THE EFFECTS OF PROPRANOLOL ON ACUTE INFLAMMATORY CYTOKINES AND MUSCLE FUNCTION FOLLOWING AN EXERCISE INDUCED MUSCLE INJURY TO THE SHOULDER IN INDIVIDUALS WITH A HIGH PAIN SENSITIVITY COMT POLYMORPHISM By WILLIAM C. HEDDERSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 201 9
201 9 W illiam C. Hedderson
3 To Mom, Dad, and Joyce
4 ACKNOWLEDGMENTS I would first like to thank a special group of people that have been with me through this entire process. My parents, Bill and Janet Hedderson, for their years of support through my journey of learning, they ha ve experienced my ups and downs and have given me guidance throughout. Joyce Joseph has been a constant source of motivation, she pushed me when I felt stuck and celebrated with me when I hit milestones, I would not have completed this dissertation without her, and for that I am forever grateful. My committee has been an incredible resource, I feel lucky to have had the opportunity to work with these extraordinary scholars throughout the past three years. Dr. Christopher Hass and Dr. Stephen Coombes have provided keen advice, suggestions and guidance. Dr. Roger Fillingim and Dr. Steven George have been an amazing resource and have allowed me the opportunity to work closely with them on a nationally funded grant while still working intimately with me to m ake me a better researcher by expanding my understanding of their specialties. Lastly, Dr. Paul Borsa has been a patient, wise and great mentor. There is no way I would have completed this PhD without him, he worked closely with me to guide me to the ans wers I was looking for and allowed me the opportunity to consider myself a scientist. His passion and genuine care for my success was evident as we progressed through th e PhD. Warren Greenfield III, Dr. Mark Bishop, Dr. Samual Wu, Yunfeng Dai, Brain Bouve rat, Katie Butera, Josh Crow and the rest of the BISP and PRICE teams were amazing to work with while I collected my data over the past 3 years. The memories of working with such a great team will last me forever.
5 Thank you so much to everyone that had a hand in helping me to get to this point, you are all amazing people that will always have a place in my heart and I wish you all the best of success in your future endeavors.
6 T A BLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 A BSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ....... 14 Background and Significance ................................ ................................ ................. 14 Specific Aims and Hypotheses ................................ ................................ ............... 20 Specific Aim 1 ................................ ................................ ................................ ... 20 Hypothesis 1 ................................ ................................ ................................ ..... 21 Specific Aim 2 ................................ ................................ ................................ ... 21 Hypothesis 2 ................................ ................................ ................................ ..... 22 Specific Aim 3 ................................ ................................ ................................ ... 22 Hypothesis 3 ................................ ................................ ................................ ..... 22 2 LITERA TURE REVIEW ................................ ................................ ............................. 23 Inflammation Cascade ................................ ................................ ............................ 23 Exercise Induced Muscle Injury ................................ ................................ .............. 25 Exercise Induced Muscle Damage and Inflammation ................................ ............. 2 6 ................... 27 Catechol O Methyltransferase, Pain, and Propranolol ................................ ............ 29 3 ME THODS & MATERIALS ................................ ................................ ........................ 34 Blinding ................................ ................................ ................................ ................. 36 Outcome Measurements ................................ ................................ ....................... 36 Experimental Injury Protocol ................................ ................................ ................. 38 Statistical Analysis ................................ ................................ ................................ 39 4 RESULTS ................................ ................................ ................................ .................. 42 Specific Aim 1 ................................ ................................ ................................ ........ 42 Specific Aim 2 ................................ ................................ ................................ ........ 44 Specific Aim 3 ................................ ................................ ................................ ........ 44 5 DISCUSSION ................................ ................................ ................................ ............ 58 Broad Overview of the Study ................................ ................................ ................ 58 Experimentally Induced Muscle Injury ................................ ................................ .. 59 Specific Aim 1 ................................ ................................ ................................ ....... 60 Specific Aim 2 ................................ ................................ ................................ ....... 64 Specif ic Aim 3 ................................ ................................ ................................ ....... 65 Summary of Study Design Limitations ................................ ................................ .. 71
7 Future Directions ................................ ................................ ................................ .. 76 Conclusion ................................ ................................ ................................ ........... 77 REFERENCES ................................ ................................ ................................ .............. 79 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 98
8 LIST OF TABLES Table P age 3 1 Study exclusion criteria ................................ ................................ ....................... 40 3 2 Participant demographics ................................ ................................ ................... 41 3 3 Study protocol ................................ ................................ ................................ ..... 41 4 1 ................................ ........ 52 4 2 Inflammatory cytokines correlated with active pain at p<0.0167 ......................... 54 4 3 Inflammatory cytokines correlated with disability at p<0.0167 ............................ 55 4 4 Inflammatory cytokines correlated with resting pain at p<0.0167 ....................... 55 5 1 Revisited study design for observing the effects of prop ranolol on i nflammatory cytokines following an EIMI ................................ ........................... 78
9 LIST OF FIGURES Figure P age 1 1 Flow chart of EIMI ................................ ................................ ............................... 22 4 1 MVIC measurements of the shoulder o ver time. ................................ ................. 47 4 2 ROM measurements of the shoulde r over time. ................................ ................. 47 4 3 Disability (QDASH) measurements of the shoulder over time. ........................... 48 4 4 Estimated marginal means of IL ................................ ................... 48 4 5 Estimated marginal means of IL 6 over time. ................................ ..................... 49 4 6 Estimated marginal means of IL 8 over time. ................................ ..................... 49 4 7 Estimated marginal means of IL 10 over time. ................................ ................... 50 4 8 ................................ .................. 50 4 9 Estimated marginal means of CRP over time. ................................ .................... 51 4 10 Estimated marginal means of IL 10 in women over time. ................................ ... 51 4 11 Scatterplot regression analysis between IL 10 blood plasma concentration and resting pain on Day 3. ................................ ................................ .................. 56 4 12 Scatterplot regression analysis between IL 10 blood plasma concentration and active pain on Day 3. ................................ ................................ ................... 56 4 13 Scatterplot regression analysis between IL 10 plasma concentration on Day 3 with active pain on Day 3. ................................ ................................ ................ 57
10 LIST OF ABBREVIATIONS APS Average pain sensitivity AROM Active range of motion ARs adrenergic receptors BPI Brief pain inventory COMT Catechol O Methyltransferase CRP C Reactive Protein DOMS Delayed onset muscle soreness ECG Electrocardiogram EDTA Ethylenediaminetetraacetic acid EIMI Exercise induced muscle injury ER External Rotation HCP Health c are provider HPS High pain sensitivity Tumor Necrotic Factor Alpha IL Interleukin 1 Beta IL 6 Interleukin 6 IL 8 Interleukin 8 IL 10 Interleukin 10 IR Internal rotation LPS Low pain sensitivity MVIC Maximal voluntary isometric contraction NO Nitrous oxide PCS Pain catas trophizing scale
11 PROM Passive range of motion QD QuickDASH RPM Revolutions per minute
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment o f the Requirements for the Degree of Doctor of Philosophy THE EFFECTS OF PROPRANOLOL ON ACUTE INFLAMMATORY CYTOKINES AND MUSCLE FUNCTION FOLLOWING AN EXERCISE INDUCED MUSCLE INJURY TO THE SHOULDER IN INDIVIDUALS WITH A HIGH PAIN SENSITIVITY COMT POLYMORPH ISM By William C. Hedderson August 201 9 Chair: Paul Borsa Major: Health and Human Performance The current pharmacological treatment options for pain are generally unsuccessful and not recommended for long term use. Treatment of chronic pain is also a huge economic burden on society. Current pharmacological interventions for pain include the use of opioids and n on steroidal anti inflammatory medication these treatments either have a low success rate or put the patient at an increased risk for complica tions following long term use. Exploring alternative pharmacological interventions to treat pain c an improve care and pain outcomes One avenue of exploration is the use of personalized medicine. Previous studies have identified the COMT gene in conjunct ion with pain catastrophizing can predict pain intensity in the shoulder after an exercise induced muscle injury. Current literature suggests that the non specific beta blocker, propranolol, may effectively mediate pain in these individuals but there is influence on pain This study identified if propranolol has any anti inflammatory properties in blood plasma following an exercise induced muscle injury.
13 54 participants that were positive for the COMT HPS polymorphism and pain catastrophizing were enrolled in the study. Participants were randomly assigned to the propranolol or the placebo group. The protocol required participants to complete baseline testing, which included a blood draw and t hen perform a fatigue protocol intended to cause an exercise induced muscle injury to the shoulder. Participants returned at 24 hour intervals for follow up measurements. Levels of inflammatory cytokines and proteins were measured in the blood plasma a t each subsequent visit these include d TNF IL 1 IL 6, IL 8, IL 10 and CRP. There was no interaction effect observed indicating that propranolol does not alter blood plasma levels of inflammatory cytokines after an acute exercise induced muscle injury.
