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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.

Permanent Link: http://ufdc.ufl.edu/UFE0041443/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.
Physical Description: Book
Language: english
Creator: Harris, Allison
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Allison Harris.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Widmer, Charles G.
Electronic Access: INACCESSIBLE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041443:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041443/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-04-30.
Physical Description: Book
Language: english
Creator: Harris, Allison
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Allison Harris.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Widmer, Charles G.
Electronic Access: INACCESSIBLE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041443:00001


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DOES AN ALTERED INFLAMMATORY RE SPONSE HAVE A ROLE IN DELAYED MASSETER MUSCLE REPAIR? By ALLISON C. HARRIS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1

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2010 Allison C. Harris 2

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To my husband, Adam, who supported me tirelessly with love and understanding throughout the adventure of receiving my post-graduate education 3

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ACKNOWLEDGMENTS I thank the members of my research co mmittee for their guidance and support. I also thank Southern Associati on of Orthodontists and the Univer sity of Florida, College of Dentistry Research Committee for their generous support. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4LIST OF TABLES............................................................................................................7LIST OF FI GURES..........................................................................................................8LIST OF ABBR EVIATIONS.............................................................................................9ABSTRACT ...................................................................................................................11 CHAPTER 1 INTRODUC TION....................................................................................................13Temporomandibular Disorder s, Bruxism, and EIMD...............................................13Gender Differences in Muscle Pain........................................................................14Inflammatory Response to Muscle Damage ...........................................................15Healing and the Influenc e of Infla mmation..............................................................17Delayed Healing in Masseter Mu scle......................................................................18Summary ................................................................................................................19Signific ance............................................................................................................20Hypothes es.............................................................................................................20Specific Aims..........................................................................................................212 MATERIALS A ND METHOD S................................................................................22Animals...................................................................................................................22Cytokine/Chemoki ne Analys is................................................................................23Histological Ev aluations..........................................................................................243 RESULT S...............................................................................................................28Histological Comparison of Muscle Damage ..........................................................28Cytokines/Chemokines in Injured Muscle Compared to Control.............................28Cytokine/Chemokine Main Effects and Inte ractions................................................304 DISCU SSION.........................................................................................................41Tibialis Anterior Reflects Normal Muscle Healing...................................................41Baseline Cytokine/Chemokine Differ ences between TA and Masseter..................41Normal Cytokine/Chem okine Res ponse.................................................................42Colony-Stimulati ng Factors.....................................................................................44Overlapping Effects between Masseter+Cromolyn and TA....................................44Effects of Cromolyn on Masse ter............................................................................46 5

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Major Inflammatory Cytokines Exhibiting No Differenc e.........................................46Directions for Futu re Res earch...............................................................................47Conclusi ons............................................................................................................48LIST OF RE FERENCES...............................................................................................50BIOGRAPHICAL SKETCH ............................................................................................55 6

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LIST OF TABLES Table page 2-1 Number of mice exami ned for each group/timepoint ..........................................26 2-2 Cytokines/chemok ines exam ined.......................................................................27 3-1 Characterization of cytokines/chemokines .........................................................36 3-2 Cytokines and chemokines exhibiti ng main effects and interacti ons..................36 7

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LIST OF FIGURES Figure page 2-1 Timeline of major ev ents....................................................................................26 3-1 Representative front al cryosections (14 m thick) of muscle samples stained with Mayers Hematoxylin from the thr ee groups at 7 days post-injury................32 3-2 Comparison of the area of damage and the cellularity between collages of TA+PBS, Masseter+cromol yn, and Masseter+PBS frontal cryosections (14 m thick).............................................................................................................34 3-3 Significant differences in cytokine/chemokine expression detected within groups at baseline (T0) and/or duri ng early stages of repair (T1).......................37 3-4 Significant differences in cytokine/chemokine expression detected within groups at later stages of repair (T4 and T7). ......................................................38 3-5 Cytokine/chemokine main effects by day...........................................................39 3-6 Cytokine/chemokine interact ions of muscl e by day ............................................40 8

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LIST OF ABBREVIATIONS ANOVA Analysis of variance AOI Area of interest EIMD Exercise induced muscle damage FGF Fibroblastic growth factor G-CSF Granulocyte colony stimulating factor GM-CSF Granulocyte macrophage co lony stimulating factor HGF Hepatocyte growth factor IFN Interferon IL Interleukin IP Interferon gamma induced protein KC Keratinocyte chemoattractant LIF Leukemia inhibitory factor LSD Least significant difference MCP Monocyte chemoattractant protein M-CSF Macrophage colony stimulating factor MHC Major histocompatibility complex MIG Monokine induced by gamma interferon MIP Macrophage inflammatory protein MMP Matrix metalloproteinases NGF Nerve growth factor PBS Phosphate buffered saline PEMS Post-exercise muscle soreness RANTES Regulated upon activation, no rmal T cell expressed and secreted SCF Stem cell factor 9

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TA Tibialis anterior muscle TGF Transforming growth factor TMD Temporomandibular disorders TNF Tumor necrosis factor VEGF Vascular endothelial growth factor 10

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Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science DOES AN ALTERED INFLAMMATORY RESPO NSE HAVE A ROLE IN DELAYED MASSETER MUSCLE REPAIR? By Allison C. Harris May 2010 Chair: Charles G. Widmer Major: Dental Sciences Background Temporomandibular disorders (TMD ) are a prevalent orofacial pain condition and consist predominately of musc le pain that can have a profound negative impact on those affected. Parafunctional habits, a behavior that is commonly associated with TMD, may cause muscle pai n and this pain may be due to exercise induced muscle damage in the masticatory mu scles. Inflammation plays a crucial role in promoting tissue repair following injury, but a balance between inflammatory cell populations and the inflammatory mediators released is r equired for the normal healing process to occur. The differences in the healing process observed between masseter and tibialis anterior muscles could be the resu lt of a varied inflammatory process within masseter. Mast cells appear to play a major role in this variation, causing delayed healing and increasing fibrotic tissue through the release of various mediators. Purpose (1) To quantify the differences in infla mmatory mediators present in the masseter muscle and the tibialis anterior muscle follo wing standardized muscle freeze injury over the first seven days of healing; and (2) To determine whether healing is improved in the masseter muscle with daily injections of a ma st cell-stabilizer following muscle injury. Methods Fifty-two female CD-1 mi ce, 6 weeks of age, were allocated to the following 11

