 TABLE OF CONTENTS Abstract ii Table of Contents iii List of Figures vi List of Abbreviations vii Acknowledgements viii Chapter 1 - Introduction 1.1 Importance of Skeletal Muscle and the Role of Microcirculation 1 1.2 Cell Communication within the Arteriolar Network 2 1.3 Intravascular Signaling and Gap Junctions 5 1.4 The Effect of Inflammation on Skeletal Muscle Health 6 1.5 Inflammation and Endothelial Integrity 10 1.6 Disruption of Nitric Oxide Balance 12 1.7 Analyzing Microvascular Function with Intravital Microscopy 15 Chapter 2 - Research Objectives 2.1 Mastering the use of IVM to Analyze Skeletal Muscle Microcirculation 19 2.2 Constructing a Vasoactive Range for Arterioles in a Locomotory Muscle 19 2.3 Observing the Effects of Inflammation on the Arteriolar Aspect of Microcirculation 20 2.4 Observing the Effects of Inflammation on Arterioles in a Locomotory Muscle 21 2.5 Investigating the Relation of the NO Pathway to Arteriolar Inflammation 22 in Chapter 3 - Methodology 3.1 Animal Care and Surgery 25 3.2 Chemicals and Reagents 26 3.3 Intravital Microscopy 27 3.4 Arteriolar Reactivity 29 3.5 Vasomotor Activity within the Arterioles 30 3.6 Characterizing Inflammatory Profiles 31 3.7 The Role of NO in the Locomotion Arterioles 31 Chapter 4 - Results 4.1 Gluteus Maximus 2A Endothelial Response to Vasodilator Application 33 4.2 Gluteus Maximus 2A Smooth Muscle Response to Vasoconstrictor Application 34 4.3 Gluteus Maximus 2A Arteriolar Response to Histamine Application 35 4.4 Gluteus Maximus 2A Arteriolar Response to PAF Application 36 4.5 Gluteus Maximus 2A Arteriolar Response to L-NAME Application 37 4.6 Gluteus Maximus 2A Arteriolar Response to Histamine in the Presence of L-NAME 38 Chapter 5 - Discussion 5.1 Validation of IVM Techniques and Tissue Integrity 40 5.2 Vasoactivity of the Gluteus Maximus 2A Arterioles 42 5.3 The Impact of Inflammatory Mediators on Gluteus Maximus 2A Arterioles 44 5.4 The Role of NO During a Physiological and Inflammatory state 47 IV Chapter 6 - Research Implications and Future Directions 6.1 Regulating Inflammation 51 6.2 Aging 54 6.3 Gender Differences 55 6.4 Physical Activity 55 Conclusion 57 References 58 v List of Figures Figure 1. Microvasculature of dissected skeletal tissue 3 Figure 2. The NO production pathway 14 Figure 3. Intravital microscope apparatus 17 Figure 4. Mouse preparation for intravital microscopy recordings 28 Figure 5. Measurement of arteriole diameter 29 Figure 6. Acetylcholine dose response curve 33 Figure 7. Phenylephrine dose response curve 34 Figure 8. Histamine dose response curve 35 Figure 9. Platelet Activating Factor dose response curve 36 Figure 10. L-NAME time response curve 37 Figure 11. The effect of histamine during inhibition of NO production 38 Figure 12. The effect of histamine before and after inhibition of NO production 39 VI List of Abbreviations Ach Acetylcholine CVD Conducted Vasodilation Cx Connexin EDHF Endothelium Derived Hyperpolarization Factor eNOS Endothelial Nitric Oxide Synthase iNOS Inducible Nitric Oxide Synthase IL-6 Interleukin-6 IVM Intravital Microscopy L-NA Nitro-L-arginine L-NAME N^-nitro-L-arginine methyl ester L-NMMA N^-monomethyl-L-arginine LPS Escherichia coli Lipopolysaccharide Endotoxin MMP Matrix Metalloproteinases NO Nitric Oxide nNOS Neuronal Nitric Oxide Synthase PAF Platelet Activating Factor PE Phenylephrine PGI 2 Prostacyclin (or Prostaglandin I2) PSS Physiological Saline Solution RBC Red Blood Cell ROS Reactive Oxygen Species SNP Sodium Nitroprusside TNF-a Tumor Necrosis Factor- a vii Acknowledgements I would like to thank Stephen Rader, Andrea Gorrell, and Roy Rea for their continuous guidance and inspiration during both my undergraduate and graduate studies. Additionally, I would like to thank Lydia Troc and Dee Jones for all their generosity and patience in the animal care facility. I am also thankful to Jordie Fraser, Stephanie Sellers, Ravinder Grewal, Heath de la Giroday and other past and present researchers of the Northern Biosciences Research Unit for sharing the laughter and tears of research with me. Furthermore, I would like to thank all my family and friends for being a never-ending source of encouragement and keeping my priorities in perspective. Most of all, I would like to thank Dr. Geoffrey W. Payne for making my studies such an enjoyable challenge and a rewarding experience. viii CHAPTER 1: INTRODUCTION Chapter 1: Introduction 1.1 Importance of Skeletal Muscle and the Role of Microcirculation Regular physical activity is crucial for maintaining a healthy body. Exercise improves the quality of the cardiovascular system, providing protection from many chronic metabolic conditions. Another benefit of exercise is an improvement in local blood flow supply, which increases in proportion to the metabolic demands of the muscle tissue (Clifford & Hellsten, 2004). Thus, having a healthy systemic circulation and locomotion abilities to negate the onset of cardiovascular disease depends on the local circulation feeding the skeletal muscles. Deficiencies in blood flow regulation will result in breakdown of muscle tissue, thus increasing susceptibility to a sedentary lifestyle (Payne, 2006). The health of skeletal muscles is controlled by their microcirculation. Whereas the macrocirculation distributes blood flow globally throughout the body, the microcirculation is the critical component for blood flow regulation and homeostasis within individual tissues (Trzeciak et al., 2007). A direct exchange of oxygen, nutrients, and waste products take place within these tissues (Chierego et ah, 2006). The tissue perfusion and vascular resistance is made possible by the high surface area of three types of microvessels: capillaries, arterioles, and venules. Capillaries have a diameter of about 5 to 10 \im, whereas arterioles and venules have diameters ranging from 10 to 25 |im (Mchedlishvili & Maeda, 2001). Together, these vessels are the largest endothelial surface in the mammalian body, encompassing more than 0.5 km2 of its total surface area (Verdant & De Backer, 2005). The majority of these microvessels are contained in skeletal muscle, an organ system that contributes to 40% of 1 CHAPTER 1: INTRODUCTION total body mass (Payne & Bearden, 2006). Therefore, a large percentage of the body will receive cardiovascular benefits if the skeletal muscle perfusion is maintained. 1.2 Cell Communication within the Arteriolar Network In order for skeletal muscle to receive its needed blood supply, molecular signals must be communicated through its feeding arterioles via intercellular communication. Oxygen and nutrients are fed to the muscle through an intertwined arteriolar network (Figure 1), which is composed of resistance segments arranged in series and in parallel (LooftWilson et ah, 2004). Through coordinated or conducted vasodilation (CVD), the signal is propagated along and between neighbouring branches to maximize the blood delivery of the network. 2 CHAPTER 1: INTRODUCTION 1A 2A 3A - M^ ( Figure 1. Microvasculature of dissected skeletal muscle tissue. Proximal arterioles (1A) branch into 2A and 3 A arterioles (in red). The 2A arterioles are of interest in microvascular research because they are the major distributing vessels and responsible for nitric oxide (NO) contribution (VanTeeffelen et ah, 2005). Note also that arterioles run in parallel to postcapillary venules (in blue). This can facilitate communication between the components of the microcirculation, such as when leukocytes accumulate during inflammation. During exercise, the contracting muscle can initiate a signal of vasodilation within the arteriolar network to respond to the increase in metabolic demand (Segal & Jacobs, 2001). Skeletal muscle can in fact be considered an endocrine organ because contraction stimulates the production and release of cytokines that can influence metabolism for both the muscle itself and other tissues and organs (Nielsen & Pederson, 2008). A common vasodilating 3 CHAPTER 1: INTRODUCTION signal produced by the muscle is through the release of acetylcholine (Ach). The Ach delivered from the neighbouring muscle fibers triggers a local increase in endothelial cell [Ca2+], which activates calcium dependent K+ channels (Kca) (Domeier & Segal, 2007). As the endothelial [Ca2+] rises, Kc a channels open for K+ to escape and the membrane hyperpolarizes, signaling the arteriole to vasodilate (Dora et al., 2003). This process continues upstream through the endothelium and the smooth muscle cell layers, relaxing the arteriole in less than a second (Payne et al, 2004). The hyperpolarizing signal is spread primarily by nitric oxide (NO), prostacyclin (PGI2), and endothelium derived hyperpolarization factor (EDHF). These three secondary messengers all travel from the endothelium to the vascular smooth muscle to induce relaxation (Matoba & Shimokawa, 2003). A specific structure of EDHF remains to be clarified, but its hyperpolarization can be inhibited by increasing the concentration of extracellular potassium ions or by the presence of potassium channel inhibitors (Takano et ah, 2005). An increase in [Ca2+] may be the initiator of vasodilation locally, but endothelial [Ca2+] did not increase at distances of 500 and 1000 \im upstream from the site of initiation (Dora et ah, 2003). It is more difficult for Ca2+ to travel along the arteriole because the arteriolar response depends on the electrical resistance of the pathway the CVD travels (Kurjiaka, 2004). To overcome these electromechanical obstacles, the endothelium must rely on other molecular messengers. Whereas Ca2+ travels slowly along the endothelium (-111 um/ second), the NO and PGI2 activated by Ca can transmit faster (~1 mm/ second) (Domeier & Segal, 2007). Secondary messengers can therefore carry the CVD signal more quickly and efficiently. The observation of vasodilation upstream of stimulation (de Wit, 2004) also illustrates that vasoactive signals are not merely flow-mediated and must travel 4 CHAPTER 1: INTRODUCTION between cells of the arteriole against the current of blood flow. Thus, having strong cell communication within the arteriole will facilitate the spread of vasodilatory signals. 1.3 Intravascular Signaling and Gap Junctions The three major intercellular junctions responsible for vascular continuity are tight junctions, adherens junctions, and gap junctions. Tight junctions prevent vascular leakage between cells, adherens junctions link the cytoskeleton, and gap junctions allow intercellular communications. Therefore, gap junctions are essential for allowing the transmission of vasoactive messages. These junctions consist of connexin subunits assembled into hexameric channels, which mediate the direct exchange of ions and small molecules (second messengers, metabolites, linear peptides, mRNA) between the cells (Chanson & Kwak, 2007). To date, 21 connexin subunits (Cx's) have been described (Tran & Welsh, 2009). In general, Cx37 and Cx40 subunits are found predominantly in endothelial gap junctions and Cx43 and Cx45 subunits are found in smooth muscle gap junctions (Figueroa et ah, 2003). These gap junctions are present in both endothelial and smooth muscle cells of the arteriole, but connexin assembly can differ (de Wit, 2004). Variation in connexin assembly has been shown to correspond to arteriole function. For example, Cx40 deficient mice were found to have a slower spread of vasodilation in skeletal muscle arterioles, as well as a higher blood pressure (de Wit et ah, 2000). Thus, there is potential for diversity of cell communication within the arteriole. 