14 CHAPTER 1 INTRODUCTION Backgro und and Significance Chronic pain is a global problem and a frequent reason for seeking medical care (1) Approxima tely 31% of the adult population in the United States has reported experiencing pain lasting for at least 6 months, with a majority of those individuals experiencing pain lasting a minimum of one year (2) Chronic pain is a huge economic burden leading to time away from work and decreased productivity (3) The total economic impact to the population because of chronic pain is a loss of approximately $635 bil lion annually (4) The societal costs of chronic pain exceed the combined costs of cancer, AIDS, and heart disease indicat ing that current pain treatments and interventions are not adequate for improving pain outc omes for the entire population. Continued investigation into chronic pain and therapeutic interventions is necessary to help improve clinical outcomes and reduce the economic burden on society. Acute musculoskeletal pain is norm ally transient and protecti ve in nature, alerting an individual that injury is present and homeostasis has been disrupted (5) If acute pain is allowed to persist be yond a normal period of time without intervention it can transition to post acute pain and eventually chronic pain. The transition to a chronic pain c ondition is no longer viewed as a protective mechanism, but instead a symptom of disease (6) Chronic pain, which is considered to be a maladaptive pain response, commonly results in physical i mpairments and disability such as decreased range of motion (7) movement speed (8) and muscular strength (9) Chronic pain also contributes to negative psychosocial behaviors, including fear avoidance and depression (10) Restoring normal function is a necessary goal for ind ividuals suffering
15 from chronic pain because it enables an injured individual to observe progress in their pain treatment programs (8) The biopsychosocial approach to pain management is thought to be more ef fective than previous biomedical models for treating pain since it recognizes that pain is an u npleasant and subjective experience that varies widely between individuals. The biopsychosocial model acknowledges that the psychological, social and biological pain, an d also that each aspect needs to be addressed for optimal recovery (11) The psychological aspect of pain addresses condi tions that play a substantial Important psychological conditions that individuals with chronic pain experience include anxiety, fear, depression, anger, catastrophizing, and loss of self efficacy (12 17) Psychological interventions have been shown to be effective in reducing chronic pain (18) Cognitive behavioral therapy and/or training is regarded as one of the most successful psychological intervent ions in the treatment of chronic pain. A primary focus of cognitive behavioral training is to ensure individuals have a positive outlook on pain and recovery (18, 19) Social based interventions address goals of integrating an individual back into work or sport participation after injury. A popular intervention to address sociological need s to individuals overcome social barriers, such as low soc io economic status; thus improving (20)
16 The biological aspect of pain targets the site of the injury and the underlying causes and physiological mechanisms that contribute to the pain experience. This i ncludes looking at a variety of vascular, cellular and molecular events that lead to increased pain sensitivity including the intensity and duration of symptoms. The biological aspect of pain also provides a therapeutic window for developing potential phy siological interventions that aid in promoting wound healing and/or reducing pain and inflammation. Popular treatments to address the biological aspects of pain include pharmacological (nonsteroidal anti inflammatory drugs, also known as NSAIDs, and/or opi oids) and physical approaches (therapeutic modalities and rehabilitation) (21, 22) Developing a treatment plan for chronic musculoskeletal pain can be extremely complicated, often because of the inability to det ermine the actual source of the pain signal. In situations where it is difficult to identify the source of chronic pain, health care professionals commonly pr escribe medications (e.g. opioids NSAIDs) to treat chronic pain. Opioid medications are commonly prescribed to individuals who experience chronic pain symptoms. Opioids are a type of narcotic pain medication and refer to a compound that binds to opiate receptors in the brain, spinal cord and other areas of the body The types of opioids currently in use to treat plain include opiates (i.e. morphine codeine) that are derived from the opium poppy, semi synthetic opiates (i.e. heroin oxycodone), and synthetic opiates (i.e. methadone fentanyl) (23) Opioid treatment is effective in ap proximately 33% of the population experiencing chronic pain (24) but prolonged use could yield a long term dependency, higher tolerance (25) and an increased risk for fractures, myocardial infarction and sexual dysfunction (26 28) Protracted use of NSAIDs is not recommended, mainly because it can negatively affect
17 platelet function and can cause gastrointestinal dysfunction, renal dysfunction and hepatic damage (29, 30) These negative factors identify a need for continued research into the biological mechanisms of pain in an effort to develop more effective and less costly therapeutic interventions. Cytokines are signaling molecules synthesized and released by immune cells that affect the communication and interac tion s between a variety of other cells (31) they work synergistically to drive the acute inflammatory response following musculoskeletal injury (32) Catecholaminergic neurotransmitters are released in response to cell injury and bind to beta 2 3 adrenergic) on immune cells, stimulating the release of pro inflammatory cytokines ( 33) These cytokines can then target a cell to synthesize and release additional inflammatory mediators, this process has been referred to as a cascade. The predominant producers of cytokines during inflammation are immune cells such as granulocytes, lym phocytes and macrophages. I mmune cells are recruited following tissue damage and play an integral role in initiati ng the inflammatory cascade. cytokines. The lines are blurred as to which cytokines are truly pro and anti inflammatory, but 6 and IL 8 have been identified as having pro inflammatory properties and have been directly linked with increased pain and disability (34 37) while other cytokines have anti inflammatory properti es such as IL 10, respond to cell injury and assist in regulating the magnitude and extent of inflammation and tissue repair (37) The sequence of events of an exercise induced muscle injury (EIMI) as proposed by Smith and Miles are as follows (38) (follow chart figure 1.1) : 1) Tissue damage from EIMI
18 2) Activation of resident neutrophils and macrophages by damaged or dying cells 3) Release of pro inflammatory cytokines (e.g. IL ) 4) Neutrophils s ynthesi ze a variety of chemoattractant agents. These agents will subsequently act to guide migrating leukocytes to injured cells 5) Pro inflammatory c ytokines stimulate local endothelial cells to release IL 6, cell adhesion molecules, and other stimulating factors They also stimulate the liver to produce and release acute phase proteins including CRP C ytokines also activate the hypothalamic pituitary adrenal axis resulting in the release of cort icosteroids 6) Leukoc ytes migrate from circulation to site of injury Termination is a result of a combination of increased circulating levels of corticosteroids, apoptosis, and inhibition of pro inflammatory factors by molecules such as IL 10 Pain signals are transmitted and processed by the central nervous system (CNS) via neurotransmitters. Epinephrine and norepinephrine are catacholaminergic neurotransmitters that have been found to play a major role in pain transmission with in the CNS These neurotrans mitters are also present in the periphery and are demonstrated in animal models to enhance pain after intraplantar injection (39) 2 3 adrenergic receptors by epinephrine and norepinephrine at the periphery have also shown to produce allodynia (40, 41) Accumulation of catecholaminergic neurotransmitters could lead to a prolonged pain response that may result in chronic pain (42) Thus, it is imperative that the body works to metabolize these neurotransmitters as effic iently as possible to avoid overstimulating these receptors ( 2 3 adrenergic )
19 G enetic factors also influence the development of chronic pain. DNA sequence variations in gene s that encode for proteins involved in pain transmission have been shown to contribute to chronic pain. One protein of importan ce is catechol O methyltransferase (COMT). COMT is a ubiquitously expressed detox ifyin g enzyme involved in a number of important biochemical pathways, particularly metabolism of catecholamines (43) The COMT gene encodes the COMT enzyme which in turn metabolizes catecholamines (norepinephrine/epinephrine). COMT polymorphisms are associated with decreased enzymatic a ctivity and have been linked to heightened pain sensitivity and increased risk of multiple musculoskeletal pain conditions (44) The impact of COMT on pain modulation occurs via multiple pathways including the beta adrenergic system (33, 43) Elevated levels of norepinephrine/epinephrine, resulting from decreased COMT 2 and 3 adrenergic receptors to promote the release of pro inflammatory cytokines 6, which in turn activate the inflammatory cascade and produce heightened pain sensitivity (40) Beta adrenergic 2 3 ) are located on mononuclear leukocytes and aid in regulating intracellular activity (45) During the acute phase of inflammation, s 2 3 adrenergic receptors induce elevated levels of pro inflammatory cytokines particularly (42) Propranolol is a non specific beta 2 adrene 3 adrenergic receptors and has been found to be effective in reducing mechanical and thermal pain in individuals with the low activity COMT genotype (41) In addition to 2 3 adrenergic rece ptors via oral ingestion of propranolol may also help control inflammation by modulating the production of pro
20 inflammatory cytokines Of importance, t hese cytokines initiat e the inflammatory cascade following a musculoskeletal injury and their p rimary duties are to activate phagocytic cells and induce vasodilation at the site of the injury A therapeutic intervention that can target specific cytokines or proteins involved in the inflammatory cascade will result in substantial pain relief as wel l as inflammatory modulation. We postulate that owing to its blockade of beta adrenergic receptors, propranolol will be effective at modulating pain and inflammation in individuals who possess the high pain sensitivity COMT polymorphism. The impact of this novel intervention may permit healthcare professionals to develop individualized medication prescribing opioid medications and risk of long term dependency. Therefore, our ov erall objective is to determine the efficacy and mechanisms of a personalized pharmaceutical intervention (60 mg of propranolol) designed to modulate pain, inflammation, and physical impairment following a single bout of high intensity resistance exe rcise in a high risk group. Specific Aims and Hypotheses Specific Aim 1 Determine whether a daily dose of orally administered propranolol (60 mg via capsule) effectively modulates blood plasma levels of biomarkers involved with the inflammatory cascade of individuals in a high risk group within the first 96 hours following a single bout of high intensity resistance exercise compared to a placebo control group. The high risk group consists of those individuals who test positive for the high pain sensitivity COMT polymorphism (rs6269 ; AA genotype ) and score a minimum
21 of 5 on the pain catastrophizing scale. The high intensity resistance exercise protocol is intended to cause a controlled muscle injury to the shoulder external rotator muscle group (infrasp inatu s and teres minor). The inflammatory biomarkers of interest include 6, IL 8, IL 10, and CRP. The placebo control group will receive a capsule filled with an inert substance that is visually indistinguishable from the propranolol capsu les. H ypothesis 1 A daily dose of 60 mg of propranolol will effectively modulate the inflammatory cascade following a single bout of high intensity resistance exercise. We hypothesize tely post i njury, with subsequent diminished levels of IL 6, IL 8, IL 10 and CRP detected at 24 and 48 hours post injury in a high risk group compared to a placebo control group. Specific Aim 2 Determine whether a daily dose of propranolol orally (60 mg via capsule ) influences subjective and objective functional measurements of the shoulder including static strength (M aximal voluntary isometric contraction (MVIC) ), active shoulder flexion, active and passive shoulder internal/external rotation and self report disa bility (QuickDASH) in a high risk group within the first 48 hours following a single bout of high intensity resistance exercise intended to cause a controlled muscle injury to the shoulder external rotator muscle group (infraspinatus and teres minor) compa red to a placebo control group.
22 Hypothesis 2 A daily dose of 60 mg of propranolol will effectively modulate functional measurements of the shoulder within in the first 48 hours following an exercise induced muscle injury in a high risk group compared t o a placebo control group. We hypothesize that functional measurements of the shoulder including static strength (MVIC), active shoulder flexion, passive shoulder internal/external rotation and self report disability (QuickDASH) in the high risk group th at received propranolol will be significantly greater at 24 hours and 48 hours compared to the placebo control group. Specific Aim 3 Determine the strength of the relationship between inflammatory biomarkers 6, IL 8, IL 10, IL 6/IL 10 ratio and CRP) and peak pain intensity as measured by the Brief Pain Inventory (BPI) and functional impairments (range of motion, MVIC, a nd disability) at 24 hours and 48 hours post exercise induced muscle injury. Hypothesis 3 6, IL 8, IL 10 IL 6/IL 10 ratio and CRP) will be positively correlated with pain inte nsity ratings and functional impairment measures (range of motion, MVIC and disability) within the 48 hours following an exercise induced muscle injury. Specifically, those individuals with higher plasma levels of inflammatory biomarkers will display high er pain intensity ratings and functional impairment.
23 CHAPTER 2 LITERATURE REVIEW Soft tissue injuries (i.e. strains contusions) are common in sports as well as from motor vehicle injuries. The body responds to these injuries by inducing inflammation for the sole purpose of healing. There are symptoms of inflammation experienced by an individual after injury occurs. One of the initial symptoms is acute pain which is necessary during the initial stages of wound healing as it acts as a protective mechan ism, ensuring that the individual protects and allows the wound to heal When the inflammation does not resolve after tissue healing has occurred, acute pain, first associated with the injury, now has the potential to transition into chronic pain. Chronic requires treatment. I nflammation Cascade Wound healing is a complex response divided into three continuous and overlapping processes inflammation, proliferation, and tissue remodeling (46) Inflammation is the initial response of the body to tissue injury. Symptoms of inflammation include redness, swelling, heat, pain and loss of function (47) although not all clinical symptoms are always detectable. The inflammatory response varies depending on the tissue involved, type of injury, and severity of the injury. The acute inflammatory respons e usually lasts between 24 48 hours but can continue for up to two weeks before transitioning to the proliferation stage (46) The inflammation response is characterized by movement of fluid, plasma proteins and leukocytes into and out of injured tissue (38) The cascade begins as soon as injury occurs with the purpose of recruiting white blood cells to the in jured tissue.