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12 groups: masseter muscle injury plus saline in jections, masseter muscle injury plus mast cell-stabilizer injections, tibialis anterior muscle injury plus saline, and unmanipulated control. A cryoprobe was applie d to the surface of the mu scle for 5 seconds causing a standardized 5 mm freeze injury in posterior s uperficial masseter or tibialis anterior and the region of injury was harvested after 1, 4 and 7 days of repair. The Milliplex Mouse Cytokine/Chemokine Kit, 96 well plat e assay was used to detect 32 different cytokines in the injured muscles and controls at each time point. Histological analysis of repair in the three groups 7 days post injury determined how well the muscle healed by measuring areas of inflammatory exudat e. Differences between groups and across time were analyzed using parametric statistics (ANOVA, LSD test). Results The area of inflammatory exudate in the injured tibial is anterior muscle at 7 days post injury more closely approximated that of masseter muscl e with cromolyn injections. Significant changes in cytokine/chemokine expression were most pronounced during repair of tibialis anterior, followed by expressi on in the masseter+cromolyn group. The masseter+PBS group exhibited no statistically significant changes in expression of any cytokine/chemokine examined at any ti me point. The majority of the cytokines/chemokines that varied across ti me were related to macrophage activation, suggesting the important role of macrophages in the inflamma tory process leading to repair. Conclusion When compared to tibialis ant erior muscle, the masseter muscle exhibits a blunted cytokine/chemokine response that is enhanced with the administration of cromolyn. The expression of cytokines/chemokines that affect the migration and activation of macrophages are increased during muscle repair in tibialis anterior but not in repair of masseter.

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CHAPTER 1 INTRODUCTION Pain associated with temporomandibular disorders (TMD) is the most common chronic orofacial pain condition, and it affects between eight and fifteen percent of women and three to ten percent of men.1 Muscle pain is a frequent characteristic of temporomandibular disorders, and the ma sseter muscle has been implicated in nearly sixty percent of cases.2 The basis for muscle pain a ssociated with temporomandibular disorders could be linked to exercise i nduced muscle damage (EIMD) which follows increased muscle contraction, as in bruxis m or clenching. Masticatory muscle pain affects women three times more than men.3 The explanation for a gender difference in the prevalence of muscle pain conditi ons in women appears to have a hormonal basis.4 5 6 In addition to hormonal differences in pain perception, the role of inflammation in the healing of damaged muscle is crucial to understand. The masseter muscle has been found to have a significant del ay in healing compared to limb muscle such as the tibialis anterior (TA).7 Morris-Wiman and Widmer have identified an increased number of mast cells in the masset er muscle after injury and mast cells could influence the decreased healing response exhibited by the masseter.8 This study has been designed to determine whether or not an altered inflammatory response has a role in delayed masseter muscle repair. Temporomandibular Disorders, Bruxism, and EIMD Temporomandibular disorders is a te rm used to describe musculoskeletal conditions in the temporomandibu lar region involving pain in the muscles of mastication (myofascial pain), in the joint it self (arthralgia), or in both.1 Bruxism is considered a risk factor, with an odds ratio of up to 4.8, for TMD myofascial pain with or without 13

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arthralgia.9 10 The high level of concentric and ecc entric muscle contract ions that occur during parafunctional habits and bruxism coul d cause exercise induced muscle damage (EIMD). During nocturnal bruxism there is a sustained, rhythmic masticatory muscle activity, and at times both jaw openers and jaw closers are co-contracting.11. The increased intensity of the jaw closers (concentric contraction) and the co-activation of the jaw openers (eccentric contraction) coul d theoretically cause muscle damage. Muscle pain following exercise is referred to as delayed onset or postexercise muscle soreness (PEMS) and has been associated with EIMD. Abno rmal muscle activity, such as nocturnal bruxism, probably causes some form of PEMS in certain individuals. PEMS is presumably caused by the infl ammatory events that accompany the mechanical breakdown of myofibrils and connective tissue in the muscle.12 Initial injury to the muscle and the accompanying inflammation and pain may form a basis for muscle pain associated with TMD. Gender Differences in Muscle Pain Females exhibit muscle pain conditions more frequently than males, and the basis for this increased prevalence in females is not completely understood. In a study examining jaw pain induced by clenching, females exhibited a different response than males to exertional pain by having in creased pain twenty-four hours later.13 One study found chewing increased masticatory muscle pain not only in patients diagnosed with temporomandibular disorders but also in female controls.14 This shows the increased susceptibility of females to muscle pain. Gender differences in muscle pain conditions are not fully understood, but it appears sex hormones could play a significant role.4,5,6 It has been suggested that estrogen plays a signi ficant role in the healing of damaged muscle by protecting against further damage, but the exact mechanisms by which this 14

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occurs have yet to be determined.5 It is also possible that the increased prevalence for muscle pain in women could be due to the influence of estrogen or progesterone on pain perception.15 The literature is inconclusive on the exact role of estrogen or progesterone, but the studies have shown that these hormones impact pain perception. This could potentially explain the increas ed prevalence of muscle pain in females versus males. Inflammatory Response to Muscle Damage Tissue injury induces inflammation in order to protect the body, and the inflammatory response involves a cascade of events. After tissue injury macrophages, mast cells, fibroblasts, and injured muscle fibers release cytokines, chemokines, and growth factors. Tumor necrosis factor (TNF), various interleukins, and monocyte chemoattractant protein (MCP1) are important inflamma tory mediators released following muscle injury.16 17 TNFis involved in the ac ute phase reaction of inflammation, and it serves to regulate immune cells by pr omoting the accumulation of neutrophils and macrophages in skeletal muscle.18 Recovery of function in injured muscle has also been shown to be impacted by TNF, which could be controlling expression of muscle regulat ory genes, including MyoD.19 In addition, TNFalso upregulates class I major histocompatibil ity complex (MHC) in myoblasts and regenerating myofibers. This could caus e the myoblasts and myofibers to become a target for inflammatory cells, which would delay healing in the muscle.20 Interleukin 1 (IL-1 ) and Interleukin 6 (IL-6) are key proinflammatory cytokines that are also involved in the acute phase reaction. IL-6 has been shown to increase significantly following eccentric exercise.21 Circulating IL-6 has even been shown to exert an antiinflammatory effect by inducing the produc tion of anti-inflammatory cytokines and 15

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reducing circulating TNF.22 MCP-1 is a chemokine t hat guides the migration and activation of macrophages in damaged tissue. Impaired muscle repair in MCP-1 deficient mice suggests macrophages and MC P-1 are required for a normal healing response in muscle.23 The ability of damaged myofi bers to express a variety of cytokines including TNF, IL-1 and IL-6, as well as MCP-1 could prolong the inflammatory response and cause further injury.24 The inflammatory mediators that are released, which is dependent on the type of muscle injury, serve to control the healing process of the tissue.17 One of the main objectives of inflammati on is to clear cellular debris and prepare the tissue for regeneration.25 Following the initial injury the tissue is infiltrated by various inflammatory cells, including neutrophils, macrophages, and mast cells. Neutrophils have been shown to quickly resp ond to muscle damage and arrive in the tissue within one hour of increased muscle use. The neutrophil population can remain elevated within the muscle tissue for up to five days and can either increase tissue injury or promote tissue repair depending on the inflammatory mediators released.17 It has been shown that following muscle injury neutrophils seem to contribute to muscle injury and may impede the healing process.26 Macrophage populations also increase following injury, with the init ial population promoting tiss ue injury and the subsequent population promoti ng tissue repair.27 Following injury, degranul ation of resident mast cells provides an instantaneous s ource of preformed mediators.28 Mast cells accumulate in injured tissue even if the injury is minor and could prolong an acute inflammation leading to chronic inflammation.29 Mast cells serve as a source of cytokines and inflammatory mediators that serve to promote inflammation in the 16