5 CHAPTER 1: INTRODUCTION Of the two cell layers within the arteriole, it is known that the endothelium is the main type driving the CVD. This is because larger levels of connexin expression were found in endothelial cells than in smooth muscle cells of skeletal muscle (Looft-Wilson et ah, 2004). The smooth muscle layer is also more heterogeneously coupled by gap junctions that are small and rare (Rummery & Hill, 2004). Myoendothelial gap junctions have been found between the two microvascular layers, but they are smaller and less numerous than the homocellular gap junctions (Griffith, 2004). The importance of each vascular layer has also been tested using selective damage. One method uses light-dye treatment to photodamage abluminal (smooth muscle) or luminal (endothelium) regions before the CVD induction. When vasodilation was induced in the hamster retractor muscle after this damage, conduction was impaired along the damaged segment; selective smooth muscle damage had no effect on conduction past the damaged site (Emerson & Segal, 2000). Therefore, the strength of a vasodilatory wave depends primarily on the cell communication within the endothelium. 1.4 The Effect of Inflammation on Skeletal Muscle Health One of the potential promoters of skeletal muscle dysfunction is inflammation (Visser et ah, 2002). Inflammation can directly impact both the skeletal muscle and its feeding microcirculation, particularly in an aging population. An increased rate in muscle atrophy found in aging individuals is associated with the presence of chronic low-grade inflammation (Hsu et al., 2009). For instance, higher plasma concentrations of IL-6 and TNF-a (inflammatory mediators) are associated with lower muscle mass and strength in wellfunctioning older men and women (Pedersen et al., 2003; Visser et ah, 2002). Chronic low6 CHAPTER 1: INTRODUCTION grade inflammation is characterized by a two- to four-fold elevation in circulating levels of inflammatory mediators, as well as minor increases in counts of neutrophils and natural killer cells (Bruunsgaard, 2005). In addition, an increased number of mast cells, a source of inflammatory mediators, are found in aging individuals (Kruger & Lagunoff, 1981). The inflammatory environment can also give rise to endothelial dysfunction, interfering with the ability of arterioles to dilate. For example, endothelium-dependent vasodilation was found to be reduced in arterioles from skeletal muscles of aged animals (Muller-Delp, 2002). Reduced blood flow diminishes the arteriole's ability to meet muscle's metabolic demands, consequently limiting the ability of older adults to perform functional tasks. Regardless of age, exercise helps lower the inflammatory state within muscles. For instance, inflammatory mediators obtained from muscle biopsies were reduced by 50% in the frail, obese elderly after exercise, whereas weight loss had no effect (Lambert et ah, 2008). However, muscle mass in the highly active elderly is still lower than that of younger individuals with sedentary lifestyles (Klitgaard, 1990). This discrepancy is due to sarcopenia, also known as age-related muscle wasting. In addition to a loss of skeletal muscle mass, this disease is characterized by a gradual decline in muscle function, such as a general slowing of contraction and relaxation (Ryall et ah, 2008). However, future damage by inflammatory mediators may be minimized with exercise training, which decreases levels of circulating inflammatory monocytes (Timmerman et ah, 2008). Exercise is clearly beneficial for the elderly (Lambert et al., 2008), but in a limited manner when there is less functional skeletal muscle to utilize. The increased metabolic demand put on the remaining muscle tissue makes microcirculation perfusion even more crucial. 7 CHAPTER 1: INTRODUCTION A prevailing inflammatory state is not necessarily restricted to the elderly. There are numerous conditions that create a chronic inflammatory state within the microvasculature of skeletal muscle. For instance, the inflammation caused by chronic obstructive pulmonary disease is linked to increased muscle wasting (Degens & Alway, 2005). Furthermore, weight gain is associated with a higher expression of inflammatory mediators in human skeletal muscle, as indicated by the similar inflammatory profiles of overweight middle-aged men and the elderly (de la Maza et al., 2006). Even a single high fat meal reduced brachial artery endothelium-dependent vasodilation for up to four hours in healthy, normocholesterolemic volunteers (Plotnick et al., 1997). An increase in weight is also tied to microvascular complications in animals. For example, a diet high in fat impaired the endotheliumdependent dilations of skeletal muscle arterioles in rats, due to the increased presence of oxidative stress (Erdei et al. 2006). Therefore, an individual's age can be considered a relative term, as the amount of circulating inflammatory mediators and lifestyle affects the physiology of the microcirculation. A low-grade chronic inflammatory state will cause microvascular dysfunction, but so will a severe, acute inflammatory state. The onset of sepsis is an excellent example of this other end of the inflammatory spectrum. During temporary states of acute inflammation, the body's defenses can usually clear minor infections while causing minimal damage to nearby tissues (Bucci et ah, 2005). During sepsis, the immune system becomes hyper-inflammatory as it attempts to destroy any challenging pathogens, upregulating inflammatory mediators and reactive oxygen species (ROS) (Rudiger et al., 2008). ROS are proinflammatory mediators that are capable of activating matrix degrading proteins (MMPs), which will induce apoptosis of nearby cells (Xiong et al., 2008). Even though this aggressive reaction 8 CHAPTER 1: INTRODUCTION will destroy pathogens, the body's own muscle and vascular cells can become collateral damage. Thus, sepsis may cause muscular atrophy through direct and indirect methods. If not reversed, systemic inflammation can lead to multiple organ failure (Sakr et al, 2004). The current challenge in treating this type of inflammation is illustrated by the impact of septic shock in hospitals. This condition remains the predominant killer throughout intensive care units, with high mortality rates (Groeneveld et al., 2008). For instance, Sakr et al. (2004) observed that among 49 intensive care unit patients suffering from septic shock, only 26 overcame their condition. Doctors respond to this condition by restoring the blood pressure and cardiac output (i.e. macrocirculation) in these patients, but septic shock was not overcome without improved microvessel perfusion (Sakr et al, 2004; Trzeciak et al, 2008). A healthy macrocirculation does not necessarily translate to a healthy microcirculation. Another validation of this concept is how patients can have normal coronary angiograms, yet also an abnormal arteriolar response to sympathetic stimulation because of prevailing systemic microinflammation (Schindler et al., 2004). If the impact of the inflammation on the microcirculation can be better understood, more effective therapies can be designed that utilize the complete cardiovascular tree. Furthermore, mortality from systemic inflammation will be decreased by repairing the microcirculation of the body's largest organ: skeletal muscle. 9 CHAPTER 1: INTRODUCTION 1.5 Inflammation and Endothelial Integrity The endothelium is a semi-permeable barrier that regulates the transport of fluids and solutes into and out of the blood. This barrier is held together by cell adhesion structuresdistinct trans-membrane proteins that assemble into arrays, strands, or focal contacts (Segretain & Falk, 2004). Vascular permeability in the microcirculation is essential for maintaining tissue perfusion. However, vascular permeability is also a characteristic of many disease states when permeability increases beyond regular limits, such as during inflammation (Nagy et ah, 2008). Within normal physiological parameters, permeability is tightly regulated and reversible. The endothelial cells will display vasodilator, anti-coagulant and anti-adhesive properties (Crimi et ah, 2007). Yet, during inflammation the disruption of the vascular barrier often results in the recruitment of platelets and leukocytes to the site of the leak (Weis, 2008). The endothelial activation during inflammation will consequently allow molecules to cross and infiltrate the intravascular space and threaten endothelial integrity (Payne, 2006). This loosened adhesion could be through inflammatory mediators disrupting tight junctions, gap junctions, and adherens junctions between endothelial cells (Payne et ah, 2004). If the disruption of endothelial integrity is considered within the realm of vasomotor signal conduction, it becomes apparent that an inflammatory state will break down cell communication. The induced vascular hyperpermeability has been found to impair CVD because the endothelial gaps allow ions and proteins to escape (Nagy et ah, 2008). Consequently, vasoactive signals will be more difficult to conduct through the uncoupled pathway to meet the demand of the skeletal muscle tissue. 10 CHAPTER 1: INTRODUCTION It is important to note that disruption of vascular integrity has already been observed in capillaries and post-capillary venules. For example, electron microscopy has shown that the addition of histamine to skeletal muscle microcirculation causes partial disconnection of the intercellular junctions between endothelial cells of venules (Majno & Palade, 1961). Yet, little research has focused on the direct effects of inflammation on the intercellular communication of the arterioles. It is also just as likely that the cytoskeleton between arteriolar subunits is becoming loosened, as proposed by Payne (2006). When researched in vitro, the application of inflammatory mediators inhibited gap junctional intercellular communication between cultured human endothelial and smooth muscle cells (Hu & Cotgreave, 1997). Also, permeability to macromolecules in arterioles was found to be lower than venules under noninflammatory conditions (Sarelius et ah, 2005). Inflammatory mediators may also affect gap junction expression by decoupling connexin coregulation, leading to altered structures and disturbed vascular heterocellular communication (Isakson et ah, 2006). Bolon et al. (2008) found that the induction of sepsis has disrupted cell communication within the endothelium of mouse skeletal muscle by phosphorylating Cx40. The decreased endothelial cell coupling during inflammation can contribute to endothelial dysfunction as well as alter vasomotor responses (Simon et al., 2004). Even if vasodilation is still possible at a local site, the supply to the rest of the muscle will be minimal without proper cell communication. Further research is required to clarify the inflammatory mechanisms that interfere with blood flow to skeletal muscle. 