24 Hemostasis occurs immediately after tissue injury with the arrival of platelets to the site of injury. Platelets are part of the inflammatory cascade; they signal the release of growth factors to promote new tissue growth (46) Shortly after th e injury, neutrophils and monocytes migrate to the injured tissue; these cells initiate the clean up process and synthesize inflammatory factors. Macrophages become the predominant cell during the latter stages of inflammation (48) Macrophages rel ease factors that attract fibroblasts to the wound area. These factors include cytokines, chemokines and cell adhesion molecules. Cytokines are signaling molecules that coor dinate between the various cells involved with the inflammation process (38) Cytokines are synthesized at the site of the injury and act on target cells by binding to specific receptors. Cytokines are functionally redundant; therefore, different cytokines can produce similar effects on cells. The action of a cytokine is dependent on the function of the targete d cell and the levels of other cytokines producing antagonistic or synergistic effects on that cell. This makes the signaling system very complex and difficult to elucidate (49) The three most important cytokin es involved with the acute inflammation cascade are IL All three are pro inflammatory cytokines, with IL 6 is found at the temporal end of the cascade thus has both pro and anti inflammatory impacts (49) An increase in an ti inflammatory cytokines takes place to terminate the inflammation cascade, which indicates that the site of injury is cleared of necrotic cells and is prepared to transition to the proliferation phase.
25 Damage incurred at the site of injury is irreversible and considered the primary injury (50) Chronic inflammation can result because of macrophages being unable to solubilize once the injured area ha s been cleared of dead and damaged cells. Their presence may continue signaling via thereby continuing the inflammation response. Healthcare professionals (HCPs) have an understanding of the healing process and treat inflammation with the use of therapeutic modalities and rehabilitative exercise Therapeutic interventions help reduce secondary injury and minimize the effects of ischemia and oxidative stress that occur as a result of the inflammation process. When successful healing occurs, symptoms associated with the inflammation cascade resolve. E xercise Induced Muscle Injury EIMI is an adaptive microtrauma that results from a combination of two proposed mechanisms. The first is based on unaccustomed eccentric muscle action; this occu rs when a muscle lengthens under tension and disrupts muscle structure (51) The second mechanism is because of muscle ischemia, which may contribute to muscle injury. Alt hough this type of injury self resolves over a period of three to 21 days, with peak pain usually occurring at 48 72 hours, it does initiate an acute inflammation response that mimics a soft tissue injury (52, 53) EIMI induced by unaccustomed eccentric muscle action also produces muscle sorene ss that is often referred to as delayed onset muscle soreness ( DOMS ) DOMS is most prevalent in individuals who have not trained or are performing an unaccustomed movement. DO MS is also experienced in individuals that have had a dramatic increase
26 in the intensity and volume of the eccentric movement compared to their current training levels (53) DOMS peaks between 24 72 h ours after the EIMI. E xercise Induced Muscle Damage and Inflammation EIMI mimics a true soft tissue injury through numerous aspects. From a broad perspective swelling, pain, decreased range of motion, point tenderness and a decrease in strength have been observed to take place with EIMI, although not all these symptoms may occur simultaneously following it (54) The acute inflammation cellular response to an EIMI is also similar to a soft tissue injury. A majority of the evidence associating EIMI with inflammatio and IL 6 being investigated thoroughly (38) M uscle injury induced through high force eccentric exercise has exhibited changes in IL 6, with changes in serum and plasma. All three of these cytokines have exhibited changes when measured with muscle biopsies (55) It is thought when they enter the blood they may not be stable in circulation, rendering detection difficult (56) These cytokines may also become elevate d and return to baseline levels rapidly after EIMI, making the timing of blood draws essential to detecting changes in blood levels. As a result of th ese cytokines being detectable in muscle post EIMI, it is assumed that there cause s the increase in IL 6 (57) Elevated level s of IL 6 have been detected within the first 24 hours of an EIMI (58, 59) Plasma increases in IL 10 have also been detected in response to EIMI but are not detectable until the inflammation process is more advan ced. IL 10 lim its the production of pro inflammatory cytokines, such as IL
27 IL 6 (60) ; it is integral during the termination of the inflammatory process, thus changes in the levels of IL 10 begin to appear af ter IL 6 have peaked. CRP also responds to soft tissue injury and elevated levels of it are detected damaged cells, which enhances uptake of these cells by macroph ages. CRP may possibly play a role in decreasing the bioavailability of endothelial nitric oxide synthase (61, 62) The rise in CRP is mediated by IL 6 as it stimulates CRP synthesis with in the hepatic system, afte r which it progresses through the blood stream to the site of injury (63) I nterleukins IL : I s a major inflammatory mediator, and one of its primary roles is to induce the synthesis of chemokines including IL 8, which is a potent neutrophil chemoattractant (64, 65) IL and is important during immune response (66) Specifically, it brings about endothelium vasodilation, which increases the permeability of blood vessels; this in turn promotes the movement of serum proteins and leukocytes to the site of infection. IL involved with initiating the inflammatory response associated with autoimmune and inflammatory diseases. IL 6 : I s a multifunctional pleiotropic cytokine that re gulates the immune response, acute phase response, hematopoiesis and inflammation (67) Its function wi thin the inflammatory response is both pro and anti inflammatory as it directs leukocyte trafficking and activation. IL
28 chemokines. It also is featured in neutrophil recruitment and regulation of T cell infiltration (68) IL 8 : I s produced by a wide variety of cells including but not limited to: monocytes, macrophages, neutrophils, skeletal and smooth muscles, and several tumor cell types. IL 8 synthesis is usually stimulated by IL With regard s to its role in inflammation, IL 8 tra nscription is a fast process as it can be detected on a Northern blot within one hour after stimulation of the cells. The major function of IL 8 is the recruitment of neutrophils to the site of injury, hence its role as a pro inflammatory cytokine (69) Neutrophil accumulation is rapid and reaches a maximum after 30 minutes (70) IL 10 : I s produced predominantly by monocytes, T cells, B cells and macrophages (71) and is a key regulator of the inflammatory response. Its immunosu ppressive effects protect the host from an exaggerated inflammatory response to infections and autoimmune diseases (72) IL 10 also inhibits the expression of many cytokines including IL 6 10 induces its broad range of anti inflammatory effects in a STAT3 dependent manner, but it is largely unknown why it has such a large effect compared to oth er cytokines, such as IL 6, that signal through STAT3 and have a more minor anti inflammatory response (73) : I s a powerful pro inflammatory cytokine that regul ates many facets of macrophage function. It is rapidly released after injury and is integral to initiating the inflammation cascade (74) ophages to migrate to a site of tissue damage after injury. as activated macrophages produce this gas as a byproduct of lysing pathogens.
29 increases lipid signal transduction mediators, such as platelet activating factor. As a result of recruitment and may have a dominant part that is played in many chronic inflammatory pain diseases (75) CRP : I s an acute phase protein f eaturing a homopentameric structure and Ca binding specificity for phosphocholine. There is a rapid rise in plasma and serum concentration s of CRP in response to infection or tissue injury within the first 24 48 hours (76) CRP is produced in the liver and regulated by IL 6 du ring acute inflammation. Further, CRP is essential during acute inflammation as it recognizes pathogens and damaged cells and helps mediate their elimination through recruiting the complement system and phagocytic cells (77) CRP also appears to regulate activation of neutrophils, inhibiting neutrophil response to IL 8. CRP is a sensitive marker of inflammation and is directly involved in the pathogenesis of arteriosclerotic lesions and tissue damage (78) C atechol O Methyltransferase, Pain, and Propranolol COMT is a major metabolizing enzyme of catecholamines, specifically epinephrine and norepinephrine, and has been linked to pain models in both humans and rodents (44) There a re currently three different types of haplotypes associated with COMT enzymatic activity. These haplotypes are HPS for high pain sensitivity, APS for average pain sensitivity, and LPS for low pain sensitivity. The HPS haplotype is associated with increase d sensitivity to inflammatory conditions that evoke pain behaviors (44) These inflammation and pain sensitivi ties have been linked with increased levels of epinephrine and norepinephrine along with
30 decreased levels of the COMT enzyme (79, 80) This COMT dependent pain is ARs (33) opioid neurotransmitter system with in the central nervous system (81) Acute pain inhibition requires blocking both of these beta receptors as only blocking one of the m still leads to non significant changes in pain. The HPS haplotype of the COMT opioid neurotransmitter system through the dopaminergic and adrenergic/noradrenergic system. Its effects on the dopaminergic system produced lower lev els of enkephalins but indicated an opioid receptors (81) This uneven balance between opioids and receptors could lead to a less effective opioidergic system, therefore reducing the bility to produce analgesia. Using morphine on these individuals produces an enhanced analgesic response compared to other individuals who possess the LPS or APS haplotype (82) and ARs are expressed in pe ripheral, spinal and supraspinal sites located within the body that are involved with pain transmission. Stimulation of these receptors at the periphery produces allodynia as well as indirectly enhancing pain transmission through the release of pro inflam matory molecules, including cytokines and NO (40, 83) NO and cytokines influence the release of one another NO drives the 6 and IL (84) while cytokines can promote NO release (85) This feedback loop may be a reason for the development and/or maintenance of pa in (84) NO is also able to elicit pain by increasing the firing rate of nociceptors (84) stimulating pro pain prostaglandins, and activating cAMP response element binding (86)
31 T 6 and IL inflammation. Under these conditions these cytokines promote healing (87) but sustained and increase d levels of the m can do damage to tissues and enhance experienced pain (33) Stimul a tion of beta receptors on the cells in the periphery and CNS 6 and IL (88) which may also exacerbate experienced pain. These cytokines downstream and probable cause of COMT dependent pain through sensitized peripheral nociceptors and by increasing the concentration of other pro inflammatory cytokines. One study showed that rats lacking epinephrine were unable to deve lop the same pain sensitivity to COMT dependent pain, indicating that a peripheral increase of catecholamines are needed for HPS haplotypes to develop COMT dependent pain (39) To summarize the pain process, elevated levels of norepinephrine and epinephrine resulting from decreased COMT enzyme activity and 6 and IL (33) In addition to decreased COMT enzyme activity, George et al (89) observed a strong interaction between the HPS haplotype of th e COMT enzyme and pain catastrophizing. Pain catastrophizing is a multidimensional construct comprising elements of rumination, magnification and helplessness. High levels of pain catastrophizing have been able to predict acute and persistent pain (90) Linking pa in catastrophizing and the HPS haplotype can predict shoulder pain and disability more accurately than either one of these variables individually (83).