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damaged tissue. The presence of mast ce lls has been shown to be imperative for neutrophil recruitment and normal tissue repair.30 Mast cells have also been shown to be a factor in tissue necrosis. The de licate balance and timing of neutrophils, macrophages, and mast cells determines whet her or not these cells cause increased damage or serve to promote healing. The di fference in the proportion and timing of these cells in masseter and limb muscle coul d provide the cellular foundation for the differences in healing. Healing and the Influence of Inflammation The process by which healing and regener ation occurs in muscle involves activation of satellite cells. Satellite cells are dormant muscle precursor cells that reside between the basal lamina of muscle fibers and the sarcolemma. Satellite cells are formed late in fetal development by the resident progenitor population. Satellite cells are characterized by the expression of Pax 7, a gene important for satellite cell specification.31 32 Upon injury to the muscle, sate llite cells are activated by several factors, the most prominent of whic h is hepatocyte growth factor (HGF).33 Satellite cells differentiate to myoblasts and fuse to form new myofibers, or th ey fuse to damaged myofibers. An important c haracteristic of satellite ce lls is self-renewal through asymmetric division, thus the healing potential of mu scle is not decreased with additional injury. The inflammatory mediator Interleukin 4, IL-4, is a potential myoblast recruitment factor that contributes to the formation of myotubes.34 Muscle precursor cells can also differentiate into myofib roblasts during regeneration leading to the formation of scar tissue, which delays rehabili tation of the muscle. Fibroblasts are activated by factors released due to muscl e damage, which could also cause increased 17

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fibrous repair. Preventing fibrosis in healing muscle would therefore involve the prevention of the activation of fibr oblasts that cause scar formation. Delayed Healing in Masseter Muscle The masseter muscle exhibits delayed healin g when compared to tibialis anterior muscle.7 The basis for this difference in healin g is not completely understood. It has been shown that in order for effective muscl e regeneration to take place necrotic tissue must be removed.35 If phagocytosis is incomplete, muscle regeneration is unsuccessful.36 Masticatory muscles have a differ ent embryonic origin than limb muscles, which develop from somites. Mast icatory muscles develop from somitomeres, presomitic mesoderm located adjacent to th e embryonic brain. Differences in the number and proliferative rate of myoblasts in masseter compared to tibialis anterior may be responsible for differences in regeneration.7 It is possible that differences in the infl ammatory process are also responsible for delayed healing in masseter, and pre liminary data has shown one of the most significant differences between masseter and ti bialis anterior following injury is the increased number of mast cells in masseter.8 Mast cells exhibit different phenotypes depending on the local environment, and this affe cts cytokine expression and functional differences.37 Chronic inflammatory conditions (e .g. psoriasis, rheumatoid arthritis, allergy, parasite infection, and inflammatory bowel disease) and fibrotic disorders (e.g. pulmonary fibrosis, liver cirrhosis, and Crohns disease) are characterized by mast cell hyperplasia. Mast cells are a source of a variety of cytokines, chemokines, growth factors, and matrix metalloproteinases (MMPs). Mast ce lls also control extracellular matrix regeneration and potentially fibr osis through the release of pro-fibrotic cytokines, 18

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including fibroblastic growth factor (FGF-2) and IL-4.38 Disodium cromoglycate, cromolyn, stabilizes mast cells by im peding calcium mobilization and degranulation.39 It has been shown that blocking degranulati on of mast cells through cromolyn administration decreases the number of neutrophils in injured muscle.30 This highlights the important role mast cells play in the mu scular inflammatory response. It has also been shown that preventing ma st cell degranulation through dai ly cromolyn injections can decrease muscle fiber necrosis.40 Summary Temporomandibular disorders are a preval ent orofacial pain condition, and the muscle pain associated with TMD can have a profound negative impact on the quality of life for those affected. Women have a higher tendency to develop myofascial pain with TMD, but the explanation for this observation is not comple tely understood. Estrogen appears to play a role in both inflammato ry events within muscle and pain perception, which could explain the gender differences dete cted in myofascial pain. Inflammation plays a crucial role in promoting tissue repair following injury, but a balance between inflammatory cell populations and the inflamma tory mediators released is required for the normal healing process to occur. Shi fting the balance can cause increased injury, prolonged healing, or fibrous repair. The di fferences in the healing process observed between masseter and tibialis anterior musc les could be the result of a varied inflammatory process within masseter. Mast cells appear to play a major role in this variation, causing delayed healing and increasing fibrotic tissue through the release of various mediators. Analysis of the in flammatory mediators present following a standardized injury to the ma sseter muscle and the tibialis anterior muscle will lead to a better understanding of the inflammatory proce ss occurring in masseter versus tibialis 19

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anterior. Quantifying the differences in cytokines present following muscle injury between the masseter muscle and the tibialis anterior muscle would help explain the reasons for different proportions of mast cells and diverse healing in the two muscles. Significance The biological basis and the etio logy of myalgia associated with temporomandibular disorders are inadequately explained by the present information available. Muscle pain in the jaw closer muscles is frequently associated with TMD, with the masseter muscle more highly affected. At this time, treatment for muscle pain conditions is palliative, and it does not address the underlying muscle damage associated with muscle injury. Models of mu scle injury to help determine the molecular basis of inflammation would be beneficial in expl aining the biological basis of myalgia. The masseter muscle has been shown to have a varied healing response following muscle injury. An increased number of ma st cells has been found in the masseter following injury, potentially explaining the delayed healing respons e. Defining the inflammatory mediators present following mu scle injury could account for the presence of an increased number of mast cells in the masseter muscle. With a better understanding of the inflammatory process, mast cells could serve as a potential target for treatment of myalgia in jaw closing mu scles. The effect cromolyn has on muscle inflammation following injury will help determi ne if it is a prospective therapeutic for muscle pain conditions. Hypotheses 1. The masseter muscle will exhibit an altered cytokine/chemokine expression compared to tibialis anterior, which will help explain the different inflammatory cell 20

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21 profile exhibited by the masseter, particula rly the increased number of mast cells, and delayed muscle repair following injury. 2. Daily systemic injections of cromolyn (a mast cell stabilizer) will improve the healing of the masseter muscle. A differenc e will be evident in the cytokine/chemokine profile between masseter muscle injected wit h cromolyn and masseter muscle injected with saline. Specific Aims 1. Evaluate the expression of inflammatory mediators following muscle injury to determine if a varied inflammatory profile, temporally and/or quantitatively, exists between the masseter muscle and the tibialis ant erior muscle that could help to explain the differences in healing exhi bited by these two muscles. 2. Improve the understanding of the role of mast ce lls in muscle inflammation following injury. Determine if cromolyn has an effect on inflammatory mediators in injured muscle and determine its potential as a therapeutic agent for myalgia associated with TMD.

