Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of peripheral NMDA receptors in nerve growth factor-induced muscle pain Wong, Hayes Ga-Hei 2015

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2015_may_wong_hayes.pdf [ 1.56MB ]
Metadata
JSON: 24-1.0135676.json
JSON-LD: 24-1.0135676-ld.json
RDF/XML (Pretty): 24-1.0135676-rdf.xml
RDF/JSON: 24-1.0135676-rdf.json
Turtle: 24-1.0135676-turtle.txt
N-Triples: 24-1.0135676-rdf-ntriples.txt
Original Record: 24-1.0135676-source.json
Full Text
24-1.0135676-fulltext.txt
Citation
24-1.0135676.ris

Full Text

     THE ROLE OF PERIPHERAL NMDA RECEPTORS IN NERVE GROWTH FACTOR-INDUCED MUSCLE PAIN    by  Hayes Ga-Hei Wong  BSc, The University of British Columbia, 1999 MSc, The University of British Columbia, 2003     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in   The Faculty of Graduate and Postdoctoral Studies  (Neuroscience)         THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2015  © Hayes Ga-Hei Wong, 2015 ii  Abstract   Intramuscular injections of nerve growth factor (NGF) into human masseter muscle induce a local mechanical sensitization that mimics the symptoms of myofascial pain in patients with temporomandibular disorders.  I hypothesize that NGF induces the myofascial mechanical sensitization in part by increasing the expression of N-methyl-D-aspartate (NMDA) receptors in primary afferent neurons.  In behavioral experiments, injection of NGF into rat masseter muscle induced a prolonged local mechanical sensitization that was greater in female rats than in male rats.  This NGF-induced sensitization was partly attenuated by a local injection of the NMDA receptor antagonist APV at 3 days post NGF injection in the male rats but not in the female rats.  Immunohistochemical studies found that this NGF-induced mechanical sensitization was accompanied by the increased expression of NMDA receptor subtype 2B (NR2B) in trigeminal ganglion neurons innervating the masseter muscle in both sexes, as well as an increase in the average soma size of NR2B-expressing neurons.  An increase in the expression of neuropeptides (CGRP/SP) was also observed in the female rats but not in the male rats.  In in vivo extracellular recordings of masseter trigeminal ganglion neurons, NGF increased NMDA-induced mechanical sensitization in the male rats but not in the female rats.  However, in the female rats, this effect was greater in slow A fibers (2-7 m/s) than fast A fibers (>7-12 m/s).  My results suggest that NGF-induced mechanical sensitization is mediated, in part, through an effect on peripheral NMDA receptors in a sexually dimorphic manner. iii  Preface A version of Chapter 2 was published as “Wong H, Kang I, Dong XD, Christidis N, Ernberg M, Svensson P, Carins BE. NGF-induced mechanical sensitization of the masseter muscle is mediated through peripheral NMDA receptors. Neuroscience 269:232-44, 2014.”  I performed all the experiments and wrote the manuscript, except for the collection of human masseter muscle samples, which were provided by Dr. Christidis and Dr. Ernberg of the Karolinska Institutet.  Ms. Kang and Dr. Dong assisted in the analyses of these samples.  Dr. Dong also assisted in the rat immunohistochemical and electrophysiological experiments.  Dr. Svensson edited the manuscript and Dr. Cairns supervised the study. A version of Chapter 3 has been accepted by the Journal of Neurophysiology as “Wong H, Dong XD, Cairns BE. Nerve Growth Factor Alters the Sensitivity of Rat Masseter Muscle Mechanoreceptors to NMDA Receptor Activation” and is currently in press.  I performed all the experiments and wrote the manuscript.  Dr. Dong assisted with the experiments and Dr. Cairns supervised the study. All the figures in Chapter 1 are original figures prepared by the author, adapted and modified from the sources stated in the corresponding figure legends. All animal procedures were reviewed and approved by the University of British Columbia Animal Care Committee (Animal Care Protocol Number: A11-0242).    iv  Table of Contents  Abstract  .............................................................................................................. ii  Preface  ............................................................................................................... iii  Table of Contents  .............................................................................................. iv  List of Tables  ..................................................................................................... vi  List of Figures  .................................................................................................. vii  List of Abbreviations  ......................................................................................... x  Acknowledgements  ........................................................................................ xiii  Dedication  ....................................................................................................... xiv  Chapter 1: Introduction  ..................................................................................... 1 1.1. Project overview  ............................................................................................ 1 1.2. Mechanisms of pain  ...................................................................................... 2 1.2.1. Peripheral sensitization  ................................................................... 3 1.2.2. Central sensitization  ........................................................................ 4 1.3. Temporomandibular disorders  ...................................................................... 5 1.3.1. Cause of TMD pain  ......................................................................... 6 1.3.2. Sex-related differences in TMD  ....................................................... 8 1.3.3. Pharmacotherapy for TMD  ............................................................ 10 1.3.4. Animal models of TMD  .................................................................. 10 1.4. Sensory innervation of the craniofacial region  ............................................ 12 1.5. NGF and nociception  .................................................................................. 15 1.5.1. NGF and its receptors  ................................................................... 17 1.5.2. Neurotrophin retrograde signaling  ................................................. 20 1.6. Peripheral glutamate receptors and nociception  ......................................... 22  1.6.1. Peripheral NMDA receptors  .......................................................... 24 1.6.2. NMDA receptor subunits  ............................................................... 26 1.7. Ectopic discharge and peripheral sensitization  ........................................... 27 1.8. NGF and peripheral NMDA receptors  ......................................................... 29 1.9. Experimental hypotheses  ............................................................................ 31  Chapter 2: NGF-Induced Mechanical Sensitization of the Masseter Muscle is Mediated through Peripheral NMDA Receptors  ........................................ 35 2.1. Overview  ..................................................................................................... 35 2.2. Introduction  ................................................................................................. 36 2.3. Methods  ...................................................................................................... 38 2.3.1. Animals  ......................................................................................... 38 2.3.2. Administration of NGF and APV  .................................................... 39 v  2.3.3. Behavioural assessment  ............................................................... 39 2.3.4. Experimental design for behavioural studies  ................................. 40 2.3.5. Tissue processing and immunohistochemistry  .............................. 41 2.3.6. Human studies  .............................................................................. 43 2.3.6.1. Participants in the microbiopsy technique group  ............. 43 2.3.6.2. Microbiopsies  ................................................................... 43 2.3.7. Statistical analysis  ......................................................................... 45 2.4. Results  ........................................................................................................ 46 2.4.1. NGF-induced mechanical sensitization  ......................................... 46 2.4.2. Effect of NGF on NR2B expression in trigeminal ganglion  neurons  ................................................................................................... 48 2.4.3. Effect of APV on NGF-induced mechanical sensitization  .............. 53 2.4.4. Effect of NGF on neuropeptide expression  ................................... 55 2.4.5. Humans  ......................................................................................... 59 2.5. Discussion ................................................................................................... 63  Chapter 3: Nerve Growth Factor Alters the Sensitivity of Rat Masseter Muscle Mechanoreceptors to NMDA Receptor Activation  ........................... 70 3.1. Overview  ..................................................................................................... 70 3.2. Introduction  ................................................................................................. 71 3.3. Methods  ...................................................................................................... 73 3.3.1. Animals  ......................................................................................... 73 3.3.2. Administration of NGF  ................................................................... 73 3.3.3. Surgical preparation  ...................................................................... 74 3.3.4. In vivo electrophysiological recording  ............................................ 75 3.3.5. Plasma estrogen concentration  ..................................................... 78 3.3.6. Data analysis  ................................................................................. 78 3.4. Results  ........................................................................................................ 79 3.4.1. Mechanical activation threshold  .................................................... 79 3.4.2. Spontaneous and NMDA-evoked discharge  ................................. 87 3.4.3. Distance  ........................................................................................ 88 3.5. Discussion ................................................................................................... 90  Chapter 4: Conclusion  ..................................................................................... 97 4.1. Peripheral NMDA receptors in NGF-induced mechanical sensitization  ...... 97 4.1.1. Alternate explanations  ................................................................... 99 4.2. Animal model of myofascial pain in TMD  .................................................. 102 4.3. Other potential limitations  ......................................................................... 104 4.4. Future studies  ........................................................................................... 105 4.5. Biomarkers of pain  .................................................................................... 108 4.6 Significance ................................................................................................ 109  References  ..................................................................................................... 111    vi  List of Tables  Table 2.1. The median cross sectional somata size of NR2B subunit-                  immunopositive trigeminal masseter ganglion neurons following                   intramuscular NGF injection  .............................................................. 51  Table 3.1. The proportion of masseter afferent fibers with spontaneous                    discharge before and after NMDA injection  ....................................... 87  Table 3.2. The mean frequency of  spontaneous discharge (spikes/min) in                                   masseter afferent fibers before and after NMDA injection ................. 87   vii  List of Figures  Figure 1.1. The components of the trigeminal nociceptor  .................................... 3  Figure 1.2. The area of innervation of the trigeminal nerve  ................................ 12  Figure 1.3. A simplified dorsal view of the brainstem  ......................................... 14  Figure 1.4. TrkA receptor signaling  .................................................................... 18  Figure 1.5. The signaling endosome  .................................................................. 21             Figure 1.6. Structures of NMDA receptor agonists and antagonist  .................... 23                      Figure 1.7. (A) Domain organization of NMDA receptor subunits (B)                    Organization of NR1-NR2 NMDA receptor  ....................................... 27                        Figure 1.8. Proposed mechanism of NGF-induced mechanical                    sensitization in masseter muscle  ..................................................... 34  Figure 2.1. Intramuscular injection of NGF into the masseter muscle                    induced a prolonged local mechanical sensitization in male                    (A) and female (B) rats  ..................................................................... 47                                         Figure 2.2. Photomicrograph showing the expression of NR2B subunits                    by masseter ganglion neurons at seven days after NGF                    injection in female rats (A, B) and at three days after NGF                    injection in male rats (C, D)  .............................................................. 49   Figure 2.3. The frequency of NR2B subunit expressing trigeminal                    ganglion neurons innervating the masseter muscle increased                    following intramuscular injection of NGF in male (A) and                    female (B) rats  ................................................................................. 50  Figure 2.4. Soma size of masseter ganglion neurons expressing the                    NR2B subunit increased following NGF injection in both male                    (A) and female (B) rats  ..................................................................... 