32 Propranolol has the ability to mediate these pain pathways. Propanol can produce eff ects physiologically as well as psychologically through its pharmacological actions. Propranolol b and which permits blockage of catecholamines on these receptors. The effect is to diminish or eliminate the positive feedback loop created by NO and cytokine release. There is clinical evidence that this type of pain modulation occurs in the peripheries as animal studies have shown that epinephrine injected intraplantarly and intramuscularly (41, 91) causes hyperalgesia were both reversed by the use of propranolol. Propranol ol may potentially influence cognitive processes as it acts to assist in the mediation of pain through the CNS, as well. Propranolol has been previously used to treat psychological conditions like anxiety and post traumatic stress disorder (92) because it has the ability to prohibit the reconsolidation of a fear memory while sparing declarative memory (93) It is suggested that this process occurs by breaking a cycle of anxiety, in which catastrophic misappraisal of bodily sensations of or thosympathetic origin can cause panic attacks (94) Propranolol may also help pain catastrophizers to suppress any poor/nega tive feelings that have been associated with prior pain experiences; this would aid in modulating stress and anxiety as well as reduce sensitivity to pain. This psychological intervention may also be reliant on the COMT genetic polymorphism as there have b een variable responses in multiple studies attempting to demonstrate the efficacy of propranolol on anxiety, psychocardiac disorders and aggressive behavior (95, 96) Participants who are high pain catastrophizers and have the HPS haplotype of the COMT gene are valid candidates to
33 and AR blocker s such as propranolol, which aids in decreasing pain sensitivity.
34 CHAPTER 3 METHODS & MATERIALS The Institutional Review Board of the University of Flo rida approved the study protocol. Participant recruitment took place at the beginning of each semester at the Reitz Union at the University of Florida. Healthy, English speaking participants between the ages of 18 and 65 w ere considered for the study. Excl usion criteria used to determine eligibility for the ex perimental muscle injury protocol and to account for the risks of administering propranolol can be found in Table 1 1. Participants that were deemed eligible for the screening process were then reques ted for their consent. Screening of participants consist ed of completion of a demographics questionnaire, the Pain Catastrophizing Scale (PCS) (13 item, four point rating scale), and a collection of a saliva sample with a buccal swab (Gentra PureGene syste m ). To be eligible for the study, participants score d a minimum of five on the PCS and test ed positive for the HPS COMT polymorphism (rs6269) Genotyping for the HPS COMT polymorphism (rs6269) w as spectr ophotometry After the initial screening process, the Research C oordinator contact ed eligible participants through email and phone to schedule a date for study enroll ment The initial visit occur red on Day 0 (Friday). This session consist ed of obtaining c onsent for inclusion into the full study and further screening to ensure safety into the clinical trial. Th e secondary screening require d participants to receive a 12 lead ECG to rule out cardiac abnormalities that may be a contraindication for propranolol Female participants receive d a pregnancy test Dr. Roland Staud was responsible for interpreting the ECG for abnormalities; participants deemed ineligible because of their
35 ECG results receive d a follow up appointment or phone call to be notified of the f indings. Eligible participants w ere then randomized (www.advancedbiostatistics.com/BISP) and enrolled into the full study (Table 3 1 ). A double blind, randomized, two arm experimental design was used to investigate the effects of propranolol on blood plas ma levels of inflammatory biomarkers. Treatment groups will be referred to as Group A and Group B unless a significant interaction effect is detected. Treatment groups were kept blinded to protect the parent study and was decided a priori that the groups would be revealed only in the case that group differences were detected. Data collection beg an on Day 1 (ensuing Monday) of the study. Day 1 consist ed of collecting baseline vitals blood pressure, heart rate, temperature, and respiratory rate followe d by a blood draw, the Brief Pain Inventory (BPI), QuickDASH (QDash), administration of either propranolol or a placebo capsule, and functional measures ( ROM, MVIC of the external rotator muscle group of the shoulder ) Participants were then required to co mplete a high intensity resistance exercise protocol for the external rotator muscle group of the shoulder. This protocol is designed to induce muscle damage characterized by delayed onset muscle soreness ( DOMS ) stiffness, and functional impairment. A sec ond blood draw was collected after completion of the exercise protocol on Day 1. Follow up assessments occurred on Days 2 through 5 These visits include d collection of vitals and blood at the beginning of each visit Follow up functional and disability m easurements occurred on Days 2 and 3, and included completing the BPI and QDash questionnaires prior to the intervention. ROM and MVIC measurements
36 w ere then collected at the end of the session. Collection of this data did occur on Days 4 and 5 but was not included in the data analysis to protect the parent study. Blinding The provider collecting the data as well the participants w ere blinded to group assignments. The University of Florida Investigational Drug Service provide d the treatment capsule that w a s administered during the sessions, and the CTRB Pharmacy w as aware of the treatment groups. The CTRB Pharmacy was available to remove the blinding of certain participants if severe adverse events were observed without revealing the blindness of the entir e study. Placebo capsules were visually indistinguishable from the active propranolol medication. Outcome Measurements Blood Biomarkers: A registered nurse or phlebotomist dr ew and collect ed blood each session using standard EDTA tubes. One t ea sp oon (tsp) w as collected at the beginning of Days 1 5, with an additional blood draw of one tsp collected at the completion of Day 1 to obtain a blood plasma sample immediately post exercise Blood w as centrifuged at 3000 rpm for 15 minutes and plasma w as stored a t 80C. Plasma samples w ere then analyzed to determine concentration levels of selected inflammatory cytokines. Analysis of IL 6, IL 8, IL as performed using a multiplex panel from Milliplore Sigma (Milliplex, Human Cytokine /Chemokine panel 1, Millipore Corporation, Billerica, USA) as per standard operating procedure. CRP w as analyzed using an ELISA (Human C Reactive Protein/CRP Quantikine ELISA Kit, R&D Systems, Minneapolis, USA). All sample results below the lower limit of quantitation were classified as missing data. All assays w ere performed by the Metabolism and
37 Translational Science Core of the Claude D. Pepper Older Americans Independence Center. QuickDash (QDash) questionnaire: This questionnaire w as used to assess upp er extremity disability. The QDash consists of 11 functional items with scores ranging from 0 (no disability) to 100 (complete disability). The QDash allows us to determine the global disability of the upper extremity shoulder pain can also affect functi oning of the arm and hand (97) Range of Motion (ROM): ROM w as used to quantify mobility limitations in the shoulder as a result of the indu ced injury ROM measurements were taken on the affected shoulder and included active shoulder flexion, active and passive shoulder internal rotation IR, and active and passive shoulder ER. Shoulder flexion w as collected by having the participant in a start ing position of standing with the arm placed at their side. IR and ER rotation w ere established with the participant supine on a plinth with the arm abducted to 90 degrees and the elbow flexed to 90 degrees as the starting position. ROM measurements w ere o btained using a standard two arm plastic goniometer. Active ROM measurements w ere collected before passive ROM measurements. Maximal Voluntary Isometric Contraction (MVIC): Assessing the strength of the external rotator muscle group of the shoulder w as com pleted by using MVIC. MVIC is the maximum force produced during an isometric muscle contraction. To collect an MVIC, participants sat in an isokinetic testing and exercise device (Biodex System 4 Pro, Shirley, USA, model #850 000) with their dominant arm a bducted to 45 degrees and elbow bent at 90 degrees of flexion. Each participant perform ed five MVICs of the dominant arm. Isometric force using the shoulder external rotators was applied to the
38 transducer for five seconds. A 30 second recovery period was t hen provided between contractions. The three highest MVICs w ere recorded and an ensuing average w as calculated to obtain peak force in Foot Pounds ( ft*lbs ). Brief Pain Inventory (BPI): The BPI is utilized to measure pain intensity and duration and it has been found to have solid test rest reliability over short intervals (98) This test makes use of an 11 point numerical rating scale, ranging from 0 (no pain) to 10 (worst pain imaginable). The BPI asks subjects to rate the intensity of their current pain and pain at i ts worst, best, and average over the past 24 hours. Experimental Injury Protocol Muscle damage to the external rotator muscle group of the shoulder w as induced using high intensity resistance exercise with an isokinetic testing and exercise device and sh oulder attachment (Biodex System 4 Pro, Shirley, USA, model #850 000). Participants s a t in the Biodex with the ir shoulder at 45 degrees of abduction and elbow bent at 90 degrees of flexion. The exercise protocol consist ed of repeated concentric and eccentr ic contractions of the shoulder external rotators at an angular velocity of 60 degrees/second for both concentric and eccentric contractions. Each participant w as instructed to exert maximal effort during the protocol while they were given verbal encourage ment by the examiner throughout it. Participants complete d four sets of 10 repetitions separated by 30 seconds of recovery. At the completion of the initial four sets, an MVIC was measured. If the MVIC was 50% or less than the initial MVIC of the partici pant, the fatigue protocol was considered complete. If the MVIC wa s higher than 50%, additional sets of 10 repetitions w ere performed until the aforementioned criteria was fulfilled.