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CHAPTER 2 MATERIALS AND METHODS Animals Fifty-two female CD-1 mice, six weeks of age, were used in this study. The mice were allocated to three groups (Table 2-1). Sample size was calculated based on data from previous studies examining immunofluorescent -labeling of inflammatory cells in masseter and tibialis anterior mu scles after injury. In those studies, an effect size of 1.5 or greater was observed for ma st cell numbers. Therefore, using the parameters of an effect size of 1.5, a power of 0.80, and an alpha level of 0.05, we required 4 subjects per group. The cromolyn and physiological saline (PBS) injections were given systemically at the time of the muscle injury and each subs equent day until sacrifice. The dose of cromolyn was 100 mg/kg in 0.1 ml IP. PBS (0.1 ml) was injected IP in masseter control animals and in the tibial is anterior animal group. Mice were anesthetized using ketamine (10-14 mg/kg body weight) and xylazine (70-80 mg/kg body weight). A small incision approximatel y 3 mm in length was made in the tissue overlying the posterior portion of the superficial layer of the masseter muscle or the central portion of the tibialis anterior muscle. A Keeler Ophthalmic cryoprobe (Broomall, PA) was applied to the surface of the muscle for 5 seconds causing a standardized 5 mm freeze injury in posterior superficial masseter or tibia lis anterior. The probe was placed midway on the posterior portion of the superficial layer of the masseter muscle, between the tendon of the superficial massete r and the posterior attachment of the masseter to the mandible. The probe was placed in the center of the tibialis anterior muscle. Skin incisions were closed using su rgical glue, cyanoacrylate. The mice were sacrificed at 1, 4, or 7 days post-injury (F igure 2-1) and the area of muscle injury was 22

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harvested using a standardized 3 mm punch biopsy. Biopsies were placed in 1.5 ml tubes and snap-frozen in acetone-cooled is opentane for analysis. For histological analyses on day seven (T7), whole massete r and tibialis anterior muscles were harvested, orientated on car dboard, embedded in Fisher TissuePrep and snap-frozen in acetone-cooled isopentane. Cytokine/Chemokine Analysis The Milliplex MAP Mous e Cytokine/Chemokine 32-pl ex kit was used for the simultaneous detection and quantification of 32 cytokines and chemokines (Table 2-2) in injured muscle and control samples. Pr otein was extracted from 3 mm biopsy muscle samples using Invitrogen Tissue Extraction Reagent I according to manufacturers instructions. Briefly, the reagent was wa rmed to room temperature and mixed well. Protease inhibitor cocktail (10 l, Halt Prot ease Inhibitor, 100x, Thermo Scientific) was added to 1 ml of Tissue Extraction Reagent I just prior to use. Tissue Extraction Reagent I (200 l) was added to each biopsy specimen. This amount was found to allow adequate extraction of protein from the 3 mm biopsy samples which ranged from 7 to 10 mg in weight. The samples were homogenized in the reagent using a batterypowered pestle and then centrifuged at 10,000 RPM for 5 minutes to pellet the tissue debris. The supernatant was collected and frozen. A 10 l aliquot of each sample was used to determine protein concentration using the Pierce BCA Protein Assay Kit according to manufacturers instructions. A 35 l aliquot of each sample was sent to Millipore Assay services for analysis ut ilizing 96 well multiplex Luminex xMAP technology. In the multiplex Luminex assay, multiplex capture antibodies are attached to 5.6 micron polystyrene beads internally dyed with red and infrar ed fluorophores of differing intensities. After an analyte from a test sample is captured by the bead or 23

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microsphere, a biotinylated detection antibody is introduced. The reaction mixture is then incubated with Streptavid in-PE conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere. With the Luminex 200 detection system, the microspheres are allowed to pass rapidly through a laser which excites the internal dyes marking the microsphere set. A second laser excites PE, the fluorescent dye on the reporter molecule. Finally, high-s peed digital-signal processors identify each individual microsphere and quantify the result of its bioassay based on fluorescent reporter. Millipore Assay services provided an excel spreadsheet of cytokine/chemokine concentrati ons for each sample as well as the precision values, MFI (mean fluorescent values) and standard curv es from which these concentrations were derived. The mean amount of each cytoki ne was calculated for the T0 samples. In order to normalize the data, concentration values for eac h individual cytokine sample were divided by the respective mean T0 value. The raw data and the normalized data were statistically analyzed. Statistical Analyses : Differences between groups and across time were analyzed with a 2 way ANO VA followed by post-hoc testing using the LSD test when appropriate. A probability level of 0. 05 was used for all statistical tests. Histological Evaluations Frontal cryosections (14 m thick) of muscle sample s from the three groups: TA+PBS; masseter+PBS; masseter+cromolyn, at 7 days post-injury were stained with Mayers Hematoxylin. Sections were viewed on a Nikon FXA microscope under brightfield and images acquir ed through the region of great est muscle damage using an Mrc5 Zeiss digital camera and AxioVision (Zei ss) software. Collages of the damaged regions were constructed using Photoshop CS2. Using Image Pro software the areas of damage on each collage were outlined, specif ying the area of interest (AOI). A 24

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standardized square AOI (10,000 m2) was placed in the uninjured area of the section. A segmentation procedure wa s then performed in order to determine the cellularity within each AOI. The histogram based segm entation procedure involved separating the inflammatory exudate pixels from the ba ckground pixels in each image based on color range. The background pixels were then set as black and the range of color intensities to be emphasized were set as white. Once the segmentation procedure was performed, a mask was applied and a 2 pass medi an filter, 5x5 kernel was completed. A median filter is used to remove random high impulse noise (spots or points that vary significantly from the backgroun d) by replacing the center pi xel with the median value in its neighborhood. Statistical Analyses : Differences between groups were analyzed with an ANOVA followed by post-hoc testing us ing the LSD test when appropriate. A probability level of 0.05 was used for all statistical tests. 25

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Table 2-1. Number of mice examined for each group/timepoint. Cytokine/Chemokine Analyses Histology Injury Groups T0 (Uninjured) T1 T4 T7 T7 Masseter plus PBS 4 4 4 4 4 Masseter plus cromolyn 4 4 4 4 Tibialis Anterior plus PBS 0 (4 TA samples from same mice as above) 4 4 4 4 Figure 2-1. Timeline of major events. Baseline Formation and Maturation Formation and maturation of new muscle fibers Anti-Inflammatory Transition Infiltration of anti-inflammatory macrophages, switch from proinflammatory cytokines to antiinflammatory cytokines and growth factors, r epai r Destruction and Activation Destruction of damaged tissue with infiltration of neutrophils and proinflammatory macrophages, activation of satellite cells T7 T4 T1 T0 26