52    Figure 2.5. Intramuscular injection APV reversed NGF-induced                    mechanical sensitization in male rats (A) but not in female rats                    (B)  .................................................................................................... 54         Figure 2.6. Photomicrograph showing the expression of the NR2B,                    CGRP and SP by masseter ganglion neurons at three days                    after NGF injection in female rats  ..................................................... 56  viii  Figure 2.7. The frequency of expression of NR2B, CGRP and SP in                    trigeminal ganglion neurons innervating the masseter muscle                    at three days following intramuscular injection of NGF in male                    (A) and female (B) rats  ..................................................................... 57   Figure 2.8. The frequency of expression of trigeminal ganglion neurons                    innervating the masseter muscle doubled-labeled with                    NR2B/CGRP or NR2B/SP at three days following                    intramuscular injection of NGF in both male (A) and female                    (B) rats  ............................................................................................. 58  Figure 2.9. Example photomicrographs of two sections of human                    masseter muscle (A-D and E-H)  ...................................................... 60      Figure 2.10. The mean frequencies of expression of NR2B, substance P,                     and coexpression of NR2B and substance P by masseter                     muscle nerve fibers  ......................................................................... 61   Figure 2.11. A comparison of values for the different mean frequencies                     for each antibody (NR2B and Substance P) in the male and                     the female masseter muscle sections  ............................................. 62   Figure 3.1. Examples of extracellular electrophysiological recordings. (A)                    collision of antidromic and orthodromic action potentials. (B)                    An example of MT measurement  ..................................................... 76    Figure 3.2. The relationship between baseline MT and CV of masseter                    mechanoreceptors three days after NGF or vehicle injection in                    male and female (B) rats  .................................................................. 81   Figure 3.3. Relative mean mechanical activation threshold (Rel MT) of                    masseter mechanoreceptors (n = 12), 10 min after                    intramuscular NMDA injection in male (A) and female (B) rats  ........ 83   Figure 3.4. The relationship between Rel MT and CV of masseter                    mechanoreceptors in male (A) and female (B) rats  .......................... 84   Figure 3.5. Comparison between the Rel MT of slow Aδ fibers and fast                    Aδ fibers after vehicle or NGF treatment in male (A) and                    female (B) rats  ................................................................................. 85      Figure 3.6. The relationship between plasma estrogen concentration and                    baseline MT (A) or Rel MT (B) of masseter mechanoreceptors                    at 10 min following NMDA injection in female rats  ........................... 86  Figure 3.7. An example of the distance between the initial NGF injection  ix                     site and the subsequent NMDA injection site in the rat                     masseter muscle  ............................................................................. 88  Figure 3.8. The relationship between this distance (mm) and the baseline                     MT (A) or Rel MT (B) of masseter mechanoreceptors at 10                     min following NMDA injection in male and female rats  ................... 89 x  List of Abbreviations  Akt Protein kinase B AMPA Non-NMDA α-amino-3-hydroxy-5-methyl-5-isoxazolepropionate ANOVA Analysis of variance APV 2R-amino-5-phosphonovaleric acid ASIC3 Acid sensing ion channel 3 ATD Amino-terminal domain ATP Adenosine triphosphate BDNF Brain derived neurotrophic factor CFA Complete Freund’s Adjuvant CGRP Calcitonin gene related peptide CNS Central nervous system CV Conduction velocity DAG Diacylglycerol DRG Dorsal root ganglion ELISA Enzyme-linked immunosorbent assay EPSC Excitatory postsynaptic current EPSP Excitatory postsynaptic potential ERK Extracellular signal-regulated kinase fMRI Functional magnetic resonance imaging FRS2 Fibroblast growth factor receptor substrate 2Grb2 Growth factor receptor-bound protein 2 Gab1 Grb2-associated binder-1 xi  GABAA Type A gamma-aminobutyric acid IP3 Inositol triphosphate LBD Ligand-binding domain MAPK Mitogen-activated protein kinase MEK Mitogen activated protein kinase MRS Magnetic resonance spectroscopy MT Mechanical activation threshold NGF Nerve growth factor  NGS Normal goat serum NK1 Neurokinin 1 NMDA N-methyl-D-aspartate NR NMDA receptors NR2A NMDA receptor subtype 2A NR2B NMDA receptor subtype 2B NSAIDs Non-steroidal anti-inflammatory drugs NT3 Neurotrophin-3 NT4 Neurotrophin-4 P Pore helix PBS Phosphate buffered saline PDK1 Phosphoinositide-dependent kinase 1 PET Positron emission tomography PGP9.5 Protein gene product 9.5 PI3K Phosphatidylinositol-3-kinase xii  PKC Protein kinase C PLC Phospholipase C PNS Peripheral nervous system PPT Pressure pain threshold Rel MT Relative mechanical threshold SFK Src family kinase Shc Src homologous and collagen-like adaptor protein SOS Son of Sevenless SEM Standard error of the mean SP Substance P  TD Transmembrane domain TG Trigeminal ganglion TMD Temporomandibular disorders TMJ Temporomandibular joint TNF Tumor necrosis factor alpha Trk Tyrosine kinase TRPV1 Transient receptor potential cation channel subfamily V1 receptor WAD Whiplash associated disorder xiii  Acknowledgements This thesis would not be possible without the help of many people. Foremost, I would like to sincerely thank my supervisor Dr. Brian Cairns, for his guidance, insights and generosity. Thank you for the opportunity and your patience throughout the years. I would like to acknowledge my committee members: Dr. Ujendra Kumar, Dr. Ernest Puil and Dr. BR Sastry, for their input and guidance.  I would like to thank each member of the lab for their help and support including Dr. Akhlaq Hakim, Ms. Sun Nee Tan, Ms. Melissa O’Brien, Ms. Isabel Kang and especially Dr. Xudong Dong, for his advice and camaraderie. I would also like to thank Dr. Kumar and members of his lab, Dr. Rishi Somvanshi and Dr. Sajad War for their help in immunohistochemistry.   Special thanks to Dr. Malin Ernberg and Dr. Nikolaos Christidis of Karolinska Institutet for providing the human masseter samples. I am also grateful to Dr. Peter Svensson of Aarhus University for his input and suggestions.      xiv  Dedication  To my wife, Rinky, without you nothing is possible.1  Chapter 1 Introduction 1.1. Project overview Healthy masticatory function is important for eating, social interaction and general well being (Westberg and Kolta 2011).  It requires a well-balanced coordination of jaws and facial muscle activities.  Temporomandibular disorders (TMD) are a collection of musculoskeletal pain conditions of the head and neck that affect a significant segment of the population and many patients do not respond well to current treatments.  Nerve growth factor (NGF) is a neurotrophin essential for the growth and survival of sympathetic and small diameter sensory afferent fibres (McMahon and Bennett 1999).  In adults, elevated levels have also been found in many chronic pain diseases (Anand 1995).  Systemic injection of NGF in humans induced a long-lasting myalgia, particularly in the jaw and eye muscles (Apfel et al. 1994; Petty et al. 1994; Apfel et al. 1998; Apfel et al. 2000).  Injection of NGF into the masseter muscle of healthy subjects resulted in a local mechanical sensitization that lasted for 1-3 weeks, with a magnitude that was greater in women than men, and was similar to the myofascial pain reported by patients of TMD (Svensson et al. 2003; Svensson et al. 2008a).  These results suggest that NGF may be an important mediator in the development of chronic muscle pain.  However, the downstream mechanism of how NGF sensitizes muscle nociceptors is currently unclear.   Intramuscular injection of NGF in humans mimics some of the symptoms of chronic muscle pain and could be an attractive animal model to study chronic 2  muscle pain development.  Previous results from our laboratory demonstrated that injection of NGF into the masseter muscle of female rats induced a prolonged tyrosine kinase (Trk)A receptor-mediated mechanical sensitization of nociceptors, lasting for at least 3 h post injection (Svensson et al. 2010).  Both NGF and glutamate are released locally during injury and inflammation, and NGF has been found to increase NMDA receptor currents in cultured neurons (Bai and Kusiak 1997; Jarvis et al. 1997; Di-Luca et al. 2001).  I hypothesize that intramuscular injection of NGF induces a prolonged myofascial mechanical sensitization in part by increasing the expression of N-Methyl-D-Aspartate (NMDA) receptor in the primary afferent neurons.  Understanding the role of peripheral NMDA receptors in NGF-induced muscle pain may provide valuable insights into the development of chronic muscle pain.    1.2 Mechanisms of pain Pain is an intensely unpleasant sensation that warns the body of potentially threatening or damaging environment inputs, and is essential to maintaining the integrity of the body (Woolf and Ma 2007).  It is mediated by high-threshold primary afferent neurons or nociceptors which are mostly small diameter thinly myelinated A fibers and unmyelinated C fibers with non-specialized endings (Woolf and Ma 2007; Jankowski and Koerber 2010).  The majority of nociceptive afferents respond to multiple types of stimuli (i.e. polymodal nociceptors) (Jankowski and Koerber 2010).  Like all primary sensory neurons, they have their cell bodies in the dorsal root or trigeminal ganglia, which 3  give rise to a single axon that bifurcates into a peripheral branch innervating the peripheral target tissue, and a central axon that synapse to nociceptive second order neurons in the central nervous system (CNS) (Woolf and Ma 2007; Jankowski and Koerber 2010) (Figure 1.1).  After tissue injury, nociceptive neurons can be sensitized by peripheral and central mechanisms.     Figure 1.1. The components of the trigeminal nociceptor include a peripheral terminal that innervates the target tissue and transduces the noxious stimuli, an axon that conducts the action potentials from the periphery to the central terminal, a cell body that resides in the trigeminal ganglion, and a central terminal that transfers information to second order neurons at central synapses. Adapted from (Woolf and Ma 2007).   1.2.1. Peripheral sensitization  Peripheral sensitization is described as the elevated excitability of afferent nociceptors at the site of injury (Cairns and Prateepavanich 2009).  Tissue damage causes the release of algogenic and inflammatory mediators from surrounding tissues, including kinins, amines, prostagandins, growth factors, chemokines, cytokines, protons, adenosine triphosphate (ATP) and the excitatory amino acid glutamate (Levine and Reichling 1999; Sessle 2005; Woolf and Ma 4  2007; Jankowski and Koerber 2010).  This “inflammatory soup” activates and sensitizes the nociceptive terminals, reducing threshold and increasing responsiveness to noxious stimuli (Woolf and Ma 2007).  Afferent nerve endings can also release neuropeptides such as substance P (SP), resulting in oedema, redness and local temperature increase (Sessle 2005).  This process is termed neurogenic inflammation.  Peripheral inflammation can also produce retrograde signals in nociceptive neurons that results in phenotypic switches with an increase in the transcription of nociceptive mediators and ion channels such as neuropeptides, brain derived neurotrophic factor (BDNF), and sodium channels (Woolf and Ma 2007).  Peripheral sensitization can also result in spontaneous action potential discharge in nociceptive fibers, leading to spontaneous pain, which is pain in the absence of any identifiable noxious stimulus.  1.2.2. Central sensitization Central sensitization is described as the abnormal amplification in the central nociceptive processing that results in the spread of the painful area beyond the original site of injury (Cairns and Prateepavanich 2009). Increased peripheral input from sensitized nociceptive afferents can lead to several changes in the properties of central nociceptive neurons. These neuronal changes can include a reduction in threshold, exaggerated response to a noxious stimulus, increase in spontaneous firing, pain after the end of a stimulus and a spread of sensitivity to normal tissue (Sessle 2005; Woolf 2011).  The release of neuropeptides (e.g. SP) and excitatory amino acids (e.g. glutamate) by central 5  endings of nociceptive afferents during prolonged and intense nociceptive stimulation appear to be important in these neuroplastic processes (Sessle 2005).  Released neurotransmitters act postsynaptically on second-order nociceptive neurons, producing a prolonged neuronal depolarization and increased exicitiability of the neurons by activating neuronkinin receptors, and ionotropic (e.g. NMDA) and metabotropic excitatory amino acid receptors (Sessle 2005).  Central sensitization may also involve the loss of central inhibitory processes and changes in microglia and astrocytes (Sessle 2005; Woolf 2011).  The net result of these neuroplastic changes is an increased central excitatory state that depends on peripheral nociceptive input for its initiation, but may not depend on peripheral afferent drive for its maintenance (Sessle 2005).  It is important to note that peripheral and central sensitization appear to be normal physiological reactions that help to protect injured tissues from repeated injury and in most situations are reversible (Sessle 2005).  The mechanisms contributing to their prolongation in chronic or persistent pain are not yet well understood. Much remains to be learned, especially the role of other factors such as genetics, environmental contributors and psychophysiological influences (Sessle 2005; Woolf 2011).    1.3. Temporomandibular disorders TMD are a collection of musculoskeletal discorders of the head and neck that result in temporomandibular joint (TMJ) pain and/or masticatory muscle pain 6  (Sessle 2009; Cairns 2010; Shaefer et al. 2013).  