39 Statistical Analysis All statistical analyses w ere performed using PASW Statistics 24.0 statistical package (IBM SPSS Inc., Armonk, USA). To control for inflated alpha levels resulting from repeated statistical comparisons, the level of significance w as adjusted a priori by dividing the original alpha of 0.05 by the number of specific aims (3) instead of correcting for the number of statistical tests, which would be too conservative considering relatively small sample size and exploratory nature of the study Thus, our adjusted alpha w as set at 0.01 6 7 (0.05/3). Specific Aim 1: A 2 (treatment: propranolol or placebo) x 5 (time: immediate, 24 hour, 48 hour, 72 hour, 96 hour) repeated mix ed measures analysis of covariance (ANCOVA) w as used to determine a significant between group by time difference of the individual inflammatory biomarkers. The baseline value obtained for each inflammatory blood biomarker was w as performed if a significant interaction effect had been established. Specific Aim 2: A 2 (treatment: pro pranolol or placebo) x 3 (time: immediate, 24 hour 48 hour) repeated mix ed measures AN COVA will be employed to determine if there was a significant between group by time difference of the functional outcome measures (ROM, MVIC and self report disability). The baseline value obtained for each functional measurement w as testing w as completed if a significant interaction effect had been established. Specific Aim 3: Pearson bivariate correlation coeffi cients w ere calculated to determine the strength of the statistical relationship between inflammatory cytokine levels and pain intensity and between functional outcome measures (ROM, MVIC and
40 self report disability) over the 48 hour time period post injury Correlation matrices w ere generated for all selected inflammatory cytokines with peak pain intensity and functional outcome measures. Absolute values were used for comparison Stepwise m ultiple linear regression analyses were then calculated on peak pain intensity and functional outcome measures that were identified to have significant relationships in the same direction with three or more inflammatory cytokines. Table 3 1 Study exclusion criteria Exclusion criteria for exerc ise induced shoulder injury protocol Chronic pain (>3 months) in any area Neurologic impairment of the upper extremity (determined by loss of sensation, muscle weakness, and reflex change) Regular participation in upper extremity weight training Currently experiencing shoulder pain Previous history of neck of shoulder pain (defined as experiencing pain longer than 48 hours or seeking medical treatment) Previous history of upper extremity surgery Currently or regularly using pain medication Exclusion crite ria for propranolol Clinically significant abnormal 12 lead ECG Cardiac failure Greater than first degree heart block Coronary heart disease Sinus bradycardia (resting heart rate of 55 bpm or less) Known hypersensitivity to propranolol Uncontrolled hypert ension (resting systolic blood pressure less than 90 mmHg) Wolff Parkinson White syndrome General exclusion criteria for study participation Bronchial asthma Diabetes Chronic obstructive pulmonary disease Dementia Pregnancy Breast feeding Non allergic bronchospasm History of recent surgery requiring general anesthesia Major depression
41 Table 3 2 Participant demographics Group N Mean Age (years) Mean Weight (kg) Mean Height (cm) A 27 (Y=12, X=15) 23.1 (SD=8.07) 69.89 (SD=13 .71) 170.72 (SD=9.82) B 27 (Y=12, X=15) 20.85 (SD=1.86) 68.49 (SD=17.04) 173.23 (SD=11.8) Table 3 3 Study protocol Visit Number Function Study Day Visit 1 Consent Day 0 Visit 2 Baseline Day 1 Visit 3 24 Hours Post Day 2 Vis it 4 48 Hours Post Day 3 Informed consent X EKG X Pregnancy test (if female) X Vital signs X X X X Blood draw X X X Pill administration X X X Pain questionnaires X X X ROM X X X MVIC X X X Should fatigue protocol X Blood draw post fatigue protocol X
42 CHAPTER 4 RESULTS All participants (N=54) met fatigue protocol requirements and were included in the statistical analys e s. Two participants were excluded from the data analysis of the inflammatory cytokines because t hey were unable to provide blood samples throughout the entire week of data collection The resulting groups were N=27 (G roup A ) and N=25 (G roup B ) A repeated measures ANOVA was completed on functional and disability measurements with both treatment grou ps collapsed into one data pool The analysis was performed to assess whether the experimental injury protocol was able to induce a muscle injury. Participants displayed a significant decrease in strength, (F=40.164, 2 =0.646), ER AROM (F=7.24, p= 2 =0.12), ER PROM (F=6.387, 2 2 =0.111), and the Q DASH 2 =0.356 ) from baseline to post injury ( 24 and 48 hours ) Figures 4 1, 4 2 and 4 3 demonstrate the change of these measurements over time. There were no significant differences in blood levels identified during an analysis of the inflammatory cytokines and CRP (p>0.05) with the groups collapsed Specific Aim 1 A 2 (treatment) x 5 (time) repeated mix ed measures ANCOVA was used to analyze data for each inflammatory cytokine and CRP. Pre injury (baseline) measurements were included in the statistical analysis as covariates to account for any individual differences at baseline. There was no significant interaction effects observed with inflammatory cytokines and CRP plasma concentrations; IL 2 =0.002), IL 2 =0.005), IL 2 =0.011), IL 10
43 2 2 =0.029), and CRP (F=0.661, p=0.42, 2 =0.013). Group vs Time g raphs of the estimated marginal means can be viewed in Figures 4 4 to 4 9. Additionally, t here were no significant within group differences observed with inflammatory cytokines and plasma CRP concentration s (p>0.0 167 ). Mean plasm a concentrations of IL 2.16 pg/ml; Group B mean = 1 pg/ml, SD = 0.81 pg/ml), IL 6 (Group A mean = 3.56 pg/ml, SD = 3.74 pg/ml; Group B mean = 2.58 pg/ml, SD = 3.24 pg/ml) and IL 10 (Group A mean = 10.76 pg/ml; SD = 8.31 pg/ml, Group B mean = 9.56 pg/ml, SD = 13.25 pg/ml) peaked on Day 3 post injury for both groups. Mean plasma concentrations of IL 8 peaked for both groups on Day 2 (Group A mean = 4.1 pg/ml, SD = 2.49 pg/ml; Group B mean = 3.89 pg/ml, SD = 3.26 pg/ concentrations peaked on Day 2 for Group A and Day 3 for Group B (Group A mean = 2.8 pg/ml, SD = 2.16 pg/ml; Group B mean = 2.31 pg/ml, SD = 1.06 pg/ml). Mean CRP plasma concentrations peaked on Day 4 for Group A and Day 2 for Grou p B (Group A mean = 2771.9 pg/ml, SD = 6896.9 pg/ml; Group B mean = 1630 pg/ml, SD = 2861.2 pg/ml). Further statistical analyse s of pl asma cytokine concentrations were conducted to determine whether propranolol affected inflammatory cytokine plasma co ncentrations specific to sex The analysis consisted of separate 2 x 5 repeated mixed measures ANCOVA s for N=22 (Group A N=11; Group B N=11 males) and N=30 females (Group A N=16; Group B N=14). There were no significant between group interactions o bserved in males or females with inflammatory cytokine and CRP measurements (p>0.0 167 ). There was a positive significant within group difference
44 observed in females with IL 2 =0.126) between post exercise (immediately following the i njury protocol) and Day 5 (Figure 4 10). Group B had increased concentrations of the anti inflammatory cytokine IL 10 on Day 5 compared to baseline measurements. phericity which was not assumed, thus a Greenhouse Gei sser correction was used to determine significance. No other within group sex differences on cytokine plasma con centrations were observed (p>0.0 167 ). Specific Aim 2 The analysis of functional measurements consisted of N=54 subjects (Group A N=27 ; Group B N=27) A 2 (treatment) x 2 (time) repeated mixed measures ANOVA was carried out to evaluate the impact of propranolol on functional measurements. There were no interactions or main effects o bserved for the functional and disability measurements (p>0.0 167 ) including ROM, MVIC, and QDash. There were non significant findings related to propranolol and functional measurements when the groups were evaluated by sex (p>0.0 167 ). Specific Aim 3 Pearson bivariate correlation s examine d the strength of relationshi p s between inflammatory cytokine concentrations, and between the inflammatory cytokine concentrations and pain, functional and disability measurements. A co rrelation matrix was formulated between the inflammatory cytokines (and CRP ). All cytokine levels had positive and weak to p<0.0167 ) relationships apart from IL 8 with IL ; which did not have a significant correlation (p>0.0167) CRP was not correlated with any of the inflammatory cytokines
45 (p>0.0167) All relationships between IL moderate and positive 0 .5). Table 4 1 lists all the strong correlations between the inflammatory cytokines There were significant correlations observed between inflammatory cytokine concentrations and functional disability and pain me asurements. In total, 54 correlations demonstrated statistical significance, with these correlations having at least a weak relationship (r 0.3), while four of them possess ed a moderate negative effect (baseline CRP CRP Day 2 and CRP Day 3 with AROM flexi on on Day 2 CRP Day 2 with ER AROM on Day 2 ) Thirty eight of the correlations were between inflammatory cytokine concentrations and pain measurements, while the remaining 16 correlations were between inflammatory cytokine concentrations and measures of d isability and function IL 6/IL 10 ratio was also investigated to determine if the proportion of pro to anti inflammatory cytokines were related to the outcome measurements, but no significant correlations were detected (p>0.0167). Pain, functional and disability measurements which had significant correlations in for further analysis using multiple linear regression. These variables included resting pain on Day 2, res ting pain on Day 3 and active pain on Day 3. Although there were multiple significant correlations between the inflammatory cytokines and functional and pain measurements with flexion, they were not selected for further analysis because flexion did not h ave a significant change in function or pain throughout the study. Tables 4 2, 4 3 and 4 4 show all significant correlations between inflammatory cytokines with the functional, disability and pain measurements.
46 Five separate multiple regression analyses of interest were identified: 1) IL 6 and IL 10 concentrations on Day 2 with resting pain on Day 2 2) IL 6, IL 3) IL 6, IL Day 3 4) IL 6, IL 5) IL 6, IL The second and fourth multiple regression analyses identified that baseline IL 10 plasma concentra tions contributed to resting pain (R 2 and active pain (R 2 Day 3. IL 10 plasma concentration on Day 3 was also identified in the fifth multiple regression as the only inflammatory cytokine (R 2 that contributed significantly to active pain in the shoulder on Day 3. The third multiple regression analysis identified that IL 6 plasma concentration on Day 3 (R 2 .28x) was the lone pro inflammatory cytokine to contribute to resting pain experienced in the shoulder on Day 3. The first multiple regression analysis did not identify a significant contribution (p>0.0167) to resting pain experienced in the shoulder on Da y 2 Sex was added as a predictor variable in all models because a significant difference in IL 10 concentrations in females over time was identified, but it did not contribute significantly to any of the 5 models (p>0.0167). IL 10 plasma concentrations a t baseline and Day 3 were shown to contribute to pain (resting & active) in three of the multiple regression analyses. Scatterplots of these IL 10 relationships with active and resting pain can be found in Figures 4 11 to 4 13.