11 CHAPTER 1: INTRODUCTION 1.6 Disruption of Nitric Oxide Balance One of the key secondary messengers studied in microvascular health research is nitric oxide (NO). This is a potent vasodilator produced by three different nitric oxide synthases (NOS) within mammalian tissue. The NO generated by the neuronal NOS (nNOS) and endothelial NOS (eNOS) isozymes has beneficial effects such as vasodilation, inhibition of platelet aggregation and leukocyte adhesion to the endothelium (Crimi et al., 2007). Thus, both the endothelium and neurons are potential sources of NO within skeletal muscle tissue that can be used during contraction (Clifford & Hellsten, 2004). The eNOS and nNOS isoenzymes generally produce low, physiological levels of NO and require an increase in intracellular calcium to stimulate activity (Gocan et al., 2000). A calcium requirement ensures vasodilatory regulation. Gap junction conductivity can also be modulated through NO acting on connexins within the arteriole (Rodenwalt et ah, 2007). The NO production pathway is a key mechanism for inflammatory agents to inhibit CVD when activated to an excess (Payne et al., 2004). Consequently, active skeletal muscles will receive a lower blood supply. For instance, the inflammatory overdrive present in sepsis causes so much NO overproduction in rats that after five hours, their skeletal muscle oxygen consumption dropped by 50% (Bateman et al., 2008). The large surplus of NO can also react with any superoxide anions to produce toxic compounds (Toda et al., 2008). For example, O2" (a ROS) will combine with NO to generate peroxynitrite (ONOO), causing DNA strand breakage within cells (Cinel & Opal, 2009). High levels of NO are also seen within the skeletal muscle of aging mice during a chronic state of low-grade inflammation (Bearden, 12 CHAPTER 1: INTRODUCTION 2007). Therefore, the health of both skeletal muscle and its embedded microvasculature will be threatened if NO is not regulated. Production of NO can become excessive when inducible nitric oxide synthase (iNOS), the third isozyme, becomes activated by inflammatory stimuli. Whereas eNOS and nNOS generate low levels of NO, iNOS activation produces a larger and more persistent concentration. This can lead to problems such as hypotension, negative inotropic effect, prooxidant properties, apoptosis, mediation of the effects of cytokines, and cytotoxic innate immunity (Crimi et al, 2007). Overproduction of NO can also lead to a dramatic decrease in red blood cell (RBC) deformability, which can lead to random blockage of the microvascular beds and reduced blood flow (Bateman et al, 2001). Thus, this imbalance will induce selfdestructive signals within the body. Also, the presence of iNOS in both myocytes and endothelial cells (Cengel & Sahinarslan, 2006) can make the enzyme very influential on skeletal muscle microcirculation. Furthermore, this isoform is calcium independent, so it can respond to various other molecular signals outside of the skeletal muscle to produced high levels of NO (Gocan et ah, 2000). An overview of NO production is illustrated in Figure 2. 13 CHAPTER 1: INTRODUCTION TM '• 11 • ' ^ L-arginine I eNOS « VD f V / + + • • • L-caullne Endothelial cell Smooth muscle cell Figure 2. The NO production pathway. Vasodilators (VD) bind to endothelial receptors to stimulate NO synthesis via a calcium dependent mechanism. The nNOS pathway is also calcium dependent within neurons. In contrast, inflammatory mediators (IM) stimulate NO synthesis in a calcium independent manner. The synthesized NO then travels to the smooth muscle layer to stimulate cyclic guanosine monophosphate (cGMP) synthesis, consequently promoting relaxation in the arteriole. Note that NO is produced by the NOS isozymes in other cell types as well. Although eNOS and nNOS have traditionally been attributed to healthy NO production, there have been documented exceptions. For instance, upregulated nNOS has also been shown to impair vasodilative responses in skeletal muscle (Gocan et al., 2000) and eNOS has been shown to be upregulated during sepsis (McKinnon et al., 2006). Thus, all three have the potential to impair cell communication with the appropriate trigger from inflammatory mediators. Regardless of the source, elevated levels of NO are hypothesized to compromise intercellular coupling during inflammation (McKinnon et al., 2006). The key to arteriolar health is keeping NO levels down to baseline levels so that the endothelial function 14 CHAPTER 1: INTRODUCTION may be maintained. Conducted vasodilation may still occur without NO release from the endothelium (Payne et ah, 2004), but it could be much weaker from having to rely on the PGI2 and EDHF messengers. Thus, microvascular efficiency in the skeletal muscle will be greater if NO levels are regulated. 1.7 Analyzing Microvascular Function with Intravital Microscopy Understanding how the microcirculation performs under pathological conditions requires observing these processes as they occur. Intravital microscopy (IVM) is the primary method used for researching the microcirculation. Using an IVM enables a visualization of active microvessels within a tissue in vivo. Tissue is mainly excised from rodents, as their small bodies can be rested under the lens of the IVM, allowing a high resolution of the microvasculature (Gavins & Chatterjee, 2004). Their thin tissue is excised and pinned onto clear silicone to facilitate light penetration through the preparation. Through these techniques the individual microvessels can become visible through the lens of the microscope. Once tissue is excised from the body, it is immersed in a flowing solution to keep it from drying out. Solutions are brought to neutral pH with the necessary salts to maintain physiological activity within the microvasculature. In addition, a constant bubbling of CO2/ N2 into all flowing solution will act as a buffer, maintaining pH at 7.38-7.44 (Bearden, 2007). Finally, all solutions are also immersed in a water bath calibrated to body temperature. Therefore, researchers can externalize skeletal muscle from the mouse while still maintaining the tissue's physiological environment. 15 CHAPTER 1: INTRODUCTION In addition to viewing the microcirculation through the microscope eyepiece, the IVM also has a video camera attachment. Video-based methods are the method of choice for obtaining blood flow measurements in the microvessels because recordings can be saved for later off-line processing and analysis (Pittman, 2000). The use of video footage also allows a complete recording of time-dependent processes, such as changes in vessel diameter or red blood cell velocity through the microvessels (Gavins & Chatterjee, 2004). To enhance visibility, fluorescent filters can be fitted to the microscope to measure the signal intensity of tagged cells within the tissue, such as detecting microvascular leakage of RBC's with fluorescein isothiocyanate-albumin (Baldwin et ah, 1998). Having the IVM camera linked to a nearby computer (Figure 3) allows both a qualitative and quantitative measurement of the microcirculation in real time. The ability to measure structural and physiological changes so effectively makes IVM a crucial tool in inflammation studies. 16 CHAPTER 1: INTRODUCTION Video camera Intravital microscope Video screen Computerized recording program Mouse preparation Video calipers Figure 3. Intravital microscope apparatus. The microvasculature image viewed through the objective lens is fed into a camera attached above. The image is fed into a video screen so that vessel diameter can be measured with video calipers. All recordings are stored on a linked computer. Another benefit of measuring inflammation with IVM is that the same muscle may be used as an internal control against future vasoactive reagents. Using the same tissue throughout the experiment creates an internal control for treatment, so no sham (euthanized controls) and treatment animals need to be compared (Gavins & Chatterjee, 2004). For instance, any anti-inflammatory treatments or interventions can be used on the same tissue source to visualize effectiveness against pre-treatment characteristics. Thus, this method minimizes animal use. The major challenge encountered during IVM studies is creating and maintaining a physiological environment for the tissue similar to the one inside the mouse before experimentation. Thin tissue can easily be seen through the microscope, but it can also be 17 CHAPTER 1: INTRODUCTION sensitive to post-excision conditions (Kumer et al, 2000). There are many physiological variables that must be simultaneously balanced by the researcher during experiments for preserving microvascular function and acquiring viable data. For example, if the tissue is not handled gently during microsurgery, microvessels will rupture and torn muscle will release inflammatory mediators (Gierer et al., 2008). Thus, when performing extended experiments on a single sample of tissue, tissue integrity must be monitored at all times. In summary, it is clear that a prevailing inflammatory state is detrimental to the functionality of the skeletal muscle's embedded microvessels. Cardiovascular deficits have been observed in both a clinical setting as well as animal models. In regards to the arteriolar network, both chronic low-grade and acute severe inflammation will disrupt its cell communication. The presence of inflammatory mediators may disrupt the transmission of cellular signals through structural alterations to the endothelial layer (such as hyperpermeability or gap junction assembly) or promoting unbalanced production of harmful cytokines (such as other mediators, ROS or NO). However, since knowledge regarding the impact of inflammation on arterioles is limited, it is important to trial individual mediators in the construction of arteriolar inflammatory profiles. Intravital microscopy allows a direct visualization of a mouse's skeletal muscle microcirculation in vivo so that these inflammatory effects can be recorded. Since inflammation is a predominant force in so many diseases today, understanding how it influences the microcirculation will clarify what it takes for these conditions to develop. 18 CHAPTER 2: RESEARCH OBJECTIVES Chapter 2: Research Objectives 2.1 Mastering the use of IVM to Analyze Skeletal Muscle Microcirculation Intravital microscopic techniques are the best method for measuring the microcirculation in vivo (Gavins & Chatterjee, 2004). However, since live animals are used, many variables need to be simultaneously balanced to produce physiological conditions in the externalized tissue and make any obtained results applicable to internal conditions. Obtaining viable tissue requires maintaining proper temperature, pH, salt concentration, solution flow rate, plane of anesthesia, and surgical efficiency (Kumer et ah, 2000). Therefore, experimental procedures must be fine-tuned and followed in a manner to obtain data from the IVM that is not only consistent within one experiment, but also agrees with results obtained from other animals. Furthermore, each mouse can have its own specific physiological challenges, so flexible methods must be designed for obtaining data. In addition, animal research protocol must be learned and followed in a manner that is safe for each mouse being used, as well as the remaining animals and personnel using the facility. Therefore, overcoming the large learning curve of animal physiological research is essential before proceeding with data collection on the arterioles of the gluteus maximus. 