TMD may be accompanied by headaches (Cairns 2010; Graff-Radford and Bassiur 2014).  It has been reported that 10% of the population may suffer from painful TMD (LeResche 1997; Graff-Radford and Bassiur 2014).  The Research Criteria for TMD introduced standardized diagnostic criteria to differentiate various manifestations of TMD (Dworkin and LeResche 1992).  It classified TMD into three diagnostic categories: myofascial pain, temporomandibular joint (TMJ) pain, and other joint conditions (arthragia, arthritis and arthrosis) (Cairns 2010).  Of these, myofascial pain is the most common diagnosis in TMD patient population (Manfredini et al. 2011).  Myofascial pain is described as a chronic dull aching pain in the masticatory muscles during palpation and function (e.g. chewing, mouth opening, speech) (Cairns 2010; Shaefer et al. 2013).   1.3.1. Cause of TMD pain Many factors may contribute to development of TMD and a universal underlying mechanism has yet to be identified (Sessle 1999; Graff-Radford and Bassiur 2014).  Pain in the TMJ may be accounted for by inflammation of the joint’s synovial lining or capsule (Graff-Radford and Bassiur 2014).   These tissues are innervated by myelinated and unmyelinated nerve fibers, some of which are peptidergic fibers containing calcitonin gene related peptide (CGRP) and SP, neuropeptides associated with pain and inflammation (Asaki et al. 2006; Cairns 2010).  Inflammation of the TMJ may result from bone degeneration, disc displacement or overloading of the joint from parafunctional behaviours, such as 7  excessive clenching or bruxing (Graff-Radford and Bassiur 2014).   These may lead to the release of inflammatory substances including pro-inflammatory cytokines (e.g. TNFα, interleukins) and pronociceptive substances such as potassium chloride, leukotrienes, histamine, glutamate and ATP (Cairns 2010).   In contrast to TMJ pain, myofascial pain in TMD is less understood and is likely caused by both peripheral and central mechanisms (Fernandes-de-las-Penas et al. 2009; Cairns 2010; Graff-Radford and Bassiur 2014).  Often there is a lack of correlation between myofascial pain and gross pathophysiological evidence in the masticatory muscles of patients (Brooks et al. 1992).  In TMD, myofascial pain is associated with trigger points, specific tender areas in the muscle that upon palpation, may reproduce the same pattern of pain as described by the patient (Fricton 2007; Cairns 2010).  It has been proposed that these areas may represent regions of micro-inflammation with elevated levels of proinflammatory cytokines, algogenic substances and neuropeptides (Fricton 2007).  These tender regions may be caused by repetitive trauma to the muscle from activities such as excessive clenching and bruxing (Fricton 2007; Cairns 2010).  In TMD patients with myofacial pain, elevated levels of algogenic substances that include glutamate and serotonin have been reported in support of this peripheral mechanism of myofascial TMD pain (Ernberg et al. 1999; Cairns 2010; Castrillon et al. 2010).  Prolonged afferent input from masticatory muscle pain could also lead to central sensitization.  This may result in pain perception long after the initiating peripheral factors have disappeared (Shaefer 8  et al. 2013).  Central sensitization may explain referred pain, pain in a site outside of the primary source of pain, in some patients with myofascial pain.  For example, headaches and migraines are common among TMD patients (Shaefer et al. 2013).  Pain in the masseter muscle has also been associated with pain in maxillary teeth (Fricton 2007).  Besides these physiological factors, genetics, stress and psychosocial facors have also been proposed to contribute to the development of TMD (Cairns 2010; Shaefer et al. 2013; Graff-Radford and Bassiur 2014).  1.3.2. Sex-related differences in TMD TMD, like many craniofacial pain conditions such as burning mouth syndrome as well as tension-type, migraine and cluster headaches, shows marked sex-related difference in their prevalence (Cairns 2007b; Cairns and Gazerani 2009).  Community studies found 2-3 times more women than men suffer TMD and approximately 80-90% of patients who seek treatment for this condition are women (Bush et al. 1993; Cairns 2010; Shaefer et al. 2013).  The mechanisms for this remain unclear and may involve multiple factors.  For example, it has been suggested that the female sex hormone, estrogen, may play a role. The onset of TMD in many women is associated with menstrual-cycle related variations of sex hormones at the beginning of puberty.  The incidence of TMD in women peaks during the third and fourth decade and then declines to levels comparable to men (LeResche 1997; Cairns 2007b).  Menstrual cycle 9  related variations in sex hormone levels are associated with the intensity of jaw muscle pain in female TMD patients (LeResche 1997; Cairns 2007b).    Estrogen may have effects on both peripheral and central nervous systems.  Receptors for estrogen are found in the spinal trigeminal nucleus, trigeminal ganglion, TMJ disc, synoviocytes, stromal cells and chondrocytes of the TMJ (Cairns 2010; Gupta et al. 2011; Meloto et al. 2011).  Peripherally, estrogen has been shown to increase the expression of cytokines in TMJ cartilage cells in mice (Yun et al. 2008).  In animal models, estrogen increases nociceptive response to glutamate injection in the masseter muscle through increasing the expression of NMDA receptors in afferent fibers innervating the muscle (Svennson et al. 2003; Dong et al. 2007).  Centrally, in vivo studies showed that the receptive field sizes of spinal trigeminal neurons increased as levels of estrogen become higher (Bereiter and Barker 1975).  Masticatory muscle reflex responses to noxious stimulation of the TMJ are also greater with elevated estrogen levels (Cairns et al. 2002b).  The expression of c-fos, an early response transcription factor associated with high intensity input including pain, were also higher in the brainstem of female rats with high levels of estrogen after stimulation of the TMJ (Bereiter 2001).  These findings from animal studies suggest that the overall higher levels of estrogen in women may predispose them to a greater incidence of TMD than men.  However, the underlying mechanism of its effects remains to be determined.  10  1.3.3. Pharmacotherapy for TMD Currently, the commonly used pharmacological agents for TMD include non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, muscle relaxants, anxiolytics, opioids, and tricyclic antidepressants (Cairns 2010; Bal-Kucuk et al. 2014).  However, evidence-based scientific studies that clearly demonstrate the efficacy and safety of these agents for TMD are limited (Cascos-Romero et al. 2009).  The main drawback of these studies is that they are most often observational studies rather than randomized controlled trials with appropriate control groups (Cascos-Romero et al. 2009; Bal-Kucuk et al. 2014).  Also, since the underlying mechanism of TMD is not well understood, the reasons for using these agents are mostly empirical, as opposed to mechanistic (Cairns 2010).  Additionally, many of these agents can lead to significant side effects with long-term use.  Because of these reasons, it is not surprising that many TMD patients (5-20%) remain refractory to current treatments (Shaefer et al. 2013).  Therefore, a greater understanding of the key mechanisms for pain in TMD is needed, so that more effective treatments with a mechanistic approach can be provided to patients suffering from TMD (Cairns 2010).  1.3.4. Animal models of TMD  Current animal models of TMD can be categorized into chemical or surgical approaches (Almarza et al. 2011).  In the chemical approaches, an inflammatory or algogenic agent, such as Complete Freund’s Adjuvant (CFA), mustard oil, formalin and carrageenan, is injected into the TMJ or perioral regions 11  (Haas et al. 1992; Swift et al. 1998; Ren 1999; Roveroni et al. 2001).  Injections of these agents induce inflammation, peripheral sensitization and/or pain-like behaviors such as flinching (Guo et al. 2010; Almarza et al. 2011).  However, these models may be limited by following: first, they only focus on the inflammatory aspect of TMD which is only likely associated with TMJ pain and there is no correlation between myofascial pain and gross pathophysiological symptoms; and secondly, most of these models have only been performed in small rodents, and translation to humans is an issue since most of these agents cannot be used in humans (Almarza et al. 2011).  Surgical approaches consist of procedures to reproduce pathophysiological symptoms associated with the onset of TMD, such as disc displacement, chodylectomy and disc perforation, which mimicks joint degeneration in the TMJ (Helmy et al. 1988; Ali and Sharawy 1994; Ueki et al. 2003).  Recently, ligation of the tendon of the masseter muscle in rats was proposed as a model for myofascial pain in TMD (Guo et al. 2010).  These models are often complicated by high subject variability and the fact that they do not replicate the gradual degeneration of tissues in TMD (Almarza et al. 2011).  Also it is unclear whether these pathophysiological symptoms are the cause of TMD pain as there is often a lack of correlation between pain and gross pathophysiological evidence of tissue injury (Cairns 2010).  There is a need for new animal models of TMD with a mechanistic approach that can be translatable to humans.   12  1.4. Sensory innervation of the craniofacial region Sensory information from craniofacial structures is transmitted to the trigeminal sensory nuclear complex in the brainstem via the trigeminal nerve.  These trigeminal primary afferents include myelinated Aβ and Aδ fibers that function as low threshold mechanoreceptors and proprioceptors as well as myelinated Aδ and unmyelinated C fibers with non-specialized free nerve endings that act as nociceptors (Sessle 2005).  The trigeminal nerve is divided into three branches innervating the ophthalmic, maxillary and mandibular divisions (Figure 1.2) (Siemionow et al. 2011).  The first two branches are purely sensory while the latter is a mixed sensory and motor nerve.  The three branches converge in the trigeminal ganglion where the cell bodies of the sensory fibers reside.     Figure 1.2. The area of innervation of the three sensory divisions of the trigeminal nerve. Adapted from (Saper 2000). 13  The trigeminal sensory nuclear complex extends from the midbrain from the pons and medulla caudally to the obex (Figure 1.3) (Westberg and Kolta 2011).  It is divided into three nuclei: the mesencephalic trigeminal nucleus, the main sensory trigeminal nucleus and the spinal trigeminal nucleus.  The mesencephalic nucleus is a ganglion that receives the proprioceptive information and is where the cell bodies of proprioceptive fibers for the masticatory muscles and periodontal tissues are located (Siemionow et al. 2011; Westberg and Kolta 2011).  The main sensory nucleus mediates light touch and pressure sensation (Siemionow et al. 2011).  The spinal nucleus is further divided into three subnuclei: oralis, interpolaris and caudalis (Ren and Dubner 2011).  The large diameter afferent fibers for discriminative touch synapse at the subnucleus oralis (Hendry and Hsiao 2012).  Evidence suggests that the subnuclei oralis and interpolaris also receive some nociceptive input especially pain of intraoral origin (Dostrovsky and Sessle 2007).  It is widely accepted that majority of the nociceptive input from the craniofacial region is relayed through the subnucleus caudalis, where small diameter afferent fibers innervating the area synapse (Sessle 2005).  The subnucleus caudalis is a laminated structure with many morphological and functional similarities to the spinal dorsal horn (Dostrovsky and Sessle 2007).  The small diameter afferents terminate mainly in caudalis laminae I, II, V and VI, while larger A-fiber low threshold mechanoreceptors terminate in laminae III – VI (Sessle 2005).  The nociceptive primary afferents in the subnucleus caudalis release excitatory amino acids (e.g. glutamate) and neuropeptides (e.g. SP, CGRP) (Sessle 2005).  From the trigeminal sensory 14  nuclear complex, second order neurons cross the midline and ascend to the thalamus through the trigeminothalamic tract to the venteroposteriormedial nucleus of the thalamus and from there to the orofacial primary sensory cortex.  Some neurons project to other brainstem or higher brain structures involved in reflex or perceptual aspects of orofacial pain such as the parabrachial nucleus and the recticular formation or the cranial nerve motor nuclei, as well as via the thalamus to other cortical regions such as anterior cortex and insula which are involved in the affective aspects of pain (Sessle 2005).    Figure 1.3. A simplified dorsal view of the brainstem shows the organization of the trigeminal sensory nuclear complex. Adapted from (Saper 2000; Siegel and Sapru 2010). 15  The masseter muscle is innervated by thinly myelinated Aδ and unmyelinated C trigeminal afferent fibers with non-specialized endings, which project to the trigeminal subnuclei interpolaris and caudalis (Cairns 2007a).  These fibers appear to function as nociceptors that respond to noxious mechanical and/or chemical stimuli (Cairns 2007a).  Previous experiments have identified predominantly Aδ fibers (Cairns et al. 2002a; Cairns et al. 2003a).  In the rat, the conduction velocity of Aδ and C fibers is 2.5 – 12 m/s and < 2.5 m/s, respectively (Lewin and McMahon 1991; Cairns 2008).  1.5. NGF and nociception NGF is a neurotrophin essential for the development, maintenance and regeneration of sympathetic and small diameter sensory afferent fibres.  Recent evidence suggests that it might also be an important mediator of nociception (Bennett 2001; Pezet and McMahon 2006; Watson et al. 2008).  Neurotrophins are a family of related growth factors comprising of NGF, BDNF, neurotrophin-3 (NTF3) and neurotrophin-4 (NTF4).  Studies have established a diverse range of functions of neurotrophins in the CNS and peripheral nervous system (PNS) in both developing and adult mammals.  Increasing evidence suggests that NGF plays an important role in nociception and acts as a peripheral pain mediator.  In contrast, BDNF appears to act as a modulator of nociception in central terminals in the spinal cord, while NTF3 and NTF4 appear to play relatively small roles in pain processing (Pezet and McMahon 2006).   