47 Figure 4 1 MVIC measurements of the shoulder (Groups collapsed) over time. Figure 4 2 ROM measurements of the shoulder (Groups collapsed) over time. 0 5 10 15 20 25 30 35 PRETEST 24 HRS 48 HRS MVIC (FT*LBS) TIME (HOURS) MEAN MVIC VS TIME 0 20 40 60 80 100 120 140 PRETEST 24 HRS 48 HRS ROM (DEGREES) TIME (HOURS) MEAN RANGE OF MOTION OF THE SHOULDER VS TIME ER arom ER prom IR arom IR prom
48 Figure 4 3 Disability ( QDASH ) measuremen ts of the shoulder (Groups collapsed) over time. Figure 4 4 Estimated marginal means of IL over time. -5 0 5 10 15 20 25 30 35 PRETEST 24 HRS 48 HRS QUICKDASH SCORE TIME (HOURS) MEAN QUICKDASH VS TIME 0 0.5 1 1.5 2 2.5 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA IL 1 (PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF IL 1 ETA VS TIME Group A Group B
49 Figure 4 5 Estimated marginal means of IL 6 over time. Figure 4 6 Estimated marginal means of IL 8 over time. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA IL 6 PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF IL 6 VS TIME Group A Group B 0 1 2 3 4 5 6 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA IL 8 (PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF IL 8 VS TIME Group A Group B
50 Figure 4 7 Estimated marginal means of IL 10 over time. Figure 4 8 Estimated over time. 0 2 4 6 8 10 12 14 16 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA IL 10 (PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF IL 10 VS TIME Group A Group B 0 0.5 1 1.5 2 2.5 3 3.5 4 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA TNF ( PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF TNF TIME Group A Group B
51 Figure 4 9 Estimated marginal means of CRP over time. Figure 4 10 Estimated marginal means of IL 10 in women over time. -3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 7000 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA CRP (PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF CRP VS TIME Group A Group B 0 2 4 6 8 10 12 14 16 POST 24 HRS 48 HRS 72 HRS 96 HRS PLASMA IL 10 (PG/ML) TIME (HOURS) ESTIMATED MARGINAL MEANS OF IL 10 IN WOMEN VS TIME Group A Group B
52 Table 4 1 Inflammatory cytokines ( set; ) Inflammatory Cytokines Correlated Cytokine Pearson Correlation IL IL 6 Pre IL 6 D2 IL 6 D3 IL IL .780 .787 .719 .958 .958 .824 .722 IL IL 6 Pre IL 6 D2 IL IL .716 .782 .958 .966 .813 .789 IL IL 6 Pre IL 6 D2 IL 6 D3 IL IL .750 .786 .773 .958 .966 .800 .835 IL 6 Pre IL 6 D2 IL 6 D3 IL IL IL .822 .827 .741 .824 .813 .800 .820 .835 Pre D3 .820 8 0 0 IL 6 D2 IL 6 D3 IL IL IL .717 .725 .722 .789 .835 .835 .800
53 Table 4 1. Continued Inflammatory Cytokines Correlated Cytokine Pearson Correlation IL 6 Pre IL 6 D2 IL 6 D3 IL 10 Pre IL 10 D2 IL 10 D3 IL IL IL .965 .932 .828 .754 .759 .780 .716 .750 .822 IL 6 D2 IL 6 Pre IL 6 D3 IL 10 Pre IL 10 D2 IL 10 D3 IL IL IL .965 .930 .771 .765 .727 .787 .782 .786 .827 .717 IL 6 D3 IL 6 Pre IL 6 D2 IL 10 Pre IL 10 D2 IL 10 D3 IL IL .932 .930 .795 .737 .826 .719 .773 .741 .725 IL 8 Pre IL 8 D2 IL 8 D 3 .915 .980 IL 8 D2 IL 8 Pre IL 8 D3 .915 .91 9 IL 8 D3 IL 8 Pre IL 8 D2 .980 .919
54 Table 4 2 Inflammatory cytokines correlated with active pain ( set; p<0.0167 ) Baseline Day 2 Day 3 Pain w/ Flexion AROM Pain w/ MVIC Pain w/ Flexion AROM Pain w/ MVIC Pain w/ F lexion AROM Pain w/ MVIC IL BL 0.462 IL 6 BL 0.368 0.390 IL 10 BL 0.366 0.365 0.434 0.449 0.332 IL 0.429 IL 6 Day 2 0.372 0.380 IL 10 Day 2 0.411 0.390 0.390 TN 0.337 IL 0.456 0.327 IL 6 Day 3 0.346 0.456 IL 10 Day 3 0.345 0.481 Day 3 0.408 0.401 Table 4.1. Continued Infl ammatory Cytokine Correlated Cytokine Pearson Correlation IL 10 Pre IL 6 Pre IL 6 D2 IL 6 D3 IL 10 D2 IL 10 D3 .828 .771 .795 .933 .941 IL 10 D2 IL 6 Pre IL 6 D2 IL 6 D3 IL 10 Pre IL 10 D3 .754 .765 .826 .933 .913 IL 10 D3 IL 6 Pre IL 6 D2 IL 6 D3 IL 10 Pre IL 10 D2 .759 .727 .826 .941 .913
55 Table 4 3 Inflammatory cytokines correlated with disabi lity ( set; p<0.0167 ) Day 2 Day 3 Flx AROM QDash MVIC Flx AROM Q Dash MVIC IL BL IL 6 BL 0.351 IL 8 BL 0.372 IL 10 BL 0.328 0.401 0.372 CRP BL 0.622 IL D2 0.369 IL 6 D2 0.362 IL 8 D2 0.394 IL 10 D2 0.328 CRP D2 0.676 IL D3 0.381 IL 6 D3 0.434 IL 8 D3 IL 10 D3 0.373 0.381 CRP D3 0.661 Table 4 4 Inflammatory cytokines correlated with resting pain ( set; p<0.0167 ) Baseline Day 2 Day 3 Current Pain Worst Pain Current Pain Worst Pain Current Pain Worst Pain IL BL 0.330 IL 6 BL 0.376 IL 10 BL 0.390 0.346 IL D2 IL 6 D2 0.376 IL 10 D2 0.373 IL D3 0.330 0.358 IL 6 D3 0.336 0.430 IL 10 D3 0.330 0.339 0.427 0.344 0.383
56 Figure 4 11 Scatterplot regression analysis between IL 10 blood plasma concentration and resting p ain on Day 3 Figure 4 12 Scatterp lot regression analysis between IL 10 blood plasma concentration and active pain on Day 3 0 1 2 3 4 5 6 7 8 9 10 0 5 10 15 20 25 30 35 40 45 Resting Pain (0 100) Concentration of IL 10 (pg/ml) Correlation between baseline IL 10 plasma concentration and resting pain on Day 3 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35 40 45 Active Pain (0 100) Concentration of IL 10 (pg/ml) Correlation between baseline IL 10 plasma concentration and active pain on Day 3
57 Figure 4 13 Scatterplot regression analysis between IL 10 plasma concentration on Day 3 with active pain on Day 3 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Active Pain (0 100) IL 10 Plasma Concentration (pg/ml) Correlation between IL 10 plasma concentration on Day 3 with active pain on Day 3
58 CHAPTER 5 DISCUSSION Broad Overview of the Study with the inflammatory process following an acute musculoskeletal injury using a high risk subgroup. This study sought to identify if propranolol could be a mediator of the inflammation process following a musculoskeletal injury and, in turn, if it would have a more general impact on functional measurements post injury. The experimental injury protocol was used to induce an acute inflammatory response. We measured inflammatory cytokine plasma concentrations, functional impairment, pain and disability outcomes. No significant interaction effects were detected in this study. Pain measurements were also collected but were on ly included in the correlational and regression analyses. The functional impairment measures indicate that the experimental injury protocol may not have caused enough tissue damage to induce a robust inflammatory response with systemic involvement. There w as statistically significant functional impairment and disability following the injury, however the small effect sizes of these variables suggest that the muscular injury sustained was mild and well localized, thus making an interaction between the inflamm atory cytokines and propranolol difficult to discern. Propranolol is a pharmacological intervention prescribed for a myriad of medical conditions including anxiety (emotional) essential tremors (neurological) and blood pressure (cardiovascular) (94, 99) The hypothesis that propranolol may act as an analgesic for a high risk subgroup characterized by pain catastrophizing and the COMT
59 SNP is a novel approach to pain management with few studies examining the thera peutic effects in human clinical populations (54, 100, 101) Previous s tudies using animal models have speculated that propranolol may produce an analgesia effect at the site of injury by modulating inflammatory cy tokine levels which are most active during the acute phase of inflammation (41, 88, 102) Although our study failed to identify any therapeutic effects of propranolol, it may still be considered a novel approach to personalized medicine for treating musculoskeletal pain and inflammation. The following analysis into the possible biological response of inflammatory cytokines to propranolol and the study design will help with in the development of future research for t his sub discipline (31, 34, 36, 42, 88) Ex perimentally Induced Muscle Injury The experimental protocol displayed evidence that a musculoskeletal injury occurred, and this was shown primarily by the significant loss of strength ( MVIC ) from baseline measurements. Previous studies have identified that a significant loss of strength within the first 48 hours post exercise is a reliable measurement of induced injury (51, 54, 1 00, 101) Th e strength loss occurs secondary to eccentric overload excitation contraction coupling failure and ultrastructural damage within the sarcomere (103) There was also a significant decrease in ROM with a concomitant increase in disability observed between baseline and 24 hours post injury, which recovered by 48 hours. Collectively, the effect s ize s for all ROM measurements was quite small The participants in this study were still within normal shoulder ROM following the experimental protocol (104) Few studies have measured shoulder ROM following an
60 experimental injury protocol, however one study did observe a decrease of ~ (105) Likewise, self report disability scores (QDash) demonstrated significant increases in disability during the 48 hour post injury period, however effect sizes produced were small Specific Aim 1 We found no interaction or main effects for the inflammatory cytokines IL 6, IL 8, IL P lasma concentrations did not appear to be significantly impacted by either the musculoskeletal injury induction or ingestion of propranolol. G roup B had fluctuations in plasma concentrations of cytokines daily, while the concentrations in Group A remained relatively constant throughout the week. Group B had a slight decrease in IL 6, IL compared to Group A, and these decreases preceded peaks in plasma concentrations of the inflammatory cytokines of Group B on Day 3. The inflammatory cytokines trended back towards baseline values by Day 4. Group B had a slight decrease in CRP plasma concentration Our results suggest that our prescribed dosing regimen for propranolol may not be significantly involved in inflammatory modulation following a musculoskeletal injury in our high risk subgroup. This is plausible due to the lack of research into the physiological response to propranolol following an acute musculoskeletal injury in the molecular level currently focuses on studies using animal injury models (91, 106 108) To the best of our knowledge, this is one of the first studies to explore the effects
61 of propranolol on plasma cytokine concentrations in humans followin g an acute musculoskeletal injury. Our experimental injury model is a safe and reproduceable model used in a laboratory setting to recreate a musculoskeletal injury. The comprehensive response of inflammatory cytokines in blood plasma due to an induced i njury are not yet fully understood (109) mainly due to the fact that most research into the physiology of this model do es not include a or natural history control group. This limits our understandin g of how inflammatory cytokines may respond to our injury protocol M ost inflammatory cytokine data is based on a pretest/posttest model with the control group typically receiving a placebo or sham intervention (110) A similar model was replicated in our study allo w ing us to make comparisons to previous studies on inflammatory cytokines and experimentally induced injury Interestingly a review of the previous literature highlights the variability of using plasma to measure infl ammatory cytokines as a response to an induced injury (31, 34, 59, 100, 111 113) Consideration should be given to the mechanism for production of cytokines as a response to a musculoskeletal injury. There is gen eral agreement that plasma cytokine s respond to an induced injury and that significant elevations in our inflammatory cytokines of interest have been observed Although there is still considerable debate as to what specifically signals the production of th ese inflammatory cytokines. Pedersen et al. noted that cytokines respond to strenuous exercise (114) increases were followed by a dramatic IL 6 increase (58) however this was obs erved after a marathon race and not an isolated resistance exercise protocol to one muscle group Elevations in plasma levels of IL 6 may be based on muscle overexertion as