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27 Table 2-2. Cytokines/chemokines examined. Hematopoietic Agents G-CSF GM-CSF M-CSF IL-3 Pro-inflammatory Cytokines IFNIL-1 IL-1 IL-2 IL-5 IL-6 IL-9 IL-12(p70) IL-15 IL-17 LIF TNFAnti-inflammatory Cytokines IL-4 IL-10 IL-12(p40) IL-13 Chemokines IP-10 MIP-2 KC Eotaxin LIX MCP-1 MIP-1 MIP-1 MIG RANTES Growth Factors VEGF IL-7

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CHAPTER 3 RESULTS Histological Comparis on of Muscle Damage Examination of histological sections revealed that TA was more advanced in the healing process than masseter+PBS; muscle fiber sizes were more uniform and larger and the original muscle architecture had been re stored. Healing in masseter+cromolyn muscle also appeared to be more advanced than in masseter+PBS and the histology of the repairing region resembled that observed in TA (Figure 3-1). The analysis of the area of muscle damage seven days post-injury in histological sections revealed statistically significant differences between the three groups examined: TA, masseter+PBS and masseter+cromolyn (ANOVA, F = 5.0, p < 0.05). Masseter+PBS exhibited a greater area of damage than ma sseter+cromolyn (p = 0.04, LSD test) and TA (p < 0.02, LSD test) (Figure 3-2A). The percentage of the damaged area occupied by an inflammatory exudate at seven days pos t-injury was also significantly different among the three groups (ANOVA, F = 18.0, p < 0.001) and was found to be significantly increased in masseter+PBS compared to massete r+cromolyn (p < 0.01, LSD test) and TA (p < 0.002, LSD test) (Figure 3-2B). The percentage of the area occupied by nonmuscle cells in the uninjured standardized area was not statistically different across any of the groups (ANOVA, F = 2.1, p = 0.18) (F igure 3-2C). Cytokines/Chemokines in Injured Muscle Compared to Control Six cytokines/chemokines were not detected at any time point or were detected at levels below the standard range of the detection system at all ti mes points (Table 3-1). Seven cytokines/chemokines were detected at levels below standards at T0 but had an 28

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increased expression as a respons e to damage and repair. Nineteen cytokines/chemokines were detected at baseline and at all time points during repair. Significant differences in baseline ex pression between uninjured masseter and TA were found for four cytokines /chemokines (ANOVA, F=103, p < 0.01; t-test, p < 0.05). IL-12(p70), IL-17 and Eotaxin all were obser ved to have a significantly increased expression in the masseter muscle when compared to the TA while VEGF had a significantly increased expression in TA compared to masseter. The four cytokines/chemokines were also found to si gnificantly change over time (Table 3-1). Ten cytokines/chemokines were determined to significantly change in expression over baseline levels during repair (Table 3-1). One cytokine, VEGF, decreas ed significantly in expres sion at both time points, one and four days post-injury, from baseline (T0) in TA (Figure 33). VEGF expression did not vary between baseline (T0) and any time point during repair for either masseter group (masseter+PBS, masseter+cromolyn). At twenty four hours post-injury (T1), t he expression of G-CSF, KC and MCP-1 was significantly increased in TA but returned to baseline levels at day 4 (Figure 3-3). Expression of IL-12(p70) also was increased significantly in TA at T1 and its expression was further increased at T4 before returni ng to baseline at T7 (Figure 3-3). No significant differences in the expression of these cytokines were observed for either masseter group at any time point examined. Four days post-injury (T4),the expression of cytokines/chemokines Eotaxin and IP10 was increased in the masseter+cromolyn group but an increase was not observed for the TA or masseter+PBS groups (Figure 3-4) MIG was also significantly increased 29

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at this time in both the TA and masseter+ cromolyn groups (Figure 3-4). The two cytokines IL-4 and M-CSF were observed to be differentially expressed between the three groups at different time points. IL-4 expression was significantly decreas ed in both TA and masseter+cromolyn at T1. Expression levels rebounded to baseline le vels at T4 in masseter+cromolyn, but not TA. Expression of IL-4 in TA remained depressed unt il T7 where expression was increased over baseline levels. Expression of IL-4 showed no significant differences at any time point for masseter+PBS (Figure 3-4). M-CSF was determined to have a greater level of expression at baseline (T0) in TA than in either masseter group. Whereas, expression levels did not significant ly change in either masseter group during repair, M-CSF expression decreased in TA at T1 and again at T4 until expression was increased to exceed baseline levels at T7 (Figure 3-4). Cytokine/Chemokine Main Effects and Interactions Values for cytokine/chemokine expressi on were normalized to baseline for each group (TA or masseter) to allow the com parison of cytokine/chemokine expression between the three groups (TA, masseter+ PBS, and masseter+cromolyn) over time. Seven cytokines/chemokines were determined to have significant main effects in expression by day, by muscle group or by muscle group and day (Table 3-2). Two cytokines/chemokines were shown to differ significantly by day with no significant interactions. Both KC and MC P-1 had an increased expression at T1 for all muscle groups (Fig 3-5). Five cytokines/chemokines: G-CSF; M-C SF; IL-4; MIG and IL-12(p70), showed significant interactions of muscle by day. Expression of G-CSF by TA at T1 was significantly increased over that observed fo r either masseter group at T1 or for any 30

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31 group at later repair time points (Fig 3-6). Interestingly, M-CSF expression by TA was significantly increased at T7 over that observed for either masseter group or TA at other time points (Fig 3-6). Differences were also observed at T4 between TA and both masseter groups for M-CSF. The expressi on of M-CSF remained relatively unchanged during repair in both masseter+PBS and masseter+ cromolyn. In cont rast to G-CSF and M-CSF, a decrease in expression at T4 was observed for IL-4 in TA that was not apparent in either masseter group or TA at other times points (Fig 3-6). MIG expression increased in both TA and masseter+cromolyn between T1 and T4 and, in both groups, expression levels returned to baseline at T7 (Fig 3-6). Significant differences in MIG levels were not observed between the TA and masseter+cromolyn groups at T4 but were present between both groups and masseter+PBS. No significant differences in MIG expression were detected in the masse ter+PBS group during repair. Finally, IL12(p70) expression was observed to be signific antly increased at bot h T1 and T4 in TA compared to expression in either masseter group at all time points and TA at T7 (Fig 36).