16  In normal tissues, limited amounts of NGF are produced by peripheral tissues. Its levels are increased in response to injury or inflammation from various sources including structural (e.g. fibroblasts, keratinocytes and glial cells) and inflammatory (e.g. lymphocytes, macrophages and mast cells) cells (Pezet and McMahon 2006; Watson et al. 2008). In clinical studies, elevated tissue levels of NGF have been found in chronic pain disease states including arthritis, interstitial cystitis, pancreatitis and migraine headaches (Anand 1995). Individuals with mutations to genes regulating NGF production or its receptors display insensitivity to pain (Anand 1995; McMahon and Bennett 1999; Capsoni et al. 2011). Intravenous injections of NGF into healthy subjects resulted in a dose-dependent myalgia, particularly in the jaw (masseter) and eye muscles, while subcutaneous injections resulted in a local hyperalgesia that occurred without inflammation at the injection site for up to 7 weeks (Apfel et al. 1994; Petty et al. 1994; Apfel et al. 1998; Apfel et al. 2000). In recent experimental pain studies, intramuscular injections of NGF into the masseter muscle of healthy subjects induced a local mechanical sensitization that began within an hour of injection and lasted for 1-3 weeks (Svensson et al. 2003; Svensson et al. 2008a). This mechanical sensitization mimics some of the symptoms of patients with myofascial TMD, a chronic jaw muscle pain condition. Sensitivity to NGF was also found to be greater in women than men, which parallels the sex-related difference found in this musculoskeletal pain condition. This evidence from human pain experiments suggests that NGF may have important peripheral sensitizing effects that contribute to the development of chronic muscle pain. 17  1.5.1. NGF and its receptors NGF is synthesized as a precursor (proNGF) of approximately 30 kDa and is cleaved to a mature form of approximately 13 kDa (Watson et al. 2008).  The mature form exists as non-covalently linked homodimers and acts through two types of cell surface receptors: the low affinity neurotrophin receptor p75 and the high affinity TrkA receptor. All neurotrophins bind to the p75 receptor with similar affinity. This receptor has been implicated in the induction of apoptosis. However, its role in nociception remains unclear (Pezet and McMahon 2006; Watson et al. 2008).  NGF binds selectively to TrkA receptors with high affinity, while BDNF and NTF4 bind to TrkB receptors and NTF3 binds to TrkC receptors.  Ligand binding initiates receptor dimerization, followed by internalization and retrograde transport to the cell body, where it regulates gene transcription via activation of a series of intracellular signaling cascades, including the Ras mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K) pathway and the phospholipase C (PLC) pathway (Figure 1.4) (Chao 2003; Chao et al. 2006; Cheng and Ji 2008). Unused NGF in the extracellular space is degraded by a protease cascade that includes matrix metalloproteinase 9 (Bruno and Cuello 2006).  18    Figure 1.4: TrkA receptor signaling. TrkA receptors mediate differentiation, survival and synaptic plasticity signaling through the MAPK pathway, the PI3K pathway and the PLC pathway. Shc, Src homologous and collagen-like adaptor protein; Grb2, growth factor receptor-bound protein 2; FRS2, fibroblast growth factor receptor substrate 2; SOS, Son of Sevenless; Gab1, Grb2-associated binder-1; Akt, protein kinase B; PDK1, phosphoinositide-dependent kinase 1; MEK, mitogen activated protein kinase; ERK, extracellular signal-regulated kinase; DAG, diacylglycerol; PKC, protein kinase C; IP3, inositol triphosphate, SFK, src family kinase. Figure adapted from (Chao 2003).   Current evidence suggests that NGF-induced nociception is mediated through the TrkA receptors. Over 45% of DRG and trigeminal ganglion neurons 19  express TrkA receptors, with the majority being peptidergic small diameter sensory fibers which also express SP and CGRP, neuropeptides that have been implicated to play important roles in inflammation and pain (McMahon and Bennett 1999; Watson et al. 2008; Svensson et al. 2010).  Also, systemic injection of NGF induces hyperalgesia in transgenic mice lacking p75 receptors with magnitudes similar to that in wild type mice, implying that NGF-mediated pain signaling occurs through TrkA receptors (Raja et al. 1999). The NGF/TrkA system is thought to regulate pain processing in part by inducing the release and increased expression of neuropeptides (e.g. SP, CGRP, BDNF) both peripherally and centrally, sensitization and/or upregulation of ion channels and receptors present on the primary afferent nociceptors (e.g. transient receptor potential cation channel subfamily V1 receptor (TRPV1), voltage-gated sodium channels, acid sensing ion channel 3 (ASIC3), purinergic receptor P2X3 (McMahon and Bennett 1999; Pezet and McMahon 2006; Cheng and Ji 2008; Watson et al. 2008).  NGF administered systemically does not penetrate into the spinal cord and expression of the TrkA receptor is largely restricted to the peripheral endings of primary afferent nociceptors (Averill et al. 1995; McMahon and Bennett 1999).  Central sensitization has been proposed to occur through increased firing and/or increased release of neurotransmitters from the central endings of sensitized primary afferents (McMahon et al. 1993; Malcangio et al. 1997).  However, the contribution of these mechanisms to NGF-induced muscle pain remains unclear.   20  1.5.2. Neurtrophin retrograde signaling During development, neurotrophins and their receptors are essential for the correct establishment of neuronal connections in the central and peripheral nervous system (Ginty and Segal 2002; Zweifel et al. 2005; da-Silva and Wang 2011).  They have roles in axon and dendrite specialization, process growth, target innervation, cell death, synaptogenesis and synaptic refinement (Zweifel et al. 2005).  In the distal axons, NGF binds to the receptor and the ligand-receptor complex is internalized and retrogradely transported to the neuronal cell bodies (Ure and Campenot 1997; Ginty and Segal 2002; Zweifel et al. 2005).  Current evidence suggests these ligand-receptor complexes are localized to a small membrane-bound organelle, a signaling endosome, and transported in a dynein-dependent manner along a microtubule network toward the cell body (Figure 1.5) (Ginty and Segal 2002).  There the signals are disseminated through a specialized endosomal pathway.  The exact mechanism of retrograde neurotropin signaling remains unclear (Ginty and Segal 2002).  In In vitro studies with compartmentalized cultures of rat sympathetic neurons, where cell bodies and axons were maintained in separate culture media, radiolabelled NGF supplied to distal axons was transported to the cell bodies at a rate of ~ 3 mm/hr.  Approximately 5-30% of internalized NGF reached the cell bodies where it had a half-life of 3 hours (Ure and Campenot 1997).     21     Figure 1.5. The signaling endosome.  TrkA receptors are activated upon binding to NGF.  The ligand-receptor complex internalizes by endocytosis and becomes a specialized endosome.  They are transported retrogradely to the cell body using a dynein and microtubule-dependent transport mechanism.  The vesicle-associated Trk receptor remains autophosphorylated and promotes a unique set of signals upon arrival to the cell body. Adapted from (Ginty and Segal 2002; Cox et al. 2008).   Interestingly, no NGF transport was observed during the first hour after NGF administration, suggesting a time lag between receptor binding and the beginning of retrograde transport (Ure and Campenot 1997).  In an earlier in vivo study in the rat, intramuscular injection of NGF into the masseter muscle induced mechanical sensitization starting at a half hour after injection (Svensson et al. 2010).  Human experimental studies also found intramuscular injections of NGF induced a local mechanical sensitization with a quick onset (~ one hour) in the masseter muscle of healthy subjects (Svensson et al. 2003; Svensson et al. 2008a).  These results suggest the rapid action of NGF may be due a localized effect on the peripheral endings of afferent fibers in the muscle, separate from 22  the retrograde signaling mechanism, which may be involved in the prolongation of NGF effects.  1.6. Peripheral glutamate receptors and nociception Glutamate is a major excitatory neurotransmitter in the central nervous system (Figure 1.6). Recent evidence suggests that it may also play a nociceptive role in peripheral tissues.  Glutamate and its receptors are found in both trigeminal and DRG neurons as well as their central and peripheral terminals (Raja et al. 1999; Lam et al. 2005). It may be an important pro-inflammatory mediator since peripheral glutamate levels are elevated during cutaneous or deep tissue inflammation produced by a variety of non-neuronal cells such as mast cells and macrophages (Newsholme and Calder 1997).  Glutamate may also be released by the peripheral endings of primary afferent fibres during nociceptive stimulation and act on the glutamate receptors on the primary afferent fibres themselves (Westlund et al. 1989; Juranek and Lembeck 1997).  This raises the possibility that peripheral glutamate may have an autocrine and/or paracrine role in a positive-feedback enhancement of nociceptor excitability (Lam et al. 2005).  23    Figure 1.6: Structures of NMDA receptor agonists, glutamic acid and NMDA, and antagonist 2R-amino-5-phosphonovaleric acid (APV).     A number of human and animal studies have provided behavioural evidence in support of a role for peripheral glutamate receptors in the transduction of masticatory nociceptive signaling.  In particular, glutamate concentrations were significantly higher in the masseter muscles of myofascial TMD patients when compared to healthy controls (Castrillon et al. 2010).  Injection of high concentration glutamate into the masseter muscle of healthy subjects induced pain and mechanical sensitization and this response was blocked by a NMDA receptor antagonist (Svennson et al. 2003; Cairns et al. 2006).  This was not due to osmotic effects as confirmed in animal studies (Cairns et al. 2002a).  Glutamate-evoked pain was also found to be greater in women than men, similar to the sex-related difference in the occurrence of TMD pain (Cairns et al. 2003c).  In rat masseter muscle, baseline interstitial glutamate concentration was found to be ~25 µM. Increasing this concentration 2-3 times via systemic injection of monosodium glutamate resulted in a decrease in the 24  mechanical threshold of masseter muscle nociceptors and this effect was inhibited by NMDA receptor antagonists (Cairns et al. 2007).  These results suggest that masseter muscle mechanical sensitivity may be regulated, in part, through peripheral glutamatergic tone.  1.6.1. Peripheral NMDA receptors There are two principal groups of glutamate receptors, the inotropic glutamate receptors, which are ligand-gated mixed cation channels, and metabotropic glutamate receptors, which are G-protein coupled receptors (Wu and Zhuo 2009).  Peripheral metabotropic glutamate receptors have been shown to modulate both peripheral and central spinal nociceptive transmission, however, their relative contribution to the trigeminal system has not been studied in detail and is currently unclear (Neugebaur and Carlton 2002; Lam et al. 2005).  Ionotropic receptors include the NMDA receptors, and the non-NMDA α-amino-3-hydroxy-5-methyl-5-isoxazolepropionate (AMPA) and kainate receptors.  NMDA receptors are distinguished from AMPA and kainate receptors by their high calcium permeability, magnesium blockade at resting membrane potential, and the requirement of a co-agonist, glycine, for activation (Wu and Zhuo 2009).  NMDA receptors in the central nervous system are thought to play pivotal roles in modulating synaptic plasticity and neuronal excitability, serving as a molecular coincidence detectors.  To activate NMDA receptors, two events must occur simultaneously: glutamate must be released and bind to the receptor and the postsynaptic membrane has to be depolarized to remove the magnesium 25  blockade.  The NMDA receptor-mediated calcium influx then triggers a series of intracellular signaling molecules leading to the induction of neuronal hypersensitivity.  Phosphorylation of NMDA receptors by the Src family of kinases can also increase excitability via prolongation of channel open time and an increase in channel open probability (Doubell et al. 1999; Liu and Salter 2010).   Peripheral NMDA receptors play an important role in glutamate-evoked sensitization of nociceptive afferent fibres (Petrenko et al. 2003).  NMDA receptor antagonists, APV and ketamine, injected locally into the masseter muscle significantly decreased glutamate-evoked afferent discharge in rats (Cairns et al. 2003b).  Intraplantar injection of a NMDA receptor antagonist suppressed formalin-induced c-fos expression (a marker for nociceptive activity) in the spinal cord (Wang et al. 1997).  Expression of NMDA receptors in trigeminal ganglion neurons has also been shown to mediate the sex-related difference in glutamate-evoked afferent discharge, with female rats showing a higher level of expression than in male rats (Dong et al. 2007).  Furthermore, in recent human pain experiments, injection of the NMDA receptor antagonist ketamine into the masseter muscle in healthy subjects decreased glutamate-induced pain and sensitization (Cairns et al. 2006).  These results support the involvement of peripheral NMDA receptors in the transduction of nociceptive signaling.    26  1.6.2. NMDA receptor subunits NMDA receptors (NR) are proposed to consist of heterotetrameric assemblies of two NR1 and two NR2 subunits (Wu and Zhuo 2009; Paoletti 2011) (Figure 1.7).  Activation requires the binding of two glycine molecules, a co-agonist, to the NR1 subunits, and two glutamate molecules, the neurotransmitter, to the NR2 subunits.  Some cells express a third subtype, NR3, which have been reported to reduce calcium permeability and magnesium sensitivity when it co-assembles with NR1 and NR2 subunits (Chatterton et al. 2002; Zhu et al. 2012).  There are four different NR2 subunits (A, B, C and D).  The NMDA receptor subtype 2B (NR2B) subunit has been proposed as the critical subunit in NMDA-dependent pain sensitivity (Wu and Zhuo 2009; Liu and Salter 2010).  Spinal knockdown of NR2B subunit reduces formalin-induced nociceptive behaviours in rats (Tan et al. 2005).  In vitro studies show that estrogen can increase the NMDA receptor-mediated neuronal responses in the central nervous system through an effect on the NR2B subunit (Woolley et al. 1997; Foy et al. 1999).  