62 injury (57, 115) IL 6 is the most commonly reported cytokine measured in the plasma that responds to a muscle injury (116) and levels of IL 6 have been reported t o stay elevated for several day s post injury C are less frequently observed following intense exercise protocols designed to induce muscular injury (100, 116) In addition function through local autocrine and paracrine pathways to initiate the inflammat ory response. Therefore, cytokine detection may be difficult systemically (37) IL 6 has multiple functions in the inflammatory response, including signaling CRP through an endocrinal pathway. This systemic response should make IL 6 the most easily detectable inflammatory cytokine in blood plasma. inflammatory may still target the cytokines involved at the initial stages of the inflammatory r esponse even though we were unable to establish any inflammatory cytokine changes in the plasma. Measuring the changes locally at the site of the injury may yield more promising findings Muscle biopsies from the injured tissue would serve as a superior m ethod for analyzing the effects of propranolol, mak ing this a more reliable and precise measurement of inflammatory cytokines following an acute injury (116) Plasma c hanges have been observed in IL 8 and IL 10 following exercise however results tend to be highly variable between individuals (116) Specifically, in situations of ecce ntric exercise, smaller changes in plasma IL 8 were observed in comparison to IL 10 (117, 118) Although we did not observe any significant differences in cytokines between groups, it is possible that propranolol i s acting at the site of injury in other ways
63 There were no significant interactions observed with CRP. CRP is produced in the liver and reacts to increases of IL 6 in the plasma. Using CRP as an indicator of muscle damage following eccentric exercise ha s had mixed results. Others have observed elevated CRP following an induced muscle injury ranging from 1 7 days (47, 57, 119, 120) but there is also some evidence that concentrations of CRP remain unchanged post injury (121, 122) This suggests that CRP could be useful to determine muscle damage at a cellular level but should be used in conjunction with other biomarkers. Sex Specific Analysis of Inflammatory Cytokines Th e clearance of propranolol ha s been shown to be more expedient in males than females (123) The longer bioavailability of propranolol in females could improve the therapeutic effect of propranolol on inflammation following a musculos keletal injury Therefore, we analyzed the data on sex specific groups. Our between sex analyse s did not show any significant between group or within group interactions in either sex except for a significant within group effect on IL 10 levels in women between immediate post injury and Day 5 IL 10 is described in the literature as an anti inflammatory cytokine that peaks near the end of the inflammation process (64) This observed increase in IL 10 may indicate a more robust anti inflammatory response for females of Group B. An interesting trend was observed in females showing that IL 10 plasm a concentrations of Group B bega n to separate from Group A near the end of the study protocol IL 1 0 does not appear have a sex specific response in the inflammatory response following an injury in a healthy population (124, 125) There is evidence
64 suggesting that women in chronic pain may experience an increase d and delayed inflammatory response relative to men when women are thinking negatively about their pain experience (126) Our participants are high risk pain catastrophizers, making it plausible that our female participants could have catastrophized before or after their induced injury. It is possible that o ne of our groups experienced a pharmacological effect from the propranolol by either increasing the anti inflammatory response or by mediating the pro inflammatory response, which may have been a result of the slower clearance rate. Specific Aim 2 MVIC, ROM of the shoulder, pain with ROM of the shoulder, and the QDash are all reliable indicators of induced injury (54, 97, 105, 109, 119, 127 129) al though reductions in MVIC are considered to be the gold standard f or an induced injury (130, 131) Our study included some of the more common measurements for determining an experimental injury though we could have included others that would have offered more clarity into what oc curred because of our experimental protocol. With regards to our ROM measurements, the positioning of our patients was ideal (132 134) and the use of a goniometer to measure ROM has robust intra rater reliability (133) We could have included other clinical outcome measures such as shoulder abduction and horizontal adducti on since these ranges of motion also decrease following an injury of the rotator cuff (105, 135) In theory, the largest deficit should have been observed with internal rotation of the shoulder. Pain evoked abduction has also been shown to correlate with pain associated with induced injury (105)
65 Non selective beta blockers do contribute to increased muscle fatigue, however this has been observed predominantly in higher doses of propranolol (~265 mg/day) (136, 137) Short te rm use and lower dosages of propranolol do not seem to have as large of an impact on muscle function and performance in healthy adults (137) In addition, a single dose of propranolol has been shown to have no effect on performance during a single bout of high intensity exercise (138) Our sample population included propranolol over the five day study. The study protocol also only consisted of only one bout o f high intensity exercise. Even in the presence of an injury, it is plausible that our propranolol dosage protocol would have had minimal impact on shoulder strength and function even following a robust exercise induced injury. Specific Aim 3 I nflammator y cytokine analysis demonstrated moderate to strong significant positive correlations with each bio marker except for IL e findings are in agree ment with current research citing positive associations among inflammatory cytokines post injury (31, 34) I ncrease s in both pro and anti inflammatory cyt okines are typically detected during an acute inflammatory response Our associations do infer that cytokine concentrations increase collectively and interactively following a n induced musculoskeletal injury During the inflammatory response it is expected that cytokines increase dynamically within the first 48 hours following an acute musculoskeletal injury ( 139) and neutralize by 96 hours.
66 The main findings for this aim of the study were that several cytokines (both pro and anti inflammatory) were associated with perceived pain (both resting and active) during the post injury recovery period following ind uced injury. The significant associations are described below: 1) IL 6, IL injury) are associated with resting pain on day 3 (48 hrs). 2) IL 6, IL injury) are associat ed with active pain on day 3 (48 hrs). 3) IL 6 and IL 10 concentrations on day 2 (24 hrs) are associated with resting pain on day 2 (48 hrs). 4) IL resting pain on day 3 ( 48 hrs) 5) IL active pain on day 3 ( 48 hrs). Elevated baseline cytokine levels and pain perception at day 3 (48 hrs post injury) The positive associations between inflammatory cytokines an d pain is consistent with previous studies (113, 140) and indicates that individuals with higher baseline plasma concentrations of inflammatory cytokines may have higher pain responses 48 hours post injury. Eleva ted IL and IL 6 are all e xpressed during the inflammatory response as pro inflammatory cytokines and have also been identified in the literature as being hyperalgesic, or pain producing cytokines (141) Furthermore, elevated levels of these cytokines have been found to contribute to the development of chronic pain
67 conditions (31) This association of increased baseline inflammatory cytok ines and heightened resting and movement evoked pain in our high risk subgroup may be a specific characteristic of our high risk subgroup. We speculate that the elevated baseline cytokine levels contributed to the elevated pain levels at 48 hours post inju ry by lowering pain thresholds and increasing nociceptive sensitivity in these individuals through some peripheral or central sensitization effect. We further speculate that the elevated cytokine concentrations were able to stimulate nociceptors at varying levels of the pain pathway or possibly of an interaction with other pain producing enzymes and neurotransmitters such as S ubstance P and cyclo oxygenase, following injury (142 144) Of particular interest was th e concentration of IL 10 at baseline. Interleukin 10 was the only cytokine identified as being significant at predicting both resting and active pain during post injury recovery period. Interleukin 10 has been characterized in the literature as an anti inf lammatory cytokine, however it appears that it also may have some association with heightened inflammatory pain, and therefore may have both pro and anti inflammatory properties. IL 10 is postulated to inhibit the production of pro inflammatory cytokines by reducing signaling of pro inflammatory cells, mainly through leukocytes such as macrophages and neutrophils (141, 145) This process occurs by blocking the up regulation of mitogen activated protein kinases p38 a nd c Jun N terminal kinases and by suppressing cytokine signaling 1 and 3 (64, 141, 146, 147) ; thus reducing (146) (145,
68 148, 149) reducing their concentration would be essential for modulating pain post injury. Few studies have investigated overe xpression of baseline IL 10 concentrations however, evidence suggests that elevated levels of IL 10 prior to injury or infection may lead to poor recovery (150, 151) There is also evidence that the timing of product ion of IL 10 is essential for proper healing and recovery post injury (60) If IL 10 is produced too early or too late during the inflammatory response, secondary tissue damage may occur. The secondary damage of health y cells is primarily a result of the reduced effectiveness of the coagulation cascade early in the inflammatory response. Pre mature cell signaling by IL 10 has been linked to the overproduction of Activated Protein C (APC) (60, 152) APC plays an integral role as an anti coagulation agent in the inflammatory response, and if activated prematurely may alter the early stages of the coagulation cascade following tissue damage, thus extending the acute inflammatory resp onse (152) Elevated baseline levels of IL 10 have been identified as a predictor of long term adverse cardiovascular outcomes in patients with acute coronary syndrome (153, 154) Overexpression of IL 10 has also been shown t o delay healing in skin lesions of murine models (155) Elevated levels of IL 10 may delay the recru itment of macrophages to the site of injury. The combination of elevated levels of IL 10 and premature signaling of APC could extend the acute inflammatory response, thus amplifying pain at rest and with movement. Additionally, systemic increases of IL 10 have been shown to mediate both chronic and movement evoked pain (156, 157)
69 Psychological and physical stress may also be considered as a cause for the overexpression of IL 10 at baseline in this study. There is ev idence that stress can cause a systemic increase of IL 10 (158) The high risk subgroup in this study has been identified as catast rophizers, which makes them at risk for an exaggerated psychological stress response that could potentially increase IL 10. The initial blood draw occurred early during the first visit while participants were unfamiliar with the study protocol. We can spec ulate that some of the participants may have experienced an elevated psychological response (catastrophizing) due to heightened levels of stress and anxiety experienced during the early stages of the enrollment period when baseline testing was being perfor med (vital signs, EKG, blood draw). Further investigation of the association between pain catastrophizing and IL 10 (and other cytokines) is warranted to confirm this speculation. In summary, we speculate that elevated IL 10 concentrations at baseline prod uces a greater risk of experiencing heightened pain following musculoskeletal injury due to its strong positive relationship with selected pro inflammatory cytokines. Individuals that profile with higher inflammatory cytokines collectively at baseline may be more at risk for a robust pain response. Increased levels of IL 10 at baseline may also act as a reserve to help counterbalance pain producing pro inflammatory cytokine levels. IL 10 is the only anti inflammatory cytokine we examined in this study, oth er major anti inflammatory cytokines include IL 4, IL 11 and IL 13 (159) Future research should investigate in greater depth the relationship and interaction between pro and anti inflammatory cytokines during recovery from musculoskeletal injury.