2.2 Constructing a Vasoactive Range for Arterioles in a Locomotory muscle Constructing a vasoactive profile of the mouse gluteus maximus arteriole network is vital measure arteriole reactivity. Two common vasoactive agents used in microcirculatory research are acetylcholine (Ach) and phenylephrine (PE). Endothelium-dependent 19 CHAPTER 2: RESEARCH OBJECTIVES vasodilation is activated when Ach binds muscarinic receptors, whereas PE evokes constriction via a-adrenoreceptors on smooth muscle cells (Hungerford et al, 2000). The vasodilation induced with Ach and vasoconstriction with PE will enable construction of a vasoactive range for the 2A arterioles. The mechanism of action of each reagent also allows arteriole reactivity to be broken down into the endothelial and smooth muscle components (Bartlett & Segal, 2000). Therefore, the use of each will provide the range of vasomotor control within the gluteus maximus. Furthermore, reactive arterioles validate the microsurgical techniques being used before inflammation is induced. 2.3 Observing the Effects of Inflammation on the Arteriolar Aspect of Microcirculation More inflammatory research has focused on post-capillary venules rather than arterioles because of clear structure changes observed. In particular, gaps between vascular units have already been noted in venules after administration of histamine (Majno & Palade, 1961). This visible increase in microvascular permeability will facilitate leukocyte transmigration into the intravascular space (Weis, 2008). To date, no leukocyte transmigration across the arteriole wall appears to have been observed (Bailey et al., 2007). However, leukocytes could still hypothetically be causing inflammatory effects within the arteriole because leukocyte adhesion along the endothelial layer has also been observed in arterioles (Bailey et al, 2007; Nolte et al., 2004). It is possible that secreted inflammatory mediators could be acting on the arteriole endothelium during this adhesion. The close proximity of each component of the microcirculation also makes it possible for inflammatory signals to be delivered to arteriolars from neighbouring venules. These signals could also be 20 CHAPTER 2: RESEARCH OBJECTIVES originating from other cells besides leukocytes, such as platelets and mast cells (Liu & Xia, 2006) as well as the endothelium itself (Trzeciak et ah, 2008). Therefore, a greater understanding of inflammation cannot be achieved without more insight into the arteriolar pathophysiology. The second-order (2A) arterioles were examined because these branches are the key distributing vessels within the mouse gluteus maximus responsible for regional control of blood flow (Bearden et ah, 2007). 2.4 Observing the Effects of Inflammation on Arterioles in a Locomotory Muscle Previous research has studied the effect of inflammation on the arteriole of the mouse cremaster muscle. The cremaster is a thin muscular extension of the abdominal wall that holds the testes and epididymus in place (Armstrong et ah, 2007a). This makes the cremaster a less than ideal candidate for describing conditions linked to physical activity. Furthermore, endothelial signaling pathways are specifically adapted to muscle-fiber type and function (Muller-Delp et al, 2002). Skeletal muscle tissue is made up of three different types of fibers: slow-twitch oxidative fibers, fast-twitch glycolytic fibers, and fast-twitch oxidative/ glycolytic fibers (Aaker & Laughlin, 2003). However, skeletal muscles have the ability to adjust their fiber composition to adapt to their physiological demands. Their phenotypic profiles are affected not only by genetics and innervations/ neuromuscular activity, but also by exercise training, hormones, and aging, causing transitions from fast-to-slow or slow-tofast fiber types (Jin et ah, 2008). Thus, the different function of each skeletal muscle in the body will determine its fiber composition and microvascular function. Since the cremaster muscle is located around the testes, the majority of microcirculatory muscle research has 21 CHAPTER 2: RESEARCH OBJECTIVES been confined to males. Therefore, using the gluteus maximus as a tissue source will remedy these problems and open the door for new avenues of research. To our knowledge, no previous studies have examined the effect of inflammation or the involvement of individual mediators on arterioles of the gluteus maximus in mice. Because each mediator may disrupt cell communication to different degrees, obtaining signal profiles of each may help in the development of future therapies. Histamine has been used to induce inflammation in mouse cremaster arterioles (Payne et ah, 2004), so its use in the gluteus maximus will allow a comparison between muscle tissues. This study will also examine the impact of platelet activating factor (PAF), another common inflammatory mediator. This potent mediator not only induces an inflammatory reaction, but also mediates synthesis and release of other mediators to aggravate the degree of inflammation (Chen et ah, 2008; Liu & Xia, 2006). Consequently, PAF also has a high potential of inducing changes to microvascular structures that will affect blood flow regulation. Better understanding these common mediators will create a more complete picture of how inflammation affects the arteriole. 2.5. Investigating the Relation of the NO Pathway to Arteriolar Inflammation Further understanding how inflammation impacts the skeletal muscle arteriolar network requires probing into how diameter changes are mediated. The NO pathway has already been confirmed in the cremaster using histamine mediated inflammation. There was little effect of this inflammatory mediator on eNOS T mice or when NOS activity was 22 CHAPTER 2: RESEARCH OBJECTIVES pharmacologically inhibited by nitro-L-arginine (L-NA) (Payne et al., 2004). However, NO may not be the only secondary messenger being used to transmit a vasodilatory signal within the skeletal muscle arteriolar network. There is the potential for other secondary messengers within the endothelium to compensate if the NO pathway is inhibited, such as EDHF or PGI2 (Hungerford et al., 2000). For example, Spier et al. (2007) found that NO inhibition triggered EDHF compensation to transmit CVD signals. Therefore, the predominant pathway being utilized by inflammation within a locomotory muscle must be clarified. Discovering the pathway used by inflammatory mediators within the gluteus maximus is important to see if the effects can be reversed. Changes in arteriolar size could potentially be reversed through inhibition of NO production by using of Nw-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor (Kumer et al, 2000). Thus, NO inhibition will help rule out one of the major pathways. The concept of reversibility is promising because it shows that treatments may be designed in the future to protect muscle function from inflammation. Especially since the systemic low-level inflammation observed in today's metabolic conditions may represent a spillover from local pro-inflammatory processes (Bruunsgaard, 2005). If skeletal muscle represents the largest organ within the body, then it only makes sense to focus on controlling local inflammatory responses within its microvascular bed. Molecular therapies are already currently being developed to minimize inflammation, such as inhibiting TNF-a in animal trials to combat muscular atrophy (Roth et al., 2006). There are also histamine receptor antagonists for both the HI (pyrilamine) and H2 (cimetidine) receptors (Van de Voorde et al., 1998). Therefore, understanding the mechanism of action of 23 CHAPTER 2: RESEARCH OBJECTIVES inflammatory mediators in the microvascular bed of skeletal muscle will lead to methods of targeted regulation in the future. In summary, the objectives of this study were: 1) To learn and master microsurgical and intravital microscopic techniques required for studying mouse microcirculation. 2) To assess endothelial and smooth muscle responsiveness within 2A arterioles of the mouse gluteus maximus after muscle exteriorization. 3) To record how inflammatory mediators affect arteriole diameter in the gluteus maximus, using histamine and PAF. 4) To gauge the role of the NO pathway in the gluteus maximus arterioles during inflammation, using histamine during pharmacological NO inhibition. 24 CHAPTER 3: METHODOLOGY Chapter 3: Methodology 3.1 Animal Care and Surgery All procedures were approved by the UNBC Animal Care and Use Committee. Mice were purchased from Jackson Laboratories (Bar Harbour, ME) and housed at 22°C over a 12 hour light/ 12 hour dark cycle with free access to fresh food and water before experimentation. Male C57BL6 mice of 8-12 weeks of age between 20-30 g were each anesthetized with a ketamine/ xylazine cocktail (1 mg ketamine/ 10 g; 0.1 mg xylazine/10 g) via intraperitoneum injection. We developed an anesthetic protocol to monitor the plane of anesthesia every 10 minutes. Additional injections were provided as needed to prevent withdrawal from toe pinch. To prepare mice for surgery, the back was shaved and any excessive hair that could interfere with muscle function and visibility was removed. Mice were then laid prone on an acrylic stage under a stereomicroscope for excision of the gluteus maximus muscle with sterile microsurgical instruments. Body temperature was maintained at 34°C with a heating lamp. A Pasteur pipette was used to keep the muscle moist with freshly prepared physiological saline solution (PSS) (pH 7.4, 34°C, 131.9 mM NaCl, 4.7 mM KC1, 1.2 mM MgS04-7H20, 2.0 mM CaCl2-2H20, and 18.0 mM NaHC03, equilibrated with 5% C02-95% N2, as used by Payne et al. (2004). The proximal edge of the muscle was freed from the spine by cutting connecting tissue, allowing the muscle to peel away. Careful attention was paid to prevent touching the muscle with surgical instruments, as well as to preserve the muscle's neurovascular supply and insertion into the femur. Furthermore, inflammatory tissue damage 25 CHAPTER 3: METHODOLOGY in the central region was minimized by handling only the outer edges of the muscle. The freed muscle was pinned down flat over a transparent Sylgard 184 mold (Dow Corning, Midland, MI) and the complete apparatus transferred to an intravital microscope for analysis. After each experiment was finished, mice were euthanized via anesthesia overdose to the intraperitoneum (Payne et ah, 2004). One 2A arteriole was examined for each mouse in the central region of the muscle for all experiments. No tissues were used for data collection if white blood cells within the microvessels or dysfunctional blood flow were detected through the IVM after microsurgery. Preliminary microsurgeries were performed (n= 17) before using any vasoactive reagents to minimize inflammatory reactions and optimize tissue exteriorization. Once the protocols were established, at least five mice were used for each IVM experiment to establish statistical significance (n = 13 for Ach experiments, 13 for PE, 13 for histamine, 13 for PAF, 14 for LNAME, and 7 for L-NAME/ histamine). Any mice that had adverse reactions to anesthetic or PSS wash were not used for data collection. A total of 51 mice were used for this study. 