Previous research has also found greater expression of the NR2B subunit than the NMDA receptor subtype 2A (NR2A) subunit in the trigeminal ganglion neurons that innervate the masseter muscle and that NMDA-evoked afferent discharge in the masseter muscle can be attenuated by ifenprodil, a NR2B-selective NMDA antagonist, in both male and female rats (Dong et al. 2007).  Messenger RNA and protein level of NR2B subunits are also increased in the DRG following inflammation with increased NMDA current density and increased sensitivity to NR2B antagonist (Li et al. 2006).  Taken 27  together, these results suggest that NR2B subunit containing NMDA receptors might play an important role in the peripheral sensitization of primary afferent fibres innervating the masseter muscle.     Figure 1.7. (A) Domain organization of NMDA receptor subunits.  ATD = amino-terminal domain, LBD = ligand-binding domain, TD = transmembrane domain, P = pore helix.  (B) NMDA receptors form tetrameric channels comprising of two copies each of NR1 and NR2.  ATD was omitted for clarity. Adapted from (Furukawa et al. 2005; Stawski et al. 2010).     1.7. Ectopic discharge and peripheral sensitization After injury, many primary afferent fibers can develop spontaneous discharge that contributes to the resulting paraesthesias and pain (Tal and Devor 1992).  In contrast, some afferent sensory cell bodies in the DRG and trigeminal ganglia have been found to produce intrinsic rhythmic firing (Devor and Seltzer 1999).  The capability of the cell bodies of sensory ganglion neurons to generate 28  self-sustaining ectopic firing leads to the speculation that it may be involved in the development of peripheral sensitization (Rappaport and Devor 1994; Devor and Seltzer 1999).        In the DRG, a small percentage of A- and C-fibers have been found to fire spontaneously, sending a low level of discharge orthodromically into the CNS and antidromically into the periphery (Wall and Devor 1983; Burchiel 1984).  The magnitude of this discharge is significantly increased by chronic nerve injury and it has been proposed as a possible reason of why local anaesthetic nerve block fails to relieve neuroparathesias and pain in some cases (Kirk 1974; DeSantis and Duckworth 1982; Wall and Devor 1983; Devor and Seltzer 1999).  The sensory ganglia are protected from mechanical stimulation and contain no synapses, and have long been assumed to be a nutritive depot for the sensory axon with no direct involvement in signal generation (Lieberman 1976).  The discovery of ectopic discharges from sensory ganglia prompted two likely explanations (Devor and Seltzer 1999).  First, it may be a part of the biophysical design of the sensory neurons to insure against conduction failure as nerve impulses propagate through the ganglion.  Second, it may serve an unsuspected function, such as providing a sensory background to the CNS body schema, similar to sympathetic tone.    In a recent study from our laboratory, intraganglionic injection of glutamate into the trigeminal ganglion induced afferent discharge and significantly reduced 29  the mechanical threshold of temporalis or masseter muscle afferents, suggesting that ectopic discharge from trigeminal sensory ganglion neurons can be a potential mechanism to peripheral sensitization of the craniofacial region (Laursen et al. 2014) .  Here I propose that the ectopic discharge from the trigeminal ganglion may be mediated by extracellular glutamate, where a basal release in the normal ganglion has been suggested (Jasmin et al. 2009; Jasmin et al. 2010), and that an increase in NMDA receptor expression induced by NGF will result in an increase in ectopic discharge, contributing to the sensitization of the peripheral muscle through the release of neuropeptides and/or glutamate from the peripheral terminals.   1.8. NGF and peripheral NMDA receptors I hypothesize that the sensitizing effects of NGF might be due, in part, to a peripheral effect on nociceptors resulting from an interaction between NGF and peripheral NMDA receptors.  Both NGF and glutamate are released during injury and inflammation and can be found in elevated levels in chronic pain diseases.  Both have been shown to induce acute mechanical sensitization in primary afferent nociceptors following injection into the masseter muscle in rats.  Results from clinical and animal models have found that NGF- or glutamate-induced mechanical sensitization in the masseter muscle both exhibit a sex-related difference with the response greater in females than in males (Cairns et al. 2003c; Svensson et al. 2003; Svensson et al. 2008a).  The difference appears to be due in part to an estrogen-mediated expression of NMDA receptors by 30  masseter nociceptors in female rats (Woolley et al. 1997; Foy et al. 1999; Dong et al. 2007).  In in vitro studies, NGF can induce glutamate to be released from cultured neurons or synaptosomes through activation of TrkA receptors (Numakawa et al. 2003; Raiteri et al. 2003).  There is also evidence showing that NGF can increase NMDA receptor currents in cultured neurons by phosphorylation and/or increasing expression of NR2B subunits (Bai and Kusiak 1997; Jarvis et al. 1997; Di-Luca et al. 2001).    Previous studies showed that NGF could activate the tyrosine kinase, Src, through the trkA receptor (Sofroniew et al. 2001). Receptor tyrosine kinases have been known to activate Src through the IP3 and PKC intracellular pathways, while Src has also been shown to interact with the PI3 and MEK pathways (Sofroniew et al. 2001; Liu and Salter 2010; Park et al. 2010).  These intracellular pathways are all activated upon trkA receptor activation (Figure 1.4).  Recent studies showed phosphorylation by Src to be critical in NMDA receptor regulation during inflammation and nerve injury (Salter and Kalia 2004).  The exact tyrosine phosphorylation sites have yet to be determined but the NR2B subunit has been implicated (Abe et al. 2005; Slack et al. 2008; Liu and Salter 2010; Peng et al. 2010).  Src-dependent phosphorylation of NMDA receptors can lead to an increase in receptor expression as well as increase in channel open time and open probability (Kalia et al. 2004; Liu and Salter 2010).  These results highlight the potential of NR2B-containing NMDA receptors as a downstream 31  target of NGF-trkA receptor activation and increased activity of NMDA receptors may contribute to NGF-induced mechanical sensitization.  Although the interaction between NGF and peripheral NMDA receptors is currently unclear, these results suggest that elevated levels of NGF could influence the excitability of masseter nociceptors by altering the activity of peripheral NMDA receptors.  Experiments in our laboratory have also shown that injection of NGF into the masseter muscle of female rats induced a prolonged TrkA receptor-mediated mechanical sensitization which began at 30 min and lasted for at least 3 hours, demonstrating the potential of using this animal model to investigate the underlying mechanism of NGF-induced chronic muscle pain (Svensson et al. 2010).  Here I propose a novel hypothesis: NGF induces chronic muscle pain in part via an upregulation of peripheral NMDA receptors.  1.9. Experimental hypotheses Based on the above-mentioned human and animal experimental evidence, I hypothesize that intramuscular injection of NGF induces a prolonged myofascial mechanical sensitization in part by increasing the expression NMDA receptor in the primary afferent neurons with the following specific experimental hypotheses (Summarized in Figure 1.8):  (1) Injection of NGF into the masseter muscle induces a prolonged mechanical sensitization in rats.  This is described in Chapter 2, where 32  behavioral studies were performed to assess the effect of intramuscular injection of NGF on masseter mechanical threshold in the rat for 14 days after injection.  (2) NGF induces mechanical sensitization by increasing expression of NMDA receptors in the primary afferent neurons innervating the masseter muscle.  This is described in Chapter 2.  Immunohistochemical studies were carried out to investigate the effect of intramuscular injection of NGF on the frequency of expression of NR2B subunit expressing masseter ganglion neurons in the trigeminal ganglion.  The median soma sizes of these NR2B subunit expressing ganglion neurons were also evaluated to assess for potential phenotypic change.  (3) Increased expression of NMDA receptors at the peripheral endings of masseter afferent neurons contributes to the increased sensitization.  In Chapter 2, behavioral studies were performed to determine where local injection of APV (Figure 1.6), a NMDA receptor antagonist, can attenuate NGF-induced mechanical sensitization.  In Chapter 3, extracellular single unit electrophysiolocal recordings on masseter mechanoreceptors were performed to evaluate the effect of NGF injection on mechanical sensitization induced by NMDA (Figure 1.6).  (4) NGF-induced NMDA receptor upregulation in masseter ganglion neurons induces ectopic discharge that invades the peripheral termination 33  of the muscle nociceptors to decrease mechanical sensitivity.  This is described in Chapter 3, where spontaneous discharges of masseter mechanoreceptors were evaluated by extracellular single unit electrophysiological recordings to determine whether pre-injection of NGF increases ectopic discharges.  (5) NGF-induced mechanical sensitization and NMDA receptor upregulation are greater in female rats than in male rats.  All the experiments were performed in both male and female rats to assess potential sex-related differences.              34                                       Figure 1.8: Proposed mechanism of NGF-induced mechanical sensitization in masseter muscle. (1) NGF binds to TrkA receptors, causing autophosphorylation; and (2) subsequent phosphorylation and increased activity of peripheral NMDA receptors. (3) NGF is retrogradely transported to the trigeminal ganglion (TG), resulting in (4) an increase in expression of NMDA receptors. (5) Anterograde transport of the NMDA receptors peripherally contributes to increased sensitivity. (6) Increased NMDA receptor expression in the TG induces ectopic discharges mediated by extracellular glutamate and (7) subsequent antidromic action potentials invade the periphery and contribute to peripheral sensitization. TG Brainstem Masseter Muscle 1 2 3 4 5 6, 7 35  Chapter 2 NGF-Induced Mechanical Sensitization of the Masseter Muscle is Mediated through Peripheral NMDA Receptors   2.1. Overview  Intramuscular injection of NGF in healthy humans mimics some of the symptoms of myofascial TMD. We hypothesized that NGF induces a prolonged myofascial mechanical sensitization by increasing peripheral NMDA receptor expression, leading to an enhanced response of muscle nociceptors to endogenous glutamate. Behavioral experiments with an injection of NGF (25µg/ml, 10µl) into the masseter muscle reduced the mechanical withdrawal threshold for 1 day in male rats and 5 days in female rats.  These results mirror the sex-related differences found in NGF-induced mechanical sensitization in humans. Intramuscular injection with the competitive NMDA receptor antagonist APV (20 mg/ml, 10µL) reversed the mechanical sensitization in male but not in female rats. NGF increased the number of NR2B-expressing rat trigeminal masseter ganglion neurons in both sexes, which peaked at 3 days post injection. There was an association between the levels of NR2B expression and NGF-induced mechanical sensitization. The average soma size of NR2B-expressing neurons increased significantly.  Increased expression of neuropeptides (CGRP and SP) was observed in NR2B-expressing masseter ganglion neurons in female but not in male rats.  In healthy men and women, comparable basal expression levels of NR2B and SP were found in peripheral fibers from masseter muscle microbiopsies. This study suggests that NGF-induced sensitization of masseter nociceptors is mediated, in part, by enhanced peripheral NMDA receptor 36  expression.  Measurement of peripheral NMDA receptor expression may be useful as a biomarker for myofascial TMD pain.  2.2. Introduction NGF is a neurotrophin essential for the growth and survival of sympathetic and small diameter sensory afferent fibers (Bennett 2001; Pezet and McMahon 2006). It may also play a role in chronic muscle pain development (Stohler 1997). Recent clinical trials found anti-NGF antibodies to be efficacious in treating osteoarthritic pain and chronic low back pain (Lane et al. 2010; Katz et al. 2011). Systemic injection of NGF induced a long-lasting myalgia including jaw muscle pain (Petty et al. 1994). NGF injected into the masseter muscle of healthy subjects induced a local mechanical sensitization that lasted for 1-3 weeks as well as pain during yawning and chewing (Svensson et al. 2003; Svensson et al. 2008a). These effects of NGF mimic some of the features of myofascial TMD, where patients suffer from a localized masseter muscle sensitivity upon palpation as well as pain during oral functions (Cairns 2010). The magnitude of these symptoms was also greater in women than men (Svensson et al. 2003; Svensson et al. 2008a).  These results suggest that intramuscular NGF injections may be useful in modeling the trigger points associated with myofascial TMD (See 1.3.1).   It is not clear how NGF injections induce prolonged mechanical sensitization of the masseter muscle. Injection of NGF into rat masseter muscle 37  induced a TrkA receptor-mediated mechanical sensitization of nociceptors, which began within 30 minutes of injection and lasted for at least 3 hours (Svensson et al. 2010).  These findings suggest that a peripheral mechanism underlies the initiation of NGF-induced mechanical sensitization but do not shed light on its long duration.   We speculate that the sensitizing effects of NGF might be due, in part, to an interaction between NGF and peripheral NMDA receptors on primary afferent nociceptors.  Results from clinical and animal studies suggest that peripheral glutamate receptors might play a role in nociceptive signaling from musculoskeletal tissues in the orofacial region. NGF and glutamate are released during injury and inflammation and can be found in elevated levels in chronic pain conditions (Anand 1995; Castrillon et al. 2010).  Intramuscular injections of glutamate induced mechanical sensitization in the masseter muscle of humans and rats; however, the duration is only a few hours (Cairns et al. 2002a; Cairns et al. 2003b; Svennson et al. 2003).  