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Figure 3-1. Representative frontal cryosections (14 m thick) of muscle samples stai ned with Mayers Hematoxylin from the three groups at 7 days post-injury. The area of damage is delineated by a dashed line. A) TA+PBS. B) Masseter+Cromolyn. C) Masseter+PBS. D) Higher magnification of TA+PBS. E) Higher magnification of Masseter+Cromolyn. F) Higher magnifi cation of Masseter+PBS. In D and E, new muscle fibers (arrows), identified by their central nuclei, are uniform in size and that there is little cellula r exudate within the damaged region. In F, new muscle fiber si ze is variable and regions between ar eas of new fibers are occupied by inflammatory exudate (arrow heads). 32

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33

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Figure 3-2. Comparison of the area of damage and the cellularity between colla ges of TA+PBS, Masseter+cromolyn, and Masseter+PBS frontal cryosections (14 m thick). A) Significant differen ces were found in the area of damage between Mass+PBS and TA and Mass+cromolyn groups (p<0.05, ANOVA, post-ho c LSD test). B) A segmentation procedure was used to determine the percent of the damaged area containing inflammatory exudate in each image or cellularity in a standardized square area in t he uninjured area of the section. Significant differences were observed between Mass+ PBS and the TA and Mass+cromolyn groups (p<0.05, ANOVA, post-hoc LSD test). C) No differences were found in the cellularity of control areas between groups. 34

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35 Mass+PBS 0% 5% 10% 15% 20% 25%%AOI area TA Mass+cromolyn Mass+PBS C 0% 5% 10% 15% 20% 25%% AOI areaTA Mass+cromolyn B Mass+PBS 0 100000 200000 300000 400000 500000 600000 700000 800000 900000Area of Damage ( m2)TA Mass+cromolyn A

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Table 3-1. Characterization of cytokines/chemokines. Cytokines/Chemokines Below Detectable Levels Cytokines/Chemokines Not Detected at Baseline Cytokines/Chemokines Detected Over Time IL-3 G-CSF GM-CSF IL-7 IL-2 M-CSF MIP-2 IL-13 IL-1 LIF MCP-1 IL-1 MIP-1 LIX IL-5 TNFIFNIL-6 RANTES IL-9 IL-12p70 IL-15 IL-17 IL-4 IL-10 IL-12p40 IP-10 KC Eotaxin MIP-1 MIG VEGF Table 3-2. Cytokines and chemokines ex hibiting main effects and interactions. Cytokine/chemokine F value p value KC 7.0 0.004 MCP-1 5.3 0.01 G-CSF 3.7 0.02 M-CSF 7.4 0.001 IL-4 2.7 0.05 MIG 3.0 0.04 IL-12(p70) 18.7 0.000001 36

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Figure 3-3. Significant differences in cyt okine/chemokine expre ssion detected within groups at baseline (T0) and/or during early stages of repair (T1). KC0 50 100 150 200 250 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 1 vs Day 0, 4, 7 MCP-10 200 400 600 800 1000 1200 1400 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 1 vs Day 0, 4, 7VEGF0 20 40 60 80 100 120 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS G-CSF0 50 100 150 200 250 300 350 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 0 vs Day 1, 4; Day 0 TA vs Masseter* *p<0.05, Day 1 vs Day 0, 4, 7 IL-12 (p70)0 2 4 6 8 10 12 14 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 4 vs All Days p<0.05, Day 1 vs All Days p<0.05, Day 0 vs Day 1, 4; Day 0 TA vs Masseter 37

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Figure 3-4. Significant differences in cyt okine/chemokine expre ssion detected within groups at later stages of repair (T4 and T7). IP-100 100 200 300 400 500 600 700 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS Eotaxin0 50 100 150 200 250 300 350 400 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 4 vs Day 0 p<0.05, Day 0 TA vs Masseter* *p<0.05, Day 4 vs Day 0, 1 or 7 IL-40 5 10 15 20 25 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS p<0.05, TA and Mass+Crom, Day 1 vs Day 0,4,7 p<0.05, Day 7 vs Day 0,1,4MIG0 200 400 600 800 1000 1200 1400 0147Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 4 vs Day 0, 1, 7* M-CSF-10 0 10 20 30 40 50 0147 Daypg/ml Mass+Crom Mass+PBS TA+PBS *p<0.05, Day 7 vs Day 1,4 38

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KC0 5 10 15 20 25 30 147 DayFold Change from T0 MCP-10 200 400 600 800 1000 1200 147 DayFold Change from T0 *p<0.05, Day 1 vs Day 4, 7**p<0.05, Day 1 vs Day 4, 7* Figure 3-5. Cytokine/chemokine main effects by day. 39

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40 G-CSF-100 0 100 200 300 400 500 600 700 147 DayFold Change from T0 Figure 3-6. Cytokine/chemokine interactions of muscle by day. Mass+Crom Mass+PBS TA+PBS M-CSF0.0 0.5 1.0 1.5 2.0 2.5 147 DayFold Change from T0 Mass+Crom Mass+PBS TA+PBS p<0.05, All Other Days, Groups* p<0.05, TA Day 4 vs All Groups, Day 4 *p<0.05, Day 1 vs Day 4, 7* IL-40.0 0.5 1.0 1.5 2.0 2.5 147 DayFold Change from T0 Mass+Crom Mass+PBS TA+PBS MIG0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 147 DayFold Change from T0 Mass+Crom Mass+PBS TA+PBS p<0.05, Day 4 vs Day 1, 7* p<0.05, Day 4 vs Mass+PBS, Day 4 p<0.05, Day 1 vs Day 7* p<0.05, TA Day 4 vs All Groups, Day 4 IL-12 (p70)0.0 1.0 2.0 3.0 4.0 5.0 6.0 147 DayFold Change from T0 Mass+Crom Mass+PBS TA+PBS p<0.05, Day vs All Days, All Groups* *

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CHAPTER 4 DISCUSSION Tibialis Anterior Refl ects Normal Muscle Healing Inflammation following inju ry to tibialis anterior muscle and subsequent healing was compared in this study to injury of masseter muscle and its inflammatory and healing process. Histological evaluation of the tibialis anterior muscle showed more advanced healing, a smaller area of dam age with less inflammatory exudate, and larger, more uniform muscle fibers by day 7. Previous studies have also shown the healing of masseter muscle is delayed and exhibi ts more fibrosis than tibialis anterior.7,8 The histological evaluation confirmed a difference between masseter+cromolyn and masseter+PBS, with masseter+crom olyn more closely approximating the healing of TA. This is suggestive of a therapeutic effect of disodium cromogl ycate, cromolyn, and its effect on various cytokines/chemokines in the masseter muscle. Baseline Cytokine/Chemokine Diffe rences between TA and Masseter The expression of cytokines/chemokines in the uninjured masseter muscle was similar to that observed in the uninjured tibialis anterior muscl e except for four cytokines: Eotaxin, IL-17, IL-12(p70) and VE GF. Eotaxin and IL-17 were both present in increased concentrations in the masseter c ontrol compared to tibialis anterior. IL-17 has been shown to induce the expression of eotaxin, a chemoattractant for mast cells.41 42 The fact that mast cells have been observed in increased numbers in masseter muscle when compared to TA may be explained by the inherent increased concentration of cytokines/chemokines specif ic for mast cell recruitment in masseter muscle. 41