3.2 Chemicals and Reagents All salts used to prepare buffers (NaCl, KC1, CaCl2-2H20, MgS04-7H20, NaHC03) were purchased from Fisher Scientific (Ottawa, ON). All reagents were purchased from Sigma-Aldrich (St. Louis, MO). This included Acetylcholine chloride (Ach), (R)-(-)Phenylephrine hydrochloride (PE), histamine, l-O-Palmityl-sn-glycero-3-phosphocholine 26 CHAPTER 3: METHODOLOGY (PAF), Nffl-nitro-L-arginine methyl ester hydrochloride (L-NAME), and sodium nitroprusside (SNP). All reagents used on the muscle in this study were suspended in fresh PSS. Working PSS stock was prepared following procedures of Payne et al. (2004) by mixing 20X of Basic Salt Buffer (2638 mM NaCl, 94 mM KC1, 23.4 mM MgS04-7H20, 40 mM CaCl2-2H20) with 20X of Sodium Bicarbonate Buffer (360 mM). Basic Salt Buffer and Sodium Bicarbonate Buffer stocks were stored at 4°C. The tubing of the IVM apparatus was flushed with 1% HC1, followed by H 2 0 in a 1:3 volume ratio. 3.3 Intravital Microscopy Water from a hot water bath was circulated through the outer jacket of a glass reservoir (Radnoti, Monrovia, CA) so that any fluids contained within the inner jacket would flow over the skeletal muscle at 34°C. Fluid volume within the inner jacket was gradually restored to 50 mL via gravity through the connection of an elevated 1 L stock bottle of fluid. C0 2 / N2 gas filled the inner jacket at a constant rate to equilibrate the reservoir. PSS was then flushed through the system, so that the first drops coming in contact with the muscle would approximate physiological conditions. All solutions would leave the inner jacket via a gravity controlled valve at 5 mL/ min through a drip line secured above the muscle. Thus, fluid gently flowed over the muscle. A wick was developed with Kimwipes on the Sylgard mold to gently pull fluid off the muscle via capillary action. This allowed a gentle withdrawal of fluid away from the washed muscle 27 CHAPTER 3: METHODOLOGY and prevented any leakage onto the IVM apparatus (Figure 4). All fluid located around the muscle or stored in the glassware was suctioned into a large waste beaker. At the end of each experiment, all tubing and glassware was flushed with 1% HC1, followed by water to remove any traces of salt precipitates or contaminants within the pumping system. Figure 4. Mouse preparation for intravital microscopy recordings. The outer corners of the gluteus maximus are pinned down flat onto the Sylgard mold. A drip line is secured to gently release PSS across the muscle, which is pulled away with Kimwipes and the connected suction line. All of the apparatus resting on the IVM is clear to allow the transfer of light through the tissue and visualization through the objective lens. Once the surgery was complete, the preparation was immediately transferred to the stage of the IVM. The muscle was equilibrated in the PSS wash for 60 minutes to allow any minimal inflammation to subside. During this time, the blood flow quality of the vasculature was observed, leukocyte presence was monitored, and an arteriole branch was selected for later investigation. Second- order (2A) branches of the network were chosen from the central region of the muscle, which was untouched during microsurgeries. The intraluminal vessel diameter of chosen 2A arterioles was measured with video calipers of a Powerlab device and 28 CHAPTER 3: METHODOLOGY the corresponding Chart program (ADInstruments, 2008). A photo of the measuring technique is shown in Figure 5. Figure 5. Measurement of arteriole diameter. A 2A arteriole with continuous blood flow in the central region of the gluteus maximus was chosen (900X). Video calipers are wired into a Powerlab measuring device and video screen. The video camera attached to the IVM is rotated to align each vessel with the video calipers (white lines) and the dials are manipulated until the lines are on the edge of the arteriole. The corresponding diameter change is routed through the Powerlab device into the Chart program to record measurements. The same region of the arteriole is measured for the duration of the experiment to minimize variability. 3.4 Arteriolar Reactivity Resting internal diameter was measured in the chosen 2A arterioles after a PSS wash to provide a baseline for any changes in vessel size. All diameter changes were calculated as the percent difference = (measured diameter- PSS base diameter/ measured diameter) x 100. Vascular diameter changes were expressed as percent differences. The same segment of the 29 CHAPTER 3: METHODOLOGY same 2A arteriole in each mouse was used for the duration of each experiment to reduce variation in results. The maximal diameter was taken at the end of each experiment by pipette application of 1 mM of sodium nitroprusside (SNP) to the muscle. This NO donor directly relaxes arteriolar smooth muscle, thus allowing measurement of maximal diameter through vasodilatation (Brooks et al., 2008). Thus, baseline and maximal diameters provide an index of vasomotor tone during each experiment (Bearden, 2007). Also, having a tone value will describe the arteriole's reactivity when exposed to each reagent. Vasomotor tone = (Maximal inner diameter - initial baseline inner diameter before the first addition of a pharmacological reagent/ maximal inner diameter) x 100. Only the data collected on arterioles that displayed vascular tone greater than 20% (Donato et al, 2007) was kept for further analysis. All data are reported as means ± standard error. 3.5 Vasomotor Activity within the Arterioles Endothelial and smooth muscle cell function was analyzed in the gluteus maximus through cumulative addition of Ach and PE, respectively. For both reagents, a concentration range of 1 nM to 100 uM was used. For each concentration, the reagent was pumped over the muscle at 5 mL/ min for 2 min and the luminal edges were measured. However, to minimize animal use and maintain consistency, the order in which Ach and PE were applied was repeatedly alternated. In half the experiments, responses to Ach were washed for 30 minutes with PSS before next evaluating the response to PE and vice versa. This method is possible 30 CHAPTER 3: METHODOLOGY because the activity of both of these agents is reversible after a wash with PSS (Bearden et al, 2004). 3.6 Characterizing Inflammatory Profiles After the gluteus maximus was washed with PSS for at least 60 minutes, an inflammatory mediator was added to the PSS stock. For each concentration, the inflammatory mediator was pumped over the muscle at 5 mL/min for 2 min. After each diameter measurement, a higher concentration was immediately administered in a cumulative manner. Histamine addition to the PSS superfusion solution ranged from 1 nM to 100 uM. A larger range was chosen for PAF (100 fM to 10 uM) to observe where minor diameter changes began. 3.7 The Role of NO in the Locomotion Arterioles To evaluate the role of the NO pathway within the 2A arterioles, L-NAME (100 uM) was pumped over the muscle after a 60 minute PSS wash. This inhibitor creates a pharmacological blockade of all three NOS isozymes (Kumer et al, 2000). Time-dependent changes in diameter were measured at 15 minute intervals for 60 minutes. The effect of this nitric oxide synthase inhibitor on the arteriolar integrity was also measured during an inflammatory state using histamine. After 30 minutes of incubation with L-NAME, a histamine dose response curve was constructed in the presence of L-NAME. Finally, diameter changes from the inflammatory state and the NO inhibited inflammatory state were 31 CHAPTER 3: METHODOLOGY statistically compared using a one-tailed independent Student's t-test, where differences were qualified as significant if p < 0.05. 32 CHAPTER 4: RESULTS Chapter 4: Results 4.1 Gluteus Maximus 2A Endothelial Response to Vasodilator Application Vasodilation was successfully initiated within the gluteus maximus arterioles with Ach. The vasodilatory range of the gluteus maximus 2A arterioles is illustrated in Figure 6. Arteriole diameter increased with an increase in acetylcholine concentration. There was a large increase in diameter (63.90%) when doses reached 10 uM. The minimum percent difference induced by Ach was 2.92% (1 nM), whereas the maximum percent difference was 89.18% (100 uM). The average vascular tone among these experiments was 46.92 ±2.88%. 120 1.00E-09 1.00E-08 1.001-07 1.00E-06 Concentration (M) 1.00E-05 1.00E-04 Figure 6. Acetylcholine dose response curve. The mean change in diameter (±SE) of the 2A arteriole is shown for each treatment concentration (n = 5). All diameter changes were based off of baseline PSS wash values. Concentration was increased cumulatively by direct application to the visceral layer of the gluteus maximus. 33 CHAPTER 4: RESULTS 4.2 Gluteus Maximus 2A Smooth Muscle Response to Vasoconstrictor Application Vasoconstriction was successfully initiated within the gluteus maximus arterioles with PE. The vasoconstrictive range of the gluteus maximus 2A arterioles in response to PE is illustrated in Figure 7. The arteriole diameter decreased steadily as PE concentration increased. The arteriolar diameter began to level off as the dose response curve reached 100 uM. The minimum percent difference induced by PE was -4.38% (1 nM), whereas the maximum percent difference was -58.03% (100 uM). The average vascular tone across these experiments was 20.45 ±9.43%. -10 I * -20 -30 -40 -SO -60 -70 1.00E-09 1.00E-O8 1.00E-07 1.00E-06 1.00E-05 1.00E-04 Concentration (M) Figure 7. Phenylephrine dose response curve. The mean change in diameter (±SE) of the 2A arteriole is shown for each treatment concentration (n = 5). All diameter changes were based off of baseline PSS wash values. Concentration was increased cumulatively by direct application to the visceral layer of the gluteus maximus. 34 CHAPTER 4: RESULTS 4.3 Gluteus Maximus 2A Arteriolar Response to Histamine Application Inflammation was induced in the 2A arteriole using histamine in a dose-dependent manner. The arteriole reaction to histamine-induced inflammation is shown in Figure 8. Histamine was shown to induce both vasodilation and vasoconstriction in these microvessels. The arterioles dilated progressively as histamine concentration rose from 1 nM to 100 nM. Then the vessel diameter gradually constricted from 1 uM to 100 uM. The highest amount of observed vasodilation was at 0.1 uM (+10.43%), whereas the largest amount of vasoconstriction was at 100 uM (-26.34%). The average vasomotor tone across these experiments was 54.25 ±5.51%. It is also important to note that blood flow became sluggish during histamine application. 1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-OS 1.00E-04 Concentration (Mj Figure 8. Histamine dose response curve. The mean change in diameter (±SE) of the 2A arteriole is shown for each treatment concentration (n = 5). All diameter changes were based off of baseline PSS wash values. Concentration was increased cumulatively by direct application to the visceral layer of the gluteus maximus. 35 CHAPTER 4: RESULTS 4.4 Gluteus Maximus 2A Arteriolar Response to PAF Application Inflammation was also induced in the 2A arteriole using PAF in a dose-dependent manner. The arteriole reaction to PAF induced inflammation is shown in Figure 9. Arterioles constricted steadily as the concentration of PAF increased from 100 fM to 10 uM. The minimum percent difference induced by PAF was -10.22% (100 fM) and the maximum difference was -32.73% (10 uM). Vasoconstriction began to peak at 10 uM. The average vasomotor tone across these experiments was 36.84 ±2.93%. Blood flow also became sluggish within the arterioles after PAF application. 