NGF-induced mechanical sensitization and glutamate-evoked discharge/pain both exhibited a sex-related difference with the greater response in females (Cairns et al. 2003c; Svensson et al. 2003; Svensson et al. 2008a).  In rats, this difference appears to be due in part to an estrogen-mediated expression of NR2B subunit containing NMDA receptors by masseter nociceptors in females (Woolley et al. 1997; Foy et al. 1999; Dong et al. 2007).  Previous research has also found that the majority of peripheral NMDA receptors contain the NR2B subunit and that NMDA-evoked muscle afferent discharge can be attenuated by ifenprodil, a NR2B-selective NMDA antagonist, in both male and female rats (Li et al. 2004; Dong et al. 2007).  In 38  cultured neurons, NGF can induce release of glutamate through TrkA receptor activation (Numakawa et al. 2003; Raiteri et al. 2003) and can also increase NMDA receptor currents by phosphorylation and/or by increasing receptor expression (Bai and Kusiak 1997; Jarvis et al. 1997; Di-Luca et al. 2001). These results suggest that NGF could influence the excitability of masseter nociceptors by altering the activity of peripheral NR2B subunit containing NMDA receptors.  This study aimed to examine whether NGF induces a prolonged mechanical sensitization of rat masseter muscle by increasing NMDA receptor activity and if so, whether this sensitization exhibits a sex-related difference.  The study further examined whether putative nociceptive fibers in the masseter muscle of healthy humans express NMDA receptors.  Understanding the role of peripheral NMDA receptors in NGF-induced muscle pain may provide further insights into chronic muscle pain development.  2.3. Methods 2.3.1. Animals Male (301–426 g, n=98) and female (234-299 g, n=95) Sprague-Dawley rats were used for all experiments.  Animals were housed in groups of two with a 12-h light/dark cycle. Food and water were given ad libitum. All animal procedures were reviewed and approved by the University of British Columbia Animal Care Committee.  39  2.3.2. Administration of NGF and APV Intramuscular injection of NGF (25 µg/ml, 10 µl, Sigma) and vehicle (phosphate buffered saline, PBS, 10 µl, Sigma) was made into the masseter muscles after the animals were briefly anesthetized with isoflurane (AERrane, Baxter Corporation, Missisauga, ON, Canada; 2-2.5%) and oxygen (97-98%).  For the behavioral studies, treatment was assigned to the left or right side in a random order, and the experimenter was blinded to the content of the injections during the testing period. The masseter muscle region was shaved prior to injection and the injection sites were marked with a permanent marker for subsequent identification. The concentration of NGF was selected based on the concentration used in previous human experimental pain studies and acute experiments in rats (Svensson et al. 2003; Mann et al. 2006; Svensson et al. 2008a; Svensson et al. 2010).  APV (Santa Cruz Biotechnology, 0.0020 and 0.020 g/ml) was administered in the same manner described above for NGF. APV was used to ensure that the effect on NMDA receptors was peripheral, since APV has been found to be poorly absorbed through the blood-brain barrier (Lodge et al. 1988; Whitten et al. 1990).  2.3.3 Behavioural assessment In vivo behavioral studies were performed to assess the mechanical withdrawal threshold at the site of injection with a rigid electronic von Frey hair (IITC Life Science, Woodland Hills, CA). For habituation, animals were restrained in a towel and mechanical threshold was measured daily for at least 5 days.   40  Prior to the start of the experiment consistent mechanical thresholds were required to be obtained for 2 consecutive days. The mechanical threshold reading recorded one day prior to injection was used as the baseline. The electronic von Frey hair was applied perpendicularly to the site of NGF or vehicle injection on the masseter muscle and force was gradually increased until the animal moved its head away from the stimulus. The mechanical test stimulus was applied at 1 min intervals for 5 min and the average was calculated for analysis.    2.3.4. Experimental design for behavioural studies To assess the acute and chronic effect of NGF injection on local mechanical withdrawal threshold, rats (n=10 male, 10 female) were injected with NGF or vehicle into the right or left masseter muscles, respectively. Mechanical threshold on both sides was measured at 1 and 3 hours, and 1, 3, 5, 7, 9, 11 and 14 days post NGF injection.  To determine the effect of APV on NGF-induced mechanical sensitization, rats (n=10 male, 10 female) were injected with NGF into the right masseter muscle and vehicle into the left masseter muscle. Mechanical threshold was tested at 3 days post NGF injection to determine NGF-induced mechanical sensitization. Rats were subsequently injected with vehicle (PBS) or APV (0.0020 and 0.020 g/ml) into the NGF-injected masseter muscle under brief anesthetization with isoflurane, and mechanical threshold was assessed at 10 41  and 30 min post injection. Treatments were assigned randomly and the experimenter was blinded to the content of injections during the testing period.  2.3.5. Tissue processing and immunohistochemistry Trigeminal ganglia were extracted and processed as previously described (Svensson et al. 2010). Briefly, the fluorescent tracer dye rhodamine-dextran (5%, 20 µl, Invitrogen) was injected bilaterally into the masseter muscle of isoflurane-anesthetized Sprague-Dawley rats (n=5 male, 5 female rats per time point) to identify trigeminal ganglion neurons innervating the masseter muscle. Rats also received injections of NGF and vehicle into the right and left masseter muscles, respectively, either 14, 7, 3 or 1 day prior to termination. Naïve rats without injection of NGF or vehicle were used as a baseline control. Animals were euthanized 7 days after rhodamine injection and then perfused with 120 ml cold saline followed by 120 ml of paraformaldehyde (4%). The right and left trigeminal ganglia were removed and cut into 10 µm sections with a cryotome.  Sections were treated with 5% normal goat serum (NGS) in PBS for 1 h and incubated overnight with primary antibody against the NR2B subunit (1:400; anti-rat mouse monoclonal, BD Biosciences) in PBS containing 1% NGS. The next morning sections were washed several times with PBS and then incubated for 1 h at room temperature in the dark in the presence of Alexa Fluor 488 secondary antibody (1:700, donkey anti-mouse, Invitrogen). After several washes in buffer, all sections were mounted on slides with covers slips and visualized with a Leica TCS SPE confocal microscope. NR2B-immunopositive cells were counted and 42  photographed for estimation of cell soma area using the ImageJ image processing freeware from the National Institutes of Health.  Neurons were considered positive when the average intensity of the fluorescent tracer signal from the soma exceeded two standard deviations (estimate of the 95% confidence interval) of the mean background intensity. Mean background intensity was calculated from a region within the ganglion tissue that was considered to be unlabeled. Only neurons with a clear nucleus were counted. The experimenter counting cells was blinded to the treatment received by the animals. The percent of masseter ganglion neurons expressing the NR2B subunit was determined. Data from each treatment group was pooled for the analysis of soma size distribution.   To determine the effects of NGF on the expression of neuropeptides, masseter trigeminal ganglia were collected and processed from male and female rats (n=5) at 3 days post NGF injection as described above. Sections were treated with primary antibodies against CGRP (1:1000, anti-rat rabbit, Abcam), SP (1:700, anti-rat guinea pig, Abcam) and NR2B subunit (1:400, anti-rat mouse monoclonal, BD Biosciences), and corresponding secondary antibodies (Alexa Fluor 405 goat anti-rabbit, Alexa Fluor 633 goat anti-guinea pig and Alexa Fluor 488 donkey anti-mouse, Invitrogen) at a concentration of 1:700. Data was analyzed as mentioned above. Removal of the primary antibodies was performed to validate antibody specificity.  43  2.3.6. Human Studies 2.3.6.1. Participants in the microbiopsy technique group Nine healthy men and eight age-matched healthy women were included in this part of the study. The inclusion criteria were age over 20 years, good general health, absence of muscle pain in the masseter and tibialis anterior muscles, and no analgesic or anti-inflammatory medication during the 24 hours preceding biopsy taking. The exclusion criteria for the healthy volunteers were systemic inflammatory connective tissue diseases (e.g. rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis), whiplash associated disorder (WAD), fibromyalgia, neuropathic pain or neurological disorders (e.g. myasthenia gravis, craniomandibular dystonia), pain of dental origin, pregnancy, frequent use of muscle relaxants, and use of analgesic or anti-inflammatory medication during the 24 hours preceding biopsy. The methods and selection of participants were approved by the regional ethical review board in Stockholm, Sweden (2007/2:6). The participants were screened for trial suitability and informed about the study protocol at a separate visit. The study followed the principles for medical research according to the guidelines of the Declaration of Helsinki as well as the Good Clinical Practice guidelines and the participants received both written as well as verbal information and gave their verbal and written consent.  2.3.6.2. Microbiopsies Microbiopsies were obtained with a disposable Monopty®Bard® biopsy instrument through the skin-surface overlaying the bulky part of the superficial 44  masseter muscle under skin surface anesthesia (5% lidocaine). For the masseter muscle a penetration depth of 11 mm and a diameter of 18G was used. This biopsy system is a current version of an automated biopsy system that has been proven to be effective i.e. for the diagnosis of musculoskeletal sarcomas (Welker et al. 2000). The biopsy instrument was guided with a Bard®TruGuide™ coaxial needle (BARD Norden, Helsingborg, Sweden), that was inserted to a depth of 10 mm, along the long axis of the muscles until the fascia was penetrated. The biopsy instrument was inserted through the coaxial needle and by pressing the trigger button the needle collected a piece of muscle tissue with a size of approximately 0.12 cm x 1.1 cm in the masseter. The biopsy instrument was then removed from the coaxial needle while the latter was maintained in place, in order to avoid repeated skin punctures. The muscle section was removed from the biopsy instrument using a sterile probe, and the biopsy instrument was rinsed with isotonic saline. This procedure was repeated twice by rotating the biopsy instrument 45° each time in order to ensure that sufficient muscle tissue was obtained. Hence, three micro-biopsies were taken from each muscle. Samples were sliced and incubated with mouse monoclonal anti-human protein gene product 9.5 (PGP9.5) antibody (Abcam), rabbit polyclonal anti-rat/human NR2B antibody (Abcam), and guinea pig anti-rat/human substance P antibody (Abcam), and visualized with fluorescent secondary antibodies (Invitrogen).   A nerve fiber was defined as any fibril-shaped fluorescence of at least 4.0 µm in length which stained positive for PGP9.5 antibody. Nerve fibers were 45  considered positively-stained when the average intensity of the fluorescent tracer signal exceeded two standard deviations (estimate of the 95% confidence interval) of the mean background intensity.  2.3.7. Statistical analysis In an unblinded pilot study in 6 male rats, we found that NGF injections reduced the mechanical threshold by 18±10% on the NGF injected side relative to the control side after 24 hours.  However, because the pilot was unblinded, we conservatively adjusted the standard deviation of the difference to be equal to the average difference found in the pilot.  A paired t test sample size estimation indicated that to detect a minimum difference of 18%, with an estimated standard deviation of the difference of 18%, and α = 0.05, and β = 0.20, a minimum of 10 animals per group were needed.  In the behavioral studies, mechanical threshold data from NGF and vehicle-injected sides were analyzed using two-way repeated measures analysis of variance (ANOVA) with time and treatment as factors. Post-hoc Sidak’s multiple comparison test was used to compare post-injection mechanical thresholds between treatments. One-way repeated measures ANOVA and post-hoc Holm-Sidak method were used to assess the effect of APV on NGF-induced mechanical allodynia. In the immunohistochemical studies, the frequency of NR2B expression in trigeminal ganglion neurons between NGF and vehicle-injected sides was analyzed using pairwise Students t-test for each time point.  Kruskal-Wallis one-way ANOVA on ranks was used to test for differences in the cross sectional area between the soma size of NR2B-immunopositive cells 46  at different time points following NGF injection. A probability level of 0.05 was set for all tests. Error bars represent SEM.  2.4. Results To determine that mechanical threshold was due to muscle stimulation and not response from the skin, 2.5% lidocaine/2.5% prilocaine cream (EMLA cream, AstraZeneca) was applied topically on the shaved skin over the masseter muscle of three male rats to block mechanical sensation from the skin. The pinch test was performed on the skin with forceps to assess the analgesic effect of lidocaine. At 30 min post treatment, no response to the pinch test was observed. Mechanical threshold was measured and compared to baseline values. No significant difference was observed between baseline and post-treatment values (p = 0.844 by paired t-test), validating that the mechanical threshold measured was due to muscle nociception only.  2.4.1. NGF-induced mechanical sensitization Intramuscular injection of NGF into the masseter muscle induced a prolonged mechanical sensitization at the site of injection in both male and female rats (Figure 2.1).  Using two-way repeated measures ANOVA with time and treatment as factors, significant differences were found between NGF and vehicle-injected sides in both sexes (male, p = 0.048; female, p = 0.001). In the male rats, NGF significantly reduced mechanical threshold compared to vehicle at 3h and 1 day post NGF injection using post-hoc Sidak’s multiple comparison 47  test (Figure 2.1A). The peak reduction (29.4 ± 5.5% of baseline mechanical threshold) occurred 3h post injection. On the contralateral side, PBS induced a transient reduction from 1 to 3 h post injection with a peak intensity of 18.2 ± 3.2% of baseline mechanical threshold. In the female rats, a longer-lasting sensitization than in the males was observed following injection of NGF.   48  Figure 2.1: Intramuscular injection of NGF into the masseter muscle induced a prolonged local mechanical sensitization in male (A; n = 10) and female (B; n = 10) rats.  