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VEGF had higher basal levels in the TA control when compared to the masseter control. VEGF decreased significantly in TA following injury, especially during the destruction and activation phase and the anti-in flammatory transition, when compared to baseline controls. It has been shown that VEGF affects dystrophin-positive regenerating fibers, endogenous muscle regeneration, microvascularization, and level of fibrosis.43 It has also been shown that VEGF ex pressing muscle-derived stem cells improve muscle repair in the animal model of muscular dystrophy.43 Macrophage recruitment, an event dependent on chemokine receptor 2, is associated with the restoration of tissue VEGF leve ls after an initial decrease.44 The masseter muscle does not have the initial decrease of VEGF typical of injured muscle, which could be evidence of an altered inflammatory response. An inverse relationship has also been shown between regenerated muscle fiber size and capillary density.44 Visual examination of the masseter muscle histological sections re vealed masseter to be highly vascularized, and this could be associated with the fact the muscle fibers are smaller in healing masseter muscle. The tem poral expression of VEGF, particularly an increased concentration at baseline, a significant decr ease following injury, and a later return to baseline concentration, appears to be important in the healing of skeletal muscle and this pattern was not observ ed for the masseter muscle. Normal Cytokine/Chemokine Response Overall, it was found that TA exhibited changes in the ex pression of eight different cytokines/chemokines during the inflammato ry process, while the masseter+PBS group did not exhibit changes in the expression of any of the cytokines/chemokines. With the administration of cromolyn, the number of cytokines/chem okines that changed during the inflammatory process in the masseter mu scle injury group was increased to four. 42

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This is evidence that the masseter muscle has a blunted cytokine/chemokine response when compared to tibialis anterior muscle and that cromolyn selectively had an effect on the expression of a subgroup of cytok ines/chemokines in the masseter. When the cytokines/chemokines were norma lized to baseline values, MCP-1 and KC were shown to increase during the des truction and activation phase in all muscle groups. MCP-1 is produced by monocytes and endothelium, and it guides chemotaxis of macrophages and also causes degranulation of mast cells. KC is produced by keratinocytes, monocytes, and macrophages and activates neutrophils. Since chemokines are important early in the infl ammatory process, it is logical that chemokines MCP-1 and KC were increased init ially to promote chem otaxis of various inflammatory cells, including neutrophils and macrophages. IL-12(p70) was increa sed during the destruction and activation phase and peaked during the anti-inflammatory transition in TA. There was a significant difference in baseline levels between TA and ma sseter (TA > masseter) and TA had a significantly higher levels on days 1 and 4 co mpared to the other muscle groups. GMCSF stimulates murine bone marrow precurso rs to form GM-BMM macrophages which preferentially secrete IL-12( p70), a pro-inflammatory cyto kine important in neutrophil stimulation.45 Other macrophage populations, e.g. BMM (stimulated by M-CSF), do not secrete IL-12(p70).46 The fact that TA produced IL-12(p70) in significant quantities, and IL-12(p70) is only produced in certain populations of macrophages suggests the macrophage populations pr esent in TA may be different than those present in masseter muscle. Histological evaluation in a prev ious study by Widmer and Morris-Wiman showed fewer macrophages in masseter muscle than TA.8 Differences in the number 43

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and type of macrophages may be crucial in ex plaining the varied healing process exhibited by masseter muscle. Colony-Stimulating Factors Colony-stimulating factors also appear to pl ay a major role in the inflammatory process of tibialis anterior muscle com pared to masseter muscl e. G-CSF and M-CSF were both elevated in tibialis anterior but not masseter. G-CSF was increased during the destruction and activation phase in TA and previous studies have shown that following injury G-CSF may increase muscle fiber diameter, inhibit inflammation, and augment muscle mass regeneration through incr eased proliferation of satellite cells.47 48 Early release of G-CSF in TA but not in masseter is suggestive of its importance in the healing process potentially by ear ly activation of satellite cells. es help cle fibers. M-CSF or CSF-1 decreased dur ing the anti-inflammatory transition and increased during the formation and maturation phase in TA, and it may have played a crucial role in the remodeling process of the damaged muscle. M-CSF regulates the survival, proliferation, and differentia tion of mononuclear phagocytes.49 Previous studies suggest M-CSF is required for normal development in tissues undergoing rapid morphogenesis or tissue remodeling, and in flammatory conditions involve macrophag that are not dependent on M-CSF.50 Since M-CSF was increased during formation and maturation of new muscle fibers, its primary role was probably related to tissue remodeling, and absence of an increase in M-CSF in injured masseter muscle may explain the aberrant architecture of the mus Overlapping Effects between Masseter+Cromolyn and TA Two cytokines, IL-4 and MIG, showed sim ilar effects in masseter+cromolyn and TA. IL-4, which is released by mast cells, was decreased during the destruction and 44

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activation phases in TA and masseter+cromol yn and also during the anti-inflammatory transition in TA when compared to their respective controls. The level of IL-4 in TA during the anti-inflammatory transition wa s lower than in all other muscle groups on each day. Following the initia l decrease, IL-4 increased in TA on day 7 above baseline values. IL-4 is a critical factor in mu scle growth and it may promote the fusion of myoblasts to myotubes.34 A decrease initially in IL-4 following injury is potentially related to the importance of inducing a normal inflammatory process and not promoting myoblast differentiation and fusion muscle until the damaged area has been cleared. IL-4 was significantly lower in TA and masse ter+cromolyn groups on day 1 compared to masseter+PBS, but this difference did not persist for the masseter+cromolyn group during the anti-inflammatory tr ansition (day 4) where IL-4 levels increased to control levels. The varied response between TA, masseter+cromolyn and masseter+PBS groups, where a decreased IL-4 expressi on was maintained in TA, transiently decreased in masseter+cromolyn and did no t change from baseline in masseter+PBS, could contribute to the varied healing response seen between the two groups, with masseter+cromolyn more cl osely approximating TA. MIG is a chemokine that is increased in masseter+cromolyn and TA during the anti-inflammatory transition. MIG is invo lved in chemotaxis of monocytes and is produced by macrophages. This may be additional evidence for the importance of macrophages in the inflammatory process in order for normal healing to occur, with TA and masseter+cromolyn potentiall y exhibiting similar number s of active macrophages. It has been shown that the presence of macrophages directly affects satellite cell 45

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proliferation, skeletal muscle regeneration, and fibrosis, and th eir presence is crucial for a normal healing process to occur.51 Effects of Cromolyn on Masseter IP-10, a chemokine that attracts monocytes and is produced by macrophages, fibroblasts, and endothelial cells, was increas ed in masseter+cromolyn during the antiinflammatory transition when compared to bas eline controls. The expression of IP-10 did not change in the masseter+PBS or TA groups It was shown in this study that, histologically, healing in masseter+cromolyn was similar to TA. It was shown previously that macrophages are more prominent in TA than in masseter during repair.8 Since IP10 is released by macrophages, an increased amount of IP-10 in masseter+cromolyn may be evidence of an increased number of ac tive macrophages present at the site of injury. Major Inflammatory Cytokin es Exhibiting No Difference Pro-inflammatory cytokines like TNFand IL-1 that were expected to increase in damaged muscle did not show an appreciable change from the baseline values and were similar for all muscle groups. Tidball (2005) provided evidence that the role of TNFmay vary in muscle injury and repair depending on the type, severity, location, and stage of injury.17 It is possible TNFis not needed for the re pair of muscles with mild damage such as after a fr eeze-injury. Another explanat ion for the lack of response could be that the response occurred outside of the time points examined. If TNFincreased immediately post-injury and decreased back to baseline values within the first 24 hours, an effect would not have been observed. Including a time point that evaluates the cytokine/chemokine response within se veral hours of muscle damage may show if there is indeed an effect in these and other cytokines. 46