0 ,- E -10 xi -1.5 -20 UI Q f^4. 5 -25 1 -30 -35 -40 1.00E-13 1.00E-11 1.00E-09 1.00E-07 1.00E-05 Concentration (M) Figure 9. Platelet Activating Factor dose response curve. The mean change in diameter (±SE) of the 2A arteriole is shown for each treatment concentration (n = 5). All diameter changes were based off of baseline PSS wash values. Concentration was increased cumulatively by direct application to the visceral layer of the gluteus maximus. 36 CHAPTER 4: RESULTS 4.5 Gluteus Maximus 2A Arteriolar Response to L-NAME Application The activity of nitric oxide synthases was blocked in the 2A arterioles over time using L-NAME. The gradual inhibition of NO production is illustrated in an L-NAME time curve (Figure 10). Arteriole diameter decreased steadily over time as L-NAME flowed over the muscle. Vasoconstriction began to level off at 60 minutes of equilibrium (-21.28%). The average vascular tone of arterioles exposed to L-NAME was 25.63 ±3.41% and there was little reactivity to the application of SNP. Figure 10. L-NAME time response curve. The mean change in diameter (±SE) of the 2A arteriole is shown for each time interval (n = 5). All diameter changes were based off of baseline PSS wash values. The gluteus maximus was continuously equilibrated in 100 uM of L-NAME over the course of the experiment. 37 CHAPTER 4: RESULTS 4.6 Gluteus Maximus 2A Arteriolar Response to Histamine in the Presence of L-NAME After a 30 min wash of the 2A arteriole with 100 uM L-NAME, inflammation was induced with histamine/ L-NAME solution in a dose-dependent manner. The effect of inhibiting histamine mediated inflammation via the NO pathway is shown in Figure 11. The average vasomotor tone of histamine/ L-NAME treated arterioles was 20.02 ±6.67%. In the presence of L-NAME, histamine maintains its arteriolar effect, shifting from vasodilation to vasoconstriction with increasing concentration. Increases in diameter peaked (-6.88%) with 100 nM of histamine. As with histamine alone, arterioles immersed in 100 uM L-NAME began vasoconstricting as histamine concentration rose from 1 uM to 100 uM. l.OOE-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05 1.00E-04 Concentration ( M ) Figure 11. The effect of histamine during inhibition of NO production. Histamine concentrations were diluted in 100 uM L-NAME in a cumulative dose response. The mean change in diameter (±SE) for each concentration were calculated for n = 5. Percent differences were based off of baseline PSS wash values. Concentration of histamine was increased cumulatively by direct application to the visceral layer of the gluteus maximus. 38 CHAPTER 4: RESULTS Arterioles were shown to behave differently towards histamine before and after NO inhibition. A comparison of both histamine dose response curves is shown in Figure 11. The presence of L-NAME attenuated the impact of histamine on arteriole diameter. However, vasodilation was found to be significantly different before and after NO inhibition. The maximal vasodilation reached by histamine/ L-NAME and histamine/ PSS was at 100 nM. In addition, both histamine solutions reached similar vasoconstriction values at 100 uM. ™#~» Histamme'PSS —+— Ifctamine/l-NAME Concentration fM) Figure 12. The effect of histamine on 2A arteriole size before and after inhibition of NO production. The mean change in diameter (±SE) of the 2A arteriole is shown for each treatment concentration (n = 5). All diameter changes were based off of baseline PSS wash values. For each inflammatory condition, concentration was increased cumulatively by direct application to the visceral layer of the gluteus maximus. Significant differences between PSS and L-NAME for each concentration are noted with an asterisk. 39 CHAPTER 5: DISCUSSION Chapter 5: Discussion 5.1 Validation of IVM Techniques and Tissue Integrity This study determined the arteriole reactivity of 2A arterioles in the mouse gluteus maximus under both physiological and pathological conditions. The acetylcholine (Ach) and phenylephrine (PE) dose response curves (Figures 6 and 7, respectively) illustrate the presence of both endothelial and smooth muscle activity. If there was any surgical damage, attenuated and inconsistent responses would have been observed. Endothelial damage has been shown to abolish responses to Ach, while smooth muscle damage inhibits responses to PE (Bartlett & Segal, 2000). The maintained arteriole reactivity in both cell layers that we observed indicates that the integrity of each layer remained in tact within the gluteus maximus. If the integrity and functionality of both endothelial and smooth muscle layers are maintained, proper cell communication can also proceed through myoendothelial junctions (Griffith, 2004). Furthermore, a healthy vascular tone persisted when we used these reagents. Donato et al. (2007) discarded any arterioles that had a vascular tone of less than 20%, but all arterioles used in our study displayed a tone of 20% or greater. Therefore, the 2A arterioles observed were appropriate for microvascular study. Coagulated blood was another hazard to avoid for preventing damaged microcirculation within the gluteus maximus. This disruption in blood flow was observed in preliminary experiments, but was absent after surgical practice. The lack of coagulation before data collection correlates to a lack of inflammation since these two pathways have 40 CHAPTER 5: DISCUSSION been shown to be linked, with the activation of one subsequently activating the other (Cinel & Opal, 2009). For example, LPS-induced endotoxemia accelerated microvascular thrombus formation in cremaster muscles of mice (Lindenblatt et ah, 2006). Impaired RBC velocity was also found in capillaries of rat extensor digitorum longus muscle once sepsis was induced (Tyml et ah, 1998), so sluggish blood flow could be a marker for inflammation. Furthermore, sluggish blood flow was observed in both histamine and PAF exposed arterioles. Therefore, observing a continuous blood flow through the IVM indirectly indicated a lack of inflammation within the arterioles. The gluteus maximus could successfully be excised using microsurgical techniques without inducing acute inflammation within the area of interest. During acute inflammation, injury or damage causes an accumulation of interstitial fluid and white blood cells throughout the microcirculation (Bucci et ah, 2005). For example, soft tissue trauma caused increased leukocyte-endothelial cell interactions and a significant reduction in capillary perfusion in rat skeletal muscle tissue (Gierer et ah, 2008). In our study, no circulating white blood cells were observed though the IVM during data collection. Therefore, careful handling of only the outer edges of the gluteus maximus during microsurgies prevented damage to the central region, preserving the microcirculation. Preventing the release of inflammatory mediators within the central region of the gluteus maximus is important to prevent any inflammatory interactions. For instance, inflammatory priming or synergistic effects have been observed in the microcirculation (Noel et ah, 1995), so induction of severe trauma in the tissue could create a completely different inflammatory environment for future measurements. The healthy microcirculation observed 41 CHAPTER 5: DISCUSSION in the 2A arterioles supports the use of surgical and physiological techniques used during intravital microscopy. However, for further confirmation molecular analysis could be employed, such as performing inflammatory mediator specific western blots of muscle biopsies and/ or isolated arterioles (Starkie et ah, 2003). These could be performed at the end of each IVM experiment to build a molecular profile to match to the arteriole vasomotor tone. By complementing the physiological with the molecular aspects of arteriole function, more inflammatory quantification will be obtained. 5.2 Vasoactivity of the Gluteus Maximus 2A Arterioles Vasodilation and vasoconstriction was induced in the gluteus maximus 2A arterioles using Ach and PE, respectively. Arteriole responsiveness to the cumulative addition of both Ach and PE began leveling off at 100 uM. This vasoactive behavior also agrees with the gluteus maximus vasoactivity seen by Bearden et al. (2004) when using Ach and PE. Therefore, a vasoactive spectrum is now confirmed for arterioles within the mouse gluteus maximus. Furthermore, these experiments provide the maximum and minimum dosages required to induce a vasoactive change in the 2A arterioles of the gluteus maximus. A greater change in arteriole diameter was induced with Ach than with PE. This response can be explained through the mechanism of action of the two vasoactive reagents. Ach induced vasodilation is endothelium-dependent, whereas PE acts directly on the vascular smooth muscle to induce vasoconstriction (Tran & Welsh, 2009). Since Ach's vasoactive signals can travel from the endothelium to the smooth muscle layer via secondary 42 CHAPTER 5: DISCUSSION messengers (Matoba & Shimokawa, 2003), the vasodilator essentially recruits two different cell types to invoke a response. The ability to utilize the endothelium and cause greater signal transmission along the arteriole wall makes Ach a good reagent to use for measuring cell communication (Diep et al., 2005). The induced dilation may be mediated through the NO pathway, but Ach was found to cause vasodilation despite inhibition of NOS (Fitzgerald et ah, 2007). Other secondary messengers, such as EDHF or PGI2, can compensate for a lack of NO in the arteriole. The variety of vasodilatory alternatives may contribute to Ach's ability to induce a greater response than PE. In our study, the arterioles used for Ach displayed higher vasomotor tone than PE, which is an important precursor for cell communication (Donato et al., 2007). Thus, the effects of this vasoactive reagent will be enhanced by preparing arterioles with higher reactivity. In the present study, the two vasoactive reagents were applied directly to the muscle to stimulate the global arteriole network. Future studies can select the appropriate dose for inducing a CVD with local concentrations derived from this reactivity range. For instance, a physiological dose of Ach can be delivered with a micropipette to initiate CVD in the gluteus maximus for these mice (Bearden et al., 2004). Initiating CVD will allow cell communication to be quantified at different sites along the 2A branches of the arteriolar tree. The physiological concentration obtained is also useful so that arterioles can regain their original diameter if multiple doses are administered to skeletal muscle (Payne et al, 2004). The physiological baseline obtained for each cell layer can also be compared with pathological processes, such as the dilator response to Ach in Cx40 knockout mice (de Wit et ah, 2000). The impact of any genetic knockouts related to cell communication may now be compared with the physiological reactions of this study. Both physiological and pathological 43 CHAPTER 5: DISCUSSION conditions can now be derived from our vasoactive range to invoke new responses within the gluteus maximus. 