Asterisks indicate thresholds significantly different between PBS and NGF treatment groups by post-hoc Sidak multiple comparison test (p<0.05). Filled and open squares denote mechanical threshold averages of head withdrawal responses for ipsilateral and contralateral masseter muscle, respectively.   Mechanical threshold was significantly reduced compared to vehicle starting at 3h, 1 day, 3 days and 5 days post NGF injection, with a peak effect (25.9 ± 3.0%) at 3h post injection (Figure 2.1B). A long-lasting sensitization was also observed on the contralateral side, with the mechanical thresholds significantly lowered compared to baseline from 1h to 7 days post PBS injection, with the peak effect (14.8 ± 4.1%) observed at 3 days post injection. No overt signs of inflammation (e.g. redness, edema) or nocifensive behaviours (e.g. swiping, guarding) were observed at the site of injection during the testing period.   2.4.2. Effect of NGF on NR2B expression in trigeminal ganglion neurons The expression of NR2B subunits by trigeminal ganglion neurons innervating the masseter muscle was assessed at 1, 3, 7 and 14 days post NGF injection to determine whether injection of NGF changed the expression of NMDA receptors in peripheral nociceptors (Figure 2.2 and 2.3).  In male rats, NGF significantly increased NR2B subunit-immunopositive neurons at 3 days after injection with an increase of 8.7 ± 2.2% compared to the PBS-injected side. No changes were observed at other time points. A longer-lasting increase in NR2B expression was observed in the female rats with significant increases at 3 and 7 days post NGF injection. The peak increase of 14.2 ± 3.7% was found to 49  occur 3 days after injection of NGF. Expression returned to baseline level 14 days after injection.  NR2B expression was significantly higher in the females than in the males at 3 days after NGF injection.    Figure 2.2: The photomicrograph shows expression of the NR2B subunits by masseter ganglion neurons at seven days after NGF injection in female rats (A, B) and at three days after NGF injection in male rats (C, D).  Masseter ganglion neurons were identified by intramuscular injection of rhodamine dextran (arrows in A, C).  Immunopositivity for the NR2B subunit is identified by arrows in B, D. Bar = 75 µm.  50             A.                       B  Figure 2.3: The bar graphs show the frequency of NR2B subunit expressing trigeminal ganglion neurons innervating the masseter muscle increased following 51  intramuscular injection of NGF in male (A) and female (B) rats. Asterisks denote significant difference between the NGF-injected side and the vehicle-injected side by paired Students t-tests (p < 0.05).     NGF treatment was also associated with changes in the cross-sectional somata area of the population of NR2B subunit-immunopositive neurons (Figure 2.4 and Table 2.1). In male rats, the median area of NR2B subunit-immunopositive cells significantly increased at 1, 3 and 7 days with the level returning to baseline at 14 days after injection (Figure 2.4A). In female rats, the median area of NR2B-immunopositive cells was found to be significantly higher at all the time points up to 14 days post injection (Figure 2.4B).  Table 2.1: The median cross sectional somata size of NR2B subunit-immunopositive trigeminal masseter ganglion neurons following intramuscular NGF injection from 5 male and 5 female rats  Data from each treatment group was pooled for analysis. Asterisks denote significant difference from pre-NGF injection baseline value by Kruskal-Wallis one way ANOVA on ranks (p < 0.05).   52    Figure 2.4: Soma size of masseter ganglion neurons expressing the NR2B subunit increased following NGF injection in both male (A) and female (B) rats. The size distribution of NR2B subunit-immunopositive neurons was determined by measuring the cross sectional area of soma of rhodamine positive trigeminal ganglion neurons.    53   2.4.3. Effect of APV on NGF-induced mechanical sensitization To determine whether NGF-induced mechanical sensitization was mediated by the increased expression of NMDA receptors in peripheral afferent neurons in the masseter muscle, the effect of intramuscular injection of APV, a competitive NMDA receptor antagonist, on NGF-induced mechanical sensitization was investigated (Figure 2.5). APV was tested 3 days after NGF injection when the highest level of NR2B subunit expression was observed in both sexes as described above. In male rats, 20 mg/ml of APV significantly reversed NGF-induced mechanical sensitization at 10 min post injection while PBS and 2 mg/ml APV had no effect (Figure 2.5A). No effect of APV on mechanical sensitization was observed 30 min after injection. APV had no significant effect on NGF-induced mechanical sensitization in female rats at either of the concentrations (2 and 20 mg/ml) tested (Figure 2.5B).      54     Figure 2.5: Intramuscular injection APV reversed NGF-induced mechanical sensitization in male rats (A; n = 10) but not in female rats (B; n = 10). Treatments (vehicle, 2 mg/ml or 20 mg/ml APV) were administered 3 days after NGF injection and mechanical thresholds were tested before, and at 10 and 30 min post APV injection. Asterisks indicate thresholds significantly lower than baseline pre-NGF injection thresholds (p < 0.05, one-way repeated measures ANOVA & post-hoc test).    55  2.4.4. Effect of NGF on neuropeptide expression Neuropeptide expression has been associated with pain sensitivity (Neumann et al. 1996; Ohtori et al. 2001).  To assess whether the NGF-induced increase in NR2B expression was associated with neuropeptide expression, NGF, CGRP and SP expression in masseter ganglion neurons was determined 3 days post NGF injection (Figure 2.6 and 2.7).   Three days after NGF injection, NR2B expression in the male rats increased but no significant changes in neuropeptide expression were observed (Figure 2.7A). In female rats, NGF induced an increase in the expression in NR2B and SP but not CGRP (Figure 2.7B). In neurons which expressed the NR2B subunit, no change of expression of CGRP and SP were found in male rats but expression of both types of ganglion neurons were increased significantly in the female rats (Figure 2.8).  No differences in neuropeptide (CGRP, SP) expression were found on the vehicle-injected side between the male and female rats.   56     Figure 2.6: The photomicrograph shows expression of the NR2B, CGRP and SP by masseter ganglion neurons at three days after NGF injection in female rats.  The masseter ganglion neuron in this picture, which was identified by intramuscular injection of rhodamine dextran (arrow in A), was also immunopositive for NR2B subunit (B), CGRP (C) and SP (D). Bar = 75 µm.       57            A                     B   Figure 2.7: The bar graphs show the frequency of expression of NR2B, CGRP and SP in trigeminal ganglion neurons innervating the masseter muscle at three days following intramuscular injection of NGF in male (A) and female (B) rats. Asterisks denote significant difference between the NGF-injected side and the vehicle-injected side by paired Students t-tests (p < 0.05).   58           Figure 2.8: The frequency of expression of trigeminal ganglion neurons innervating the masseter muscle double-labeled with NR2B/CGRP or NR2B/SP at three days following intramuscular injection of NGF in both male (A) and female (B) rats. Asterisks denote significant difference between the NGF-injected side and the vehicle-injected side by paired Students t-tests (p < 0.05).    59  2.4.5. Humans Masseter muscle microbiopsies were collected from healthy men and women and the expression of NR2B and SP by muscle nerve fibers was determined (Figure 2.9 and 2.10). NR2B and SP were found to be expressed by 44 ± 10 % and 16 ± 8 % of muscle nerve fibers, respectively. This level of expression is comparable to that found in the rats (Figure 2.3 and 2.7). The comparison between sexes is shown in Figure 2.11. No difference in NR2B subunit expression was observed between men and women, while the differences in SP expression and dual expression of NR2B and SP did not reach statistical significance.              60   Figure 2.9: Example photomicrographs of two sections of human masseter muscle (A-D and E-H). Nerve fibers were identified by PGP9.5 antibody (A, E), and tested for expression of NR2B (B, F), and Substance P (C, G). D and H are composite of all three labels. The arrows identify the fibers which are immunopositive for the antibody. The inset shows the magnified area (3x) identified by the arrow. M: Myocyte, B: Blood vessel, C: Connective tissues. Bar = 75 µm.  D M H M C B E F G A B C 61       Figure 2.10: The bar graph shows the mean frequencies of expression of NR2B, substance P, and coexpression of NR2B and substance P by masseter muscle nerve fibers. Data from both men and women was combined for analysis (n=17 biopsies). The error bars represent standard error.         62         Figure 2.11: A comparison of values for the different mean frequencies for each antibody (NR2B and Substance P) in the male and the female masseter muscle sections. No statistical difference was found between each group (NR2B: p=0.0925, Substance P: p=0.062, Dual: p=0.177). Error bars are representative of standard error. 63  2.5. Discussion NGF is an important peripheral pain mediator and may contribute to the development of chronic muscle pain (Bennett 2001; Chao 2003; Pezet and McMahon 2006).  Intramuscular injections of NGF into the masseter muscle of healthy subjects induce a local mechanical sensitization that begins within hours of the injection and lasts for 1-3 weeks, and is more pronounced in women than in men (Svensson et al. 2003; Svensson et al. 2008a).  Injection of NGF into the rat masseter muscle induced a mechanical sensitization comparable to that reported in human experimental pain studies. In rats, one consequence of NGF injection was an increase in the number of NMDA receptor-expressing masseter muscle afferent fibers, which peaked at 3 days after NGF injection in both sexes. Local administration of the NMDA receptor antagonist APV resulted in a short term attenuation of mechanical sensitization in male but not in female rats. Subsequent investigation revealed that NGF induced a greater increase in SP expression in masseter ganglion neurons in female rats, suggesting that SP may contribute to the sex-related differences in response to NGF injection. These findings on the mechanism of NGF action in rats may be translatable, as the frequency of expression of NR2B and SP in rats and healthy humans was comparable.   Greater expression of SP and dual expression of NR2B and SP were also observed in women.  These results suggest that this animal model may be used to study the mechanism of chronic muscle pain development and that the expression of NMDA receptors in the masseter muscle could be further explored as a biomarker of myofascial TMD pain. However, injection of NGF is 64  not equivalent to myofascial TMD and no association between NGF level in the muscle and myofascial TMD has currently been found (Basi et al. 2012).  Therefore, while this model may produce the symptoms of myofascial TMD, it may not replicate the pathogenesis of this chronic pain condition.   The current study demonstrates that there is a relationship between increased expression of NMDA receptors and the maintenance of NGF-induced mechanical sensitization in the rat. Glutamate and its receptors are found in trigeminal and DRG neurons as well as their central and peripheral terminals (Lam et al. 2005; Dong et al. 2007; Wang et al. 2012).   In the rat masseter muscle, the basal interstitial concentration of glutamate is 20-30 µM.  Systemic administration of 50 mg/kg of monosodium glutamate increased masseter muscle interstitial glutamate concentrations by 2-3 times and resulted in a NMDA receptor-mediated decrease in the mechanical threshold of masseter muscle nociceptors (Cairns et al. 2007). Conversely, intramuscular injection of botulinum neurotoxin A into the rat masseter muscle significantly decreased interstitial glutamate concentration and increased nociceptor mechanical threshold (Gazerani et al. 2010).  These findings suggest that peripheral NMDA receptors contribute to the modulation of muscle nociceptor sensitivity to mechanical stimuli.  Increasing the expression of NMDA receptors on muscle afferent fiber endings should also increase sensitivity of muscle nociceptors to glutamate, which may explain the association between NMDA receptor expression and mechanical withdrawal threshold in rats. 65  In this study, we found an increase in NR2B subunit containing NMDA receptors in trigeminal ganglion neurons associated with NGF-induced local mechanical sensitization in the masseter muscle. This suggests an increased expression of these receptors in the peripheral and central terminals of the afferent fiber. An alternative explanation for the observed NGF-induced mechanical sensitization is that it may be mediated by the increased expression of NMDA receptors at the central terminals of the primary afferents in the trigeminal nuclear complex.  Previous research suggested that these presynaptic NMDA receptors may facilitate the release of glutamate and SP in the dorsal horn, which may contribute to sensitization after injury (Liu et al. 1997; Yan et al. 2013; Chen et al. 2014).  However, in vivo results are controversial with conflicting reports.   In some cases, intrathecal NMDA failed to evoke SP release in the dorsal horn (Nazarian et al. 2008; Bardoni 2013). It remains to be determined whether this mechanism plays a role in NGF-induced sensitization.  In healthy humans, the frequency of NR2B subunit expression by masseter muscle nerve fibers appeared comparable to that found in the rat.  It is not known if peripheral NMDA receptor expression is altered in the masseter muscle of myofascial TMD patients.  In healthy humans, injection of glutamate into the masseter muscle induces a mechanical sensitization that can be attenuated by local injection of the NMDA receptor antagonist ketamine, which indicates that it is mediated, in part, through activation of peripheral NMDA receptors (Svennson et al. 2003; Cairns et al. 2006).  