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Directions for Future Research It was beyond the scope of this projec t to evaluate the level of every cytokine/chemokine predicted to play a role in muscle repair. Several cytokines/chemokines that could be exam ined in future studies are SCF, TGF and NGF. Stem cell factor (SCF) is a growth fa ctor unique to mast cells, shown by the fact that mast cells do not exist in the absence of SCF. SCF causes local proliferation of mast cells and enhanced production of SCF by fibroblasts could lead to fibrotic processes. TGF and nerve growth factor (NGF) ar e also responsible for mediating chemotaxis of mast cells.52,53 Mast cells have been shown to have a direct effect on inflammation and healing, but they are also involved in neuroimmune interactions. The association between mast cells and nerves could serve as a means of amplifying inflammation in muscle and causing increased pain. In addition to being activated by neuropeptides and possibly nerve stimulation, mast cells release nerve growth factor (NGF) and express trk-A, a high affinity receptor for NGF.54 NGF is a neurotrophin responsible for causing sensitization of nociceptors, and in animal and human studies, inflammation has been shown to cause in creased levels of NGF resulting in hyperalgesia.55,56 NGF can cause degranulation of ma st cells as well as increased cytokine expression. The relationship betw een mast cells and nerve associated factors, including neuropeptides and NGF, could be significant in the inflammation and healing process in muscle and the accompanyin g pain experienced by the individual.57 Macrophages and the cytokines/chemoki nes associated with them have been shown to be important in the inflammatory process. GM-BMM ma crophages express an integrin, CD11c, that is not expressed by BMM macrophages.58 Evaluating the specific populations of macrophages present in TA and masseter through antibody labeling of 47

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the CD11c integrin may also help explain heali ng differences. It is possible masseter is deficient in certain factor s, therefore macrophages do no t migrate properly into the injured area. Evaluating the subtypes of macrophages present in the injured muscle would help determine if this is a reason for the delayed healing seen in masseter muscle. Conclusions Temporomandibular disorders commonly include myofascial pain, and the masseter muscle is one of the primary mu scles affected. Following damage, the masseter muscle has been shown to exhibit del ayed healing and a varied inflammatory response when compared to limb muscle. W hen compared to tibialis anterior muscle, the masseter muscle exhibits a blunted expression of cytokines/chemokines that is enhanced with the administration of cromolyn. The reasons for this may be explained by the fact: 1. Certain cytokines/chemokines are present in significantly different concentrations in the control TA and masseter muscles, e.g. Eotaxin and IL-17, and this may prime the condition of the muscle to lead to a varied inflammatory response. 2. Cytokines related to macrophage f unction and recruitment are increased in TA, emphasizing the importance of macrophages for normal healing to occur. 3. The temporal expression of VEGF is also different in TA and masseter and could explain differences in healing. When the degranulation of mast cells is blocked through administration of cromolyn, the healing of masse ter muscle appears to normalize to TA histologically and 48

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49 through the release of IL-4 and MIG in similar concentrations to that of TA. IP-10 is also increased in masseter+cromolyn which suggests the normalized healing could be occurring due to an increas ed number of macrophages.

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LIST OF REFERENCES 1. LeResche L. Epidemiology of temporom andibular disorders: Im plications for the investigation of etiologic factors. Crit Rev Oral Biol Med 1997; 8: 291-305. 2. Dworkin SF, Huggins KH, Le Resche L, Von Korff M, Howard J, Truelove E, Sommers E. Epidemiology of sign s and symptoms in temporomandibular disorders: clinical signs in cases and controls. J Am Dent Assoc 1990; 120: 273281. 3. Velly AM, Gornitsky M, Philippe P. C ontributing factors to chronic myofascial pain: a case-control study. Pain 2003; 104: 491-499. 4. Dao TTT, Knight K, TonThat V. Modulation of myofascial pain by the reproductive hormones: A preliminary report. J Prosthet Dent 1998; 79: 663-670. 5. Kendall B, Eston R. Exercise -induced muscle damage and the potential protective role of estrogen. Sports Med 2002; 32: 103-123. 6. McClung JM, Davis JM, Wilson MA, Gold smith EC, Carson JA. Estrogen status and skeletal muscle recovery from disuse atrophy. J Appl Physiol 2006; 100: 2012-2023. 7. Pavlath GK, Thaloor D, Rando TA Cheong M, English AW, Zheng B. Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities. Dev Dyn 1998; 212: 495-508. 8. Morris-Wiman, J. and Widmer C.G. Inflammatory respons e to injury in masseter muscle. J. Dent Res 2006; 85 (Spec Iss A): 0446. 9. Huang GJ, LeResche L, Critchlow CW, Ma rtin MD, Drangsholt MT. Risk factors for diagnostic subgroups of painful tempor omandibular disorders (TMD). J Dent Res 2002; 81: 284-288. 10. Roda RP, Bagan JV, Fernandez JMD, Ba zan SH, Soriano YJ. Review of temporomandibular joint pathology. Part I: Classification, epidemiology and risk factors. Med Oral Patol Or al Cir Bucal 2007; 12: 292-298. 11. Lavigne GJ, Kato T, Kolta A, Sessle BJ. Neurobiological mechanisms involved in sleep bruxism. Crit Rev Or al Biol Med 2003; 14: 30-46. 12. Lund JP. Pain and the control of muscl es. In: Fricton JR Dubner R (eds). Orofacial Pain and Tempor omandibular Disorders. New York: Raven Press, 1995: 103-115. 13. Plesh O, Curtis DA, Hall LJ, Miller A. Gender difference in jaw pain induced by clenching. J Oral Rehabil 1998; 25: 258-263. 14. Karibe H, Goddard G, Gear RW. Sex differences in masticatory muscle pain after chewing. J Dent Res 2003; 82: 112-116. 15. Cairns BE. The influence of gender and se x steroids on craniof acial nociception. Headache 2007; 47: 319-324. 50

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BIOGRAPHICAL SKETCH Allison C. Harris received her Bachelor of Science in agricultural and biological engineering in 2003 from the Univ ersity of Florida. She continued her education at the University of Florida College of Dentistry and earned her Doctorate of Dental Medicine in 2007. This thesis is a partial requirement for the degree Master of Science in Dental Sciences, Orthodontics. She received her M.S. from the University of Florida in the spring of 2010. 55