5.3 The Impact of Inflammatory Mediators on Gluteus Maximus 2A Arterioles This was the first study to our knowledge that induced inflammation in the 2A arterioles of the mouse gluteus maximus. Histamine displayed both vasodilator and vasoconstrictor properties in the 2A arterioles of the gluteus maximus (Figure 8). The mechanism of action for histamine is based on its concentration. Obtaining an inflammatory dose response curve is essential because histamine is vascular bed specific (Payne, 2006). Consequently, this mediator has the potential to behave differently within each of the body's tissues. Histamine can cause vasoconstriction, vasodilation, or a combination of these responses, depending on the dose, route of administration, animal species, anatomic region, caliber and pre-existing tone of the vessel (Van de Voorde et ah, 1998). We have illustrated some of the arteriolar effects of this specific inflammatory mediator. Interestingly, histamine displayed a similar arteriolar effect within the gluteus maximus as other mouse skeletal muscle. The histamine dose response curve used by Payne et al. (2003) for the mouse cremaster was found to peak in arteriole vasodilation at 1 uM before constricting. In both skeletal muscles the arteriole reaches a vasodilatory peak, followed by gradual vasoconstriction. These similar diameter changes illustrate that even though histamine has the potential to vasodilate and/ or vasoconstrict in a multitude of ways between tissues (Van de Voorde et ah, 1998), that there can be similar effects within skeletal 44 CHAPTER 5: DISCUSSION muscle tissues. However, there can still be subtle differences. There was less of a change in diameter observed in the gluteus maximus of this study than that seen by Payne et al. (2004) when the cremaster was used. The regular locomotion utilized by the gluteus maximus may provide protective effects against inflammatory attacks, as physical activity has antiinflammatory effects (Pedersen & Fischer, 2007). The large diversity of physiology observed in tissue types, vascular beds, and branching order warrants further exploration of the microcirculation. Our study shows that even differences in tissue functionality/ muscle fiber composition can create minor differences in the microvascular response to inflammation. Having the information on how each mediator will behave at a specific concentration is useful for when its arteriolar effect is not uniform. Both vasodilation and vasoconstriction were observed with varying dosages of histamine in our study, as well as by Payne et al. (2003). Dosages can also be linked to other physiological processes besides arteriolar size, such as cellular behaviour. For example, Asako et al. (1994) found that only a certain dosage of histamine caused recruitment of rolling leukocytes to rat mesenteric venules. Although no video recordings were made in this study, analyzing video footage of fluorescently labeled leukocytes could allow correlations to be made between the diameter and cellular effects of each inflammatory mediator on the gluteus maximus arterioles (Gavins & Chatterjee, 2004). In addition, intlammatory mediators may also induce different effects between branches of the arteriolar network. For example, when TNF-a was applied to rat cremaster tissue, only the third and fourth order arterioles were dilated significantly as a continuous phenomenon after three hours (Adanali et al., 2001). Thus, the complete arteriolar network of the gluteus maximus may be examined with PAF and histamine to build a response profile. 45 CHAPTER 5: DISCUSSION This study demonstrated vasoactive differences between inflammatory mediators in the gluteus maximus. Unlike histamine, PAF plays a strictly vasoconstrictor role within the vascular bed of the gluteus maximus (Figure 9). Arteriolar vasoconstriction was also observed in the hamster cheek pouch by Kim et al. (2000). It is possible that PAF may not be as tissue dependent as histamine. When comparing the vasoconstrictive impact between these two mediators in the gluteus maximus, PAF was found to exert a greater effect than histamine. Whereas a 100 ^M dilution of histamine induced a 26.34% decrease in arteriole diameter, a greater decrease (28.00%) was caused with a 10 uM dilution of PAF. If PAF produces such a prevailing vasoconstrictive environment, it could be more difficult for the CVD to travel along skeletal muscle arterioles, leading to hypertension (de Wit et al., 2000). However, this is the first study to use PAF mediated inflammation in the gluteus maximus, so further work must be performed to probe into its mechanism of action. Obtaining the inflammatory range for each mediator is beneficial because individual concentrations can be selected to simulate specific inflammatory conditions in skeletal muscle. For example, a high concentration may be used to induce severe acute inflammation, a medium concentration for an acute physiological response, or a low concentration for a low-grade inflammatory state. Techniques that elicit chronic low-grade inflammation have recently been developed using slow-release endotoxin pellets. Implanting these pellets beneath the skin enables controlled inflammatory dosing over extended periods of time with limited animal handling and stress (Smith et ah, 2009). This method is modeled after the acute inflammation induced in mice for septic studies (Gocan et al., 2000). Physiological concentrations of histamine have also been used to create a reversible state of inflammation with the mouse cremaster (Payne et al, 2004). Thus, having inflammatory profiles for both 46 CHAPTER 5: DISCUSSION histamine and PAF can allow a spectrum of inflammatory responses to be researched in mice. 5.4 The Role of NO During a Physiological and Inflammatory State Vasoconstriction escalated with time in the 2A arterioles as L-NAME washed over the gluteus maximus. All NOS isozymes are competitively inhibited by L-NAME, so over time it will increasingly fill more active sites until the NO supply to the arterioles becomes severely depleted (Kumer et al, 2000). Our results indicate that the NO pathway plays a key role in maintaining arteriolar diameter within the gluteus maximus. Murphy et al. (2007) also found that washing rat cremaster muscles with 100 uM of L-NAME induced arteriolar constriction. The predominant vasoconstriction seen in skeletal tissue after an NO blockade will reduce the amount of vasodilator signals being sent along the arteriole (Kumer et al, 2000). Also, the sluggish blood flow observed may be a result of increased RBC coagulation from a lack of NO regulation (Crimi et al, 2007). Therefore, NO is an essential secondary messenger to maintain the health of skeletal muscle microcirculation. As noted by Sundrani et al. (2000), L-NAME's ability to induce vasoconstriction could be therapeutically useful if chronic inflammatory states were causing prevalent hypotension. Overproduction of NO can lead to a dramatic decrease in RBC deformability, which can lead to random blockage of the microvascular beds and reduce blood flow (Bateman et al, 2001). However, caution must also be taken to not completely deplete NO levels required for maintaining arteriole health. When a nonselective nitric oxide synthase 47 CHAPTER 5: DISCUSSION inhibitor treatment regimen was used on patients suffering from septic shock, mortality actually increased (Lopez et al, 2004). Thus, a NO deficiency is just as threatening to the tissues as a NO surplus in the vasculature. A selective NO blockade will also alter blood flow regulation within the skeletal muscle. Blocking eNOS as well as calcium-activated potassium channels in mouse cremaster arterioles has been shown to inhibit the CVD (Figueroa et al, 2007). It was found that eNOS (7) mice have a defective post-contraction hyperemic response due to limb ischemia, which can be reversed with local delivery of an adenovirus containing an active form of eNOS (Yu et ah, 2005). The ability to restore blood flow in these knockouts provides further evidence that the NO pathway is essential to intercellular communication within the arteriole. Furthermore, selectively inhibiting iNOS after the induction of sepsis actually increased capillary RBC velocity and local oxygen consumption in rat skeletal muscle (Bateman et al., 2008). Thus, NO is a key secondary messenger to regulate in the skeletal muscle microcirculation. Interestingly, histamine-induced vasodilation was attenuated in the presence of LNAME, when compared to the effect of histamine alone. However, vasoconstriction proceeded similarly under both conditions. The attenuated arteriolar response to histamine in the presence of L-NAME illustrates that NO is a key requirement for an inflammatory impact on the arteriole network. The ability of histamine to spread vasodilatory signals between cells of the arteriole was attenuated without the presence of NO. Therefore, this study identified a key inflammatory mechanism within the arteriolar network of a locomotory muscle. Pharmacological blockade of NO production also attenuated the effect of histamine on the 48 CHAPTER 5: DISCUSSION CVD in arterioles (Payne et al, 2004). Treatment of the rat mesentery venules with LNAME lead to increased vascular leakage and altered endothelial cytoskeleton (Baldwin et ah, 1998). The inhibition of NO could therefore be affecting the arteriolar integrity by loosening the cytoskeleton and attenuating cell communication. In this study, histamine was chosen to investigate the role of the NO pathway during inflammation. However, it is still possible that PAF is affected by NO levels. When PAF binds to its receptor, it activates a pathway which generates the secondary messengers diacylglycerol and inositol 1,4,5-triphosphate (IP3) (Liu & Xia, 2006). Since these messengers both signal Ca2+ release within the endothelium, the NO pathway can still be activated indirectly. Production of NO by PAF was found in rat venular mesenteries, which could be inhibited by NOS inhibitor L-NMMA (Zhu & He, 2005). In the future, the relationship of the NO pathway to PAF function in the gluteus maximus 2A arterioles could be tested with the use of a pharmacological blockade, as in this study, or through NOS isozyme gene knockout mice, as used by Payne et al. (2004) for histamine. Although numerous mice were required to perform these experiments, we have now gained a better understanding of skeletal muscle microcirculation. Extensive physiological and pathological details simply cannot be obtained without animal models, as existing technology for direct visualization of arteriole function is currently lacking for human subjects (Gavins & Chatterjee, 2004; Sakr et al, 2004). Not only was arteriole function broken down into endothelial and smooth muscle components within the arteriole, but also arteriolar changes were recorded for two of the most potent mediators of inflammation (Chen et al., 2008; Payne et al., 2004). Furthermore, all this information was collected in a 49 CHAPTER 5: DISCUSSION locomotory muscle, so more microvascular research may now use our findings to probe into the links between inflammation and skeletal muscle arteriole dysfunction. Cardiovascular disease is currently the leading cause of mortality for both men and women in Canada and