70 Elevated cy tokine levels on day 2 and 3 post injury and pain perception at day 3 (48 hrs post injury) All inflammatory cytokines analyzed except IL 8, were significantly associated with resting and active pain measurements at the 24 and 48 hour post inju ry period The 48 hour time point is important because post injury symptoms (DOMS, stiffness, and weakness) ha ve been shown to peak at approximately this time after musculoskeletal injury (54, 100, 148, 160) As pai n intensified the relationship with cytokine levels became stronger as shown by the inclusion of IL 10 at 48 hours post injury. Since all cytokines were positively associated with resting and movement evoked pain, we also examined whether the proportion o f anti inflammatory cytokines to pro inflammatory cytokines had a relationship with heightened pain. Individuals that have higher anti inflammatory than pro inflammatory cytokine levels they may have the ability to mediate inflammation more efficiently by counteracting the effects of pro inflammatory cytokines. There is evidence to suggest that IL 6/IL 10 ratio may be the best indication of a balance of anti and pro inflammatory cytokines following tissue trauma (161 164) ; however we were unable to detect any significant correlations between IL 6/IL 10 ratio and pain, functional and disability measurements. This finding, may suggest that heightened pain in our population may be more dependent on the total amount of a nti inflammatory cytokines in the plasma as opposed to the ratio between pro and anti inflammatory cytokines. IL 6 was the only pro inflammatory cytokine identified by our multiple regression analyses to consistently predict pain. IL 6 is a known hyperalg esic cytokine (165 167)
71 and is important in signaling the recruitment of IL 10 to the site of injury (34) It is not surprising that IL 6 concentrations on day 3 post injury was the best predictor of 3, as IL 6 is active throughout the inflammation response. IL 6 is also important for activating CRP during acute inflammation. CRP did not correlate with any of the pain measurements or the inflammatory cytokines, but it was our only blood biomarker that had a negative relationship with multiple disability measurements specifically with our ROM measures Elevated levels of CRP were associated with deficits in shoulder ROM. Current research on CRP suggests that this protein is negatively related to muscle function (168) which supports our findings There were other relationships identified but no trends were identified, therefore we did not investigate those relationships further. We can speculate that these may h ave occurred due to chance and may not have significant meaning in this study. Summary of Study Design Limitations With this study being novel and serving as an arm of a larger study, it was not effects with the inflammatory cytokines. This may be one of the major reasons we were unable to detect any interaction effects with propranolol following our experimentally induced muscle injury ay be best studied on a population experiencing clinical pain and inflammation. A second consideration is that the injury model may not have been the most efficient to use to study the inflammatory cytokines, using clinical pain or a different experimental injury model may produce different results. With that in mind, there were many design flaws that should be
72 considered in future research effects can be found in Table 5 1. The nature of the clinica l trial allowed for a number of differences in blood sampling collection and study design that could have contributed to the outcome measurements : The circadian rhythms of participants may have had an impact on the inflammatory cytokines. Blood draws wer e collected approximately +/ 2 hours from the 24 hour interva l, but there were some adjustments made to accommodate some participants to ensure study enrollment diurnal variations that peak early in the morning (169) These naturally occurring subjects that began the study early in the morning and may have had follow up appointm ents scheduled for mid morning. This may have contributed to the group P were not controlled in the study and Zhou et al. suggest that participants should fast before collect ing blood to analyze cytokines (112) High fat meals elevate IL (170, 171) Any participant that came in for data collection directly afte r ingesting a meal may have had higher than expected inflammatory cytokine concentrations. The half life for propranolol following an oral dose is 5.5 hours ( 172) with approximately 25% of the dosage being absorbed into the system. Therefore, with an initial dosage of 40 mg, we can assume that our participants receiving
73 propranolol are absorbing 10 mg into their system. A higher dosage may be necessary to obse rve changes in plasma concentrations of inflammatory cytokines. The blood draw taken after the experimental injury protocol was the only draw taken dosage may have been too close to this blood draw to observe the full effects of propranolol as its highest concentration in blood plasma is detected between two to three hours post dosage (172) Exercise increase s the clearance rate of propranolol (173) and this may diminish the effect of the initial dose of propranolol as it was administered prior to the fatigue protocol. Other blood biomarkers could have been included into the study to help identify whether our experimental injury model produced a large enough injury to cause an inflammatory response. These include creatine kinase (54, 118, 119, 174 181) lactat e dehydrogenase (174, 181) and myoglobin (100, 128, 181) E mploying creatine kinase would have been the most beneficial as a measurement to discern muscle damage, however our study team was unable to find an ELISA that could produce reliable results after freezing the plasma samples. This biomarker increases in concentration with muscle damage and inflammation and peaks between 48 72 hours post injury (54, 119, 131, 179) Measuring inflammatory cytokines was the primary aim and took precedence over including these secondary markers. Inclusion of these blood biomarkers in a future study could yield more insight into the amount of muscle d amage that is associated with our fatigue protocol.
74 General study limitations include the lack of a natural history control. Both groups received identical treatments, and the only difference was whether the capsule that was ingested by the participants c ontained propranolol or an inert substance this could lead our participants to be influenced by the placebo effect (182) Expectatio ns of treatme nt demonstrated the ability to diminish pain associated with an EIMI ( 183) and such an analgesic effect may have influenced our outcome measurements in our control group, allowing them to suppress any pain related movement to perform as well as the propranolol group with the ROM measurements. If both groups believed they w ere receiving the active treatment, there is a possibility that the inflammatory cytokine response could be similar in both groups. Individuals with the HPS COMT polymorphism also had an enhanced placebo response (184) Further, th e control group featured participants with this genotyping, so it is plausible that this population may inflammatory cytokines and functional measurements. The labor atory setting, interaction with nurses, professionalism of the staff, and the description of the study through the informed consent all had the potential to influence the behaviors of the participants. Hence, the participants may have acted differently kno wing the importance of the clinical trial (185, 186) and this effect is more commonly known as the Hawthorne effect (185, 187) Controlling for this effect would involve t participants in this study were screened to ensure that they met the criteria to be at high risk of pain response, which agreed with the aim of the study, but this left the study weak in terms of discerning whether t here was any difference between high risk and
75 low risk groups. As previously mentioned, this high risk group may have a more robust placebo response, therefore they could also have a more robust Hawthorne response. The addition of a low risk group that wen t through the exact same protocol would permit us to determine if there is a treatment effect specific to the high risk group. The inclusion of a natural history control group would have allowed us to observe any between group differences between the no t reatment and treatment groups. Being enrolled in the study itself may have produced a response that masked any intervention, which we w ould be able to detect between natural hist ory group and treatment groups. This would be accomplished by having a group only receive the EIMI, we would then track their recovery throughout the week and compare them to the groups that were continuously coming in for treatment in the laboratory. Factors outside of the study could have also played a role For instance, our pro tocol excluded participants if they engaged in resistance training regularly, but we did not control for other types of physical activities throughout the week. There is the possibility that certain participants were more active than others, which could ha ve influenced the cytokine response. This process was ideal in a clinical trial because people experiencing an EIMI still participate in active daily living activities. Further, we could observe the effects of propranolol on a representative sample populat ion, but this does introduce confounding variables to the study that may have affected our outcome measurements.
76 Future Directions This was a novel study and the information obtained is very useful as we determined changes in the inflammatory cytokin es at our time points within blood plasma was undetectable. Research indicates that propranolol should work to mediate inflammation for this high risk population (41) therefore developing a study to specifically observe the inflammatory response a t ideal time points would be worthwhile along with the inclusion of tissue samples following an EIMI. Our investigation into the pharmaco kinetics of propranolol did not include blood draws at ideal time points. One suggestion would be to continue with b lood plasma analysis, but to change the time points for the blood draws to coincide with previously determined peaks of inflammatory cytokines. These time points would be immediately after the EIMI, and at two hour intervals for the first 24 hours followin g it. This would ensure that we collect peak plasma concentrations of the cytokines in the blood as we may have missed the optimum collection window in our previous study. This would also ascertain that we collect data when propranolol is at its peak level s within the individual. Propranolol may not have been absorbed enough in the system during our protocol to observe an interaction. With propranolol having a half life of 5.5 hours and being completely flushed from the system after 24 hours, we may not hav e detected any influence that propranolol could have had on the inflammatory cytokines. It would be intriguing to collect muscle biopsies of the injured tissue from the injury site following an injury as the inflammatory cytokines that have been discussed may act locally at the site of the injury and are not released into the circulatory system.
77 The inclusion of a muscle biopsy could provide more accuracy when detecting changes of cytokine levels rather than using blood measurements. C ontinuing the study further may be valuable We noticed the trend of CRP starting to separate from each other near the end of the study, so being able to track those numbers back to baseline would be advantageous for understanding pos sible changes of muscle injury. Although we identified IL 6 and IL 10 as having predictive properties to pain, they only contributed minimally to the overall explanation of pain. It would be beneficial to examine other cytokines and pain biomarkers as they may have a larger contribution to pain o utcomes. It would also be valuable to examine the relationships of the cytokines further by identifying possible ratios between pro and anti inflammation cytokines, as it is plausible that these may have been more affected by the propranolol than the indi vidual cytokines alone. We may want to consider including psychological factors, biopsychosocial pain models have identified these factors as being pain predictors (188) therefore it is plausible that including IL 10 may strengthen these models. IL 10 is a measurable blood biomarker that can address the biological aspect in the biopsychosocial pain model and may be valuable in identifying individuals at risk for chronic pain Conclusion Baseline plasma levels of IL 10 may play a significant role in identifying the pain response 48 hours following muscle injury in individuals who test positive for the high pain sensitivity COMT polymorphism IL 6 plasma concentrations 48 hours post injury
78 may be more useful at predicting resting pain 48 hours post injury. Resting and active pain 48 hours post injury has a weak to moderate and positive correlation with baseline concentrations of IL 6, and IL 10, where IL 10 would be the strongest predictor of pain. Propranolol does not appear to sufficiently mediate inflammatory cytokines following an experimental muscl e injury in a population of individuals that are pain catastrophizers and have the HPS COMT polymorphism. Moreover propranolol did not influence functional measurements, including ROM, MVIC, and QDash following an induced muscle injury Table 5 1 Revisited study design for observing the effects of propranolol on inflammatory cytokines post injury five groups (HPS propranolol, HPS placebo, LPS propranolol, LPS placebo, natural history) Plasma Inflammatory Cytokines MVIC QDa sh ROM Pill Administration (60 mg) Baseline X X X X immediately post injury X X X 2 hrs post X 4 hrs post X 6 hrs post X 8 hrs post X 10 hrs post X 12 hrs post X 24 hrs post X X X X 36 hrs post X 48 hrs post X X X X 60 hrs post X 72 hrs post X X X X 84 hrs post X 96 hrs post X X X X 120 hrs X X X X
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98 BIOGRAPHICAL SKETCH William Hedderson majored in h ealth and h uman p erformance with a concentration in b iobehavioral s cience at the University of Florida graduating in August 201 9 William previo usly completed his Master of Science at the University of Florida in 2011 in a thletic t raining