becomes more prevalent with our aging population (Public Health Agency of Canada, 2008). Any insights into how inflammation disrupts the skeletal muscle microcirculation will lead to methods of preservation for the muscle and greater potential for increased blood flow throughout the cardiovascular tree. 50 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS Chapter 6: Research Implications and Future Directions 6.1 Regulating Inflammation This study illustrates how inflammation can be attenuated within skeletal muscle microcirculation, but these regulatory techniques can also be applied to other cardiovascular conditions, such as diabetes and hypertension. For instance, decreased muscle mass has been found in aging individuals with type 2 diabetes (Pedersen et ah, 2003). Furthermore, diabetic patients with microvascular complications had longer duration of diabetes and worse glycemic control than diabetic patients without complications (Brooks et al., 2008). In addition, weakened vasodilation was found in the skeletal muscle of hypertensive animal models through reduced expression of Cx43 (Kurjiaka et ah, 2005). A slower spread of vasodilation in skeletal muscle arterioles and higher blood pressure was also seen in Cx40 deficient mice (de Wit et al, 2000). More treatment methods may be used for these conditions once the underlying mechanisms become known. For example, using L-NAME mediated chronic NOS inhibition over four weeks in diabetic mice enhanced the contribution of EDHF, providing an alternative for endothelium-dependent relaxation in isolated mesenteric arteries (Fitzgerald et al, 2007). Since inflammation is present in the vasculature of subjects with both diabetes and hypertension (Brooks et al, 2008; Erdei et al, 2006), finding other ways to reverse the effects of inflammation will increase the health of the cardiovascular system. Maintaining blood flow regulation will thus prevent/ protect skeletal muscle from the development of debilitating chronic conditions. 51 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS Regulatory techniques must be designed to minimize the impact of inflammation on the cardiovascular system. However, regulating inflammatory mediators is a challenge because they are generated and released from numerous cell types, such as platelets, mast cells, monocytes, or macrophages (Weis, 2008). For example, PAF alone can be produced by monocytes/ macrophages, polymorphonuclear leukocytes, eosinophils, basophils, platelets, mast cells, endothelial cells, and lymphocytes (Liu & Xia, 2006). Each mediator also has the potential to activate other inflammatory cascades. PAF is potent enough to not only induce inflammation, but also mediate synthesis and release of other mediators to aggravate the degree of inflammation (Chen et ah, 2008). The difficulty in controlling one mediator among numerous inflammatory cascades and cellular sources makes finding an overlapping mechanism of action crucial. Further complicating matters is the reality that inflammation can have beneficial or detrimental effects, depending on the context. For instance, TNF-a is secreted predominantly by monocytes and macrophages and has wide ranging biological effects on lipid metabolism, coagulation and endothelial function (de Luca & Olefsky, 2008). Moreover, a different subset of monocytes may be recruited during acute inflammation than that utilized for chronic diseases (Arnold et al., 2007). Therefore, regulating inflammation can have other vascular consequences. The combination of diverse capabilities with a large resource pool provides inflammatory mediators with a powerful influence over the microcirculation, creating a challenge for regulation. It is implausible to reduce inflammation by targeting all the different cells and mediators for regulation. However, if the most influential mediators can be found, targeted 52 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS interventions could be used. PAF antagonists have already been designed to reduce the impact of acute pancreatitis (Liu & Xia, 2006). Mice strains with genetic knockouts of TNFa have already been used to study the cremaster post-capillary venules during inflammation (Norman et ah, 2005). Also, if a common mode of action on the microcirculation is found for a collection of mediators, future therapies will become more successful. For instance, if the NO pathway is being used for inflammatory action, NO regulation techniques may be used. This could include chronic inhibition of NO with minimal levels of a non-selective inhibitor (Fitzgerald et al., 2007) or targeting specific isozymes with inhibitors during their time of maximal activity (Lidington et al., 2007). This study found different modes of action in the arteriole for two inflammatory mediators. Expanding the methods we used for application to other inflammatory mediators may lead to a further understanding of the inflammatory process. Our study is the first attempt at arteriolar inflammation in the gluteus maximus in mice and is a crucial new avenue to explore, since the majority of animal research has focused on the cremaster as the major tissue to describe skeletal muscle microcirculation. Eventually, inflammatory interactions may be observed within the arteriole. For example, primed and synergistic effects between histamine and PAF have already been investigated within hamster cheek pouch venules (Noel et al., 1995; Tomeo et al., 1991). Knowing the separate actions of these mediators will thus allow a comparison between the individual mediators, so that they may be compared with combinatorial effects. 53 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS 6.2 Aging The responsiveness of arterioles towards vasoactive reagents has been shown to change as skeletal muscle becomes older. For instance, aging is associated with a greater aadrenergic vasoconstriction in arterioles from high oxidative soleus skeletal muscle in rats (Donato et al., 2007). The reduced capacity of arterioles (as well as conduit arteries) to dilate may contribute to a reduction in muscle blood flow and ultimately limit the ability of older adults to perform functional tasks (Payne, 2006; Taddei et ah, 2001). The significant inflammatory biomarkers associated with poor physical performance in the elderly are already being sought out (Hsu et al, 2009). For example, NO bioavailability is lower in the circulation of aging subjects (Taddei et al, 2001). Similar CVD profiles were also found in the mouse cremaster when comparing aged and histamine-exposed, adult mice (Payne et al, 2004). Therefore, certain mediators may cause similar microvascular deficits between different age groups in both large and small vessels. In our study, the function of the 2A arterioles was measured in adult mice. However, the arteriolar network architecture in the gluteus maximus appears to be maintained during aging (Bearden et al, 2004). Thus, the 2A arterioles of adult mice could be compared with older mice to observe how vasomotor and inflammatory functionality differs between these similar structures. 54 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS 6.3 Gender Differences Being able to successfully perform microcirculatory measurements on the gluteus maximus proves that this is a prospective new tissue to be used in skeletal muscle studies of mice. Unlike the cremaster muscle, the gluteus maximus allows gender comparisons to be made. Gender differences have already been seen in the macrocirculation. Unlike women, men were shown to have similar leg hemodynamic responses between young and older age groups after an exercise regimen (Parker et al., 2008). Furthermore, the femoral artery dilated to a greater extent after dynamic leg exercise in young women vs. men (Parker et ah, 2007). A slower rate of vasodilation in gluteus maximus 2A arterioles has already been observed in male mice when compared to females (Bearden, 2007), suggesting similar vasomotive reactions in the microcirculation. The greater function of circulation in the skeletal muscle of females needs further clarification. There was enhanced NO mediated endotheliumdependent relaxation in female rat mesenteric arterioles when compared to males (White et ah, 2000). This greater NO utilization could also extend to the skeletal muscle, which could be tested out using the L-NAME as in our study. 6.4 Physical Activity Another major benefit of being able to observe the microcirculation in the gluteus maximus is that the impacts of physical activity can be researched. Physical activity is known to not only increase blood flow during skeletal muscle contraction (Armstrong et ah, 2007b) and after endurance training sessions (McAllister et al, 2008), but also to increase 55 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS microvascular density (Laughlin et al, 2006). However, anti-inflammatory effects can also be investigated, such as identifying the myokines that are released from skeletal muscle during exercise (Pedersen & Fischer, 2007). Skeletal muscle is now being examined as an endocrine organ to find what pathways connect physical activity to cardiovascular health. Even three hours of exercise weakened the effect of low-grade inflammation. When this exercise regimen was followed in humans before endotoxin injection, any induced increases in TNF-a were attenuated (Starkie et al, 2003). Furthermore, assigning aging individuals to both endurance and resistance training reduced the expression of active monocytes circulating in the blood (Timmerman et al, 2008). Physical activity must somehow mediate anti-inflammatory pathways. This may also explain why a locomotory muscle would be more resistant to the effects of histamine when compared to a nonlocomotory muscle. It is quite possible that there are higher levels of anti-inflammatory myokines circulating throughout the microcirculation of the gluteus maximus. This may create an anti-inflammatory potential for protection against inflammatory attacks. Some mechanisms of activity-mediated increases in physical activity have been investigated. For instance, it is possible that the activity of the NOS isozymes becomes physiologically altered with physical activity. Chronic exercise in dogs caused an increase in eNOS expression of the microcirculation (Sessa et al., 1994). Thus, exercise may shift NO production towards levels healthier for the arteriole beds supplying the active muscle. In addition, exercise training was shown to prevent hypoxia-induced increases in leukocyteendothelial adherence and in vascular permeability in the cremaster muscle of rats (Orth et ah, 2005). This impact even persisted for another month after training was discontinued. 56 CHAPTER 6: RESEARCH IMPLICATIONS AND FUTURE DIRECTIONS However, the cremaster is not a locomotor muscle, so testing the effects on other muscles to see direct rather than secondary effects may provide insights into what exercise regimen is most beneficial for microvascular health. Future studies could include using an exercise regime for mice, as either a preventative or intervention method for inflammation, to further clarify the microvascular effect of exercise on the gluteus maximus. Conclusion In this study, we performed numerous experiments on the mouse gluteus maximus. 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