In painful chronic 66  tendinopathy, increases in NMDA receptor expression and glutamate concentrations have been reported (Alfredson et al. 2001; Alfredson and Lorentzon 2002; Schizas et al. 2010). Glutamate concentrations were reported to be significantly elevated in myofascial TMD patients compared to healthy controls (Castrillon et al. 2010). Taken together, these findings suggest that peripheral NMDA receptors may play a role in myofascial TMD pain.  Prolonged tissue inflammation causes primary sensory neurons to undergo a phenotypic switch (Woolf and Ma 2007; Latremoliere and Woolf 2009).  Previous studies have found NGF-dependent increases in the average soma size of CGRP and SP expressing DRG neurons following peripheral inflammation, which may indicate the expression of these neuropeptides in larger, low threshold mechanoreceptive Aβ fibers that normally do not express them (Neumann et al. 1996; Ohtori et al. 2001). This phenotypic switch is associated with an enhancement in response to normally innocuous mechanical stimuli (Latremoliere and Woolf 2009).  An increase in the release of glutamate and SP by Aδ fibers at their central synapses, in addition to that normally released by nociceptive Aδ and C fibers, has been proposed to underlie this sensitization (Latremoliere and Woolf 2009). In the current study, we have found an increase in the average soma size of NR2B-expressing muscle afferent neurons following NGF injection, indicating that a novel population of masseter afferent fibers now express peripheral NMDA receptors and can respond to glutamate (Cairns et al. 2007). This phenotypic switch may play a role in the NGF-related sensitization.  67  NMDA receptor and neuropeptide expression were elevated 3 days after NGF injection, however, sensitization was observed 1 hour after injection, which suggests that different mechanisms may mediate the initial development of the mechanical sensitization. NGF can induce mechanical sensitization of masseter muscle nociceptors through TrkA receptor activation within 30 minutes of injection (Svensson et al. 2010). TrkA receptors are expressed in a majority of peptidergic small diameter sensory neurons in the DRG and trigeminal ganglion (Watson et al. 2008; Svensson et al. 2010). Activation of TrkA receptors leads to an increased calcium influx and phosphorylation of the NMDA receptor, which increases the period of time the channel remains open (Bai and Kusiak 1997; Jarvis et al. 1997; Chao 2003). Based on these findings, we speculate that the early component of NGF-induced mechanical sensitization is due, in part, to a combination of these alterations in neuropeptide release and NMDA receptor function.  Increased expression of NMDA receptors and neuropeptides appear to be important for maintaining mechanical sensitization following NGF injection.  We found a sex-related difference in the sensitivity of NGF-induced mechanical sensitization to peripheral NMDA receptor antagonism in male and female rats.  The frequency of expression of NMDA receptors by masseter ganglion neurons is greater in female than in male rats, an effect shown to be due to estrogen (Dong et al. 2007).    Estrogen can also increase NMDA receptor-mediated neuronal responses through an effect on the NR2B subunit (Woolley et al. 1997; Foy et al. 1999).   Thus, a larger and longer-lasting increase 68  in NMDA expression in female rats following NGF injection might explain the lack of effect of APV on NGF-induced mechanical sensitization.  Alternatively, the increased expression of neuropeptides by masseter muscle ganglion neurons in the females may have resulted in a greater release of neurotransmitters at central synapses, which, as discussed above, could lead to greater sensitization centrally (Latremoliere and Woolf 2009).   A similar sex-related difference in sensitivity to peripheral NMDA receptor antagonism has been noted in humans, where it has been found that lower concentrations of ketamine are required to attenuate glutamate-evoked masseter muscle pain in men than in women (Cairns et al. 2006; Castrillon et al. 2007).   However, in the present study NMDA receptor and neuropeptide expression by masseter nerve fibers in men and women was not significantly different, although the small sample size may not have provided sufficient statistical power to resolve this difference.   It is also possible that different receptors are important for sex-related differences in NGF-induced sensitization in humans than in rats.    Afferent fibers that express neuropeptides are thought to comprise one subgroup of nociceptors and increased expression of neuropeptides has been associated with pain sensitivity (Neumann et al. 1996; Ohtori et al. 2001). We found increased SP expression following NGF injection in female rats but not in male rats.  As NGF-induced mechanical sensitization could be attenuated by APV in the male but not in the female rats, altered expression of neuropeptides including SP may contribute to the greater sensitization in the female rats.   69  Whether SP alone is responsible for this difference is not clear, since glutamate-induced mechanical sensitization of nociceptors in the temporalis muscles of female rats was not affected by a SP receptor antagonist (Gazerani et al. 2010).  Additional work is needed to elucidate whether one or more neuropeptides are responsible for the observed sex-related differences in NGF-induced mechanical sensitization.  Current evidence suggests that NGF may contribute to the development of chronic muscle pain (Bennett 2001; Chao 2003; Pezet and McMahon 2006). Recent clinical trials have found anti-NGF antibodies to be efficacious in treating several chronic pain conditions (Garber 2011). In this study, a relationship between the expression of peripheral NMDA receptors and the maintenance of NGF-induced mechanical sensitization in the rat masseter muscle has been found.   This finding suggests that chronic pain conditions amenable to treatment with anti-NGF antibodies may also respond to treatments that target the peripheral NMDA receptor.   The relationship between NMDA receptor expression and mechanical sensitization and the finding that both rats and humans have similar baseline expression of the NMDA receptors, suggest the possibility of using the frequency of peripheral NMDA receptor expression as a marker of muscle sensitization. These results encourage further investigation into the potential of peripheral NMDA receptors as biomarkers for myofascial TMD pain as well as the use of this animal model to study the mechanism of chronic muscle pain development.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    70  Chapter 3 Nerve Growth Factor Alters the Sensitivity of Rat Masseter Muscle Mechanoreceptors to NMDA Receptor Activation   3.1. Overview Intramuscular injection of NGF into rat masseter muscle induces a local mechanical sensitization that is greater in female than in male rats.  The duration of NGF-induced sensitization in male and female rats was associated with an increase in peripheral NMDA receptor expression by masseter muscle afferent fibers that began 3 days post injection.  Here we investigated the functional consequences of increased NMDA expression on the response properties of masseter muscle mechanoreceptors.  In vivo extracellular single unit electrophysiological recordings of trigeminal ganglion neurons innervating the masseter muscle were performed in anesthetized rats 3 days after NGF injection (25 µg/ml, 10µl) into the masseter muscle.  Mechanical activation threshold was assessed before and after intramuscular injection of NMDA.  NMDA injection induced mechanical sensitization in both sexes that was increased significantly following NGF injection in the male rats but not in the female rats.  However, in female, but not male rats, further examination found that pre-administration of NGF induced a greater sensitization in slow Aδ fibers (2-7 m/s) than fast Aδ fibers (>7-12 m/s).  This suggests that pre-administration of NGF had a different effect on slowly conducting mechanoreceptors in the female rats compared to the male rats.  Although previous studies have suggested an association between estrogenic tone and NMDA activity, no correlation was observed between NMDA-evoked mechanical sensitization and plasma estrogen level.  This study 71  suggests NGF alters NMDA-induced mechanical sensitization in the peripheral endings of masseter mechanoreceptors in a sexually dimorphic manner.    3.2. Introduction Glutamate is the major excitatory neurotransmitter in the central nervous system (Platt 2007).  Evidence suggests that it may also play a role in peripheral nociception (Carlton et al. 1995; Lam et al. 2005; Haas et al. 2010; Miller et al. 2011).  Glutamate and its receptors are found in the cell bodies of trigeminal and DRG neurons, and at their central and peripheral terminals (Carlton et al. 1995; Cairns et al. 2003b; Li et al. 2004; Lam et al. 2005; Dong et al. 2007; Wong et al. 2014).  Peripheral glutamate levels are elevated during cutaneous or deep tissue inflammation, produced by a variety of non-neuronal cells such as mast cells and macrophages (Newsholme and Calder 1997).  Glutamate may also be released by the peripheral endings of the primary afferent fibers during nociceptive stimulation and acts on the glutamate receptors on the primary afferent fibers themselves (Westlund et al. 1989; deGroot et al. 2000).  This raises the possibility that peripheral glutamate may have an autocrine and/or paracrine role in a positive-feedback enhancement of nociceptor excitability (Lam et al. 2005).   A number of clinical and animal studies have provided behavioral evidence in support of a role for peripheral glutamate receptors in the transduction of masticatory muscle nociceptive signaling.  In particular, glutamate concentrations were significantly higher in the masseter muscles of TMD patients 72  when compared to healthy controls (Castrillon et al. 2010).  Injection into the masseter muscle of healthy subjects induced pain and mechanical sensitization and this response was blocked by a NMDA receptor antagonist (Svennson et al. 2003; Cairns et al. 2006; Castrillon et al. 2012).  Glutamate-evoked pain was also found to be greater in women than men, similar to the sex-related difference in the occurrence of TMD pain (Cairns et al. 2003c).  In rat masseter muscle, baseline interstitial glutamate concentration was found to be ~25 µM and increasing this concentration 2-3 times via systemic injection of monosodium glutamate resulted in a decrease in the mechanical activation threshold of masseter muscle nociceptors (Cairns et al. 2007).  These results suggest that masseter muscle afferent mechanical sensitivity may be regulated, in part, through peripheral NMDA receptors.   The neurotrophin NGF has recently been found to be an important peripheral mediator of nociception.  Elevated levels of NGF have been found in many chronic pain conditions (Anand 1995).  Injection of NGF into the masseter muscle of healthy subjects resulted in a local mechanical sensitization that lasted for 1-3 weeks, with a greater magnitude in women than men (Svensson et al. 2003; Svensson et al. 2008a).  In a previous study, we demonstrated that injection of NGF into the rat masseter muscle induced a prolonged local mechanical sensitization, which was also greater in female rats than male rats (Wong et al. 2014).  The duration of this sensitization was associated with an increased expression of NMDA receptors in masseter muscle afferent fibers, 73  suggesting that NGF-induced sensitization may be maintained by increased peripheral NMDA receptor expression.    In the present study, we examined whether pre-administration of NGF alters the response of masseter muscle afferent fibers to peripheral NMDA receptor activation in rats. We also examined whether this effect exhibits a sex-related difference. The results from this study indicate a significant interaction between NGF and the ability of peripheral NMDA receptors to modulate the mechanical sensitivity of masseter muscle afferent fibers.  3.3. Methods 3.3.1. Animals Male (310–480 g, n=22) and female (230-300 g, n=21) Sprague-Dawley rats were used for the experiments.  Animals were housed in groups of two with a 12-h light/dark cycle.  Food and water were given ad libitum.  All animal procedures were reviewed and approved by the University of British Columbia Animal Care Committee.  3.3.2. Administration of NGF  Intramuscular injection of NGF (25 µg/ml, 10 µl, Sigma) or vehicle (phosphate buffered saline, PBS, 10 µl, Sigma) was injected into the right masseter muscle after the animal was briefly anesthetized with isoflurane (AERrane, Bazter Corporation, Missisauga, ON, Canada; 2-2.5%) and oxygen 74  (97-98%).  The experimenter was blinded to the content of the injections.  The masseter muscle region was shaved prior to injection and the injection site was marked with a permanent marker for subsequent identification.  The concentration of NGF was selected based on the concentration used in previous human experimental pain studies and acute experiments in rats (Svensson et al. 2003; Mann et al. 2006; Svensson et al. 2008a; Svensson et al. 2010; Wong et al. 2014).   Injections of NGF or saline were made 3 days before electrophysiological recording experiments in anesthetized rats.  3.3.3. Surgical preparation    Rats were anesthetized with isoflurane (AErrane, Baxter Corporation, Mississauga, ON, Canada; 2-2.5% in oxygen 97-98%).  Blood pressure was monitored via a cannula inserted into the carotid artery and connected to a pressure transducer.  A trachea tube was inserted to ventilate the animal throughout the experiment and body temperature was maintained at 37.0 ± 0.2 °C with an electric heating pad controlled from a rectal thermometer.  The heart rate and blood pressure were monitored throughout the experiment.  The hair of the right side of the face was shaved before the animal’s head was positioned in a stereotaxic frame.  An incision was made to the skin over the dorsal surface of the skull to expose the skull bone and a small trephination was made in the bone to allow lowering of a microelectrode through the brain into the trigeminal ganglion for recording.  An incision was also made to the skin over the neck to expose the brainstem and the dura was removed to allow a stimulating electrode 75  to contact the brainstem.  Animals were euthanized at the end of the experiments (100 mg/kg; Nembutal, Abbott Laboratories, Chicago, IL, USA).  3.3.4. In vivo electrophysiological recording In vivo extracellular single unit recording was performed 3 days after NGF injection, which was previously found to be the time of peak NGF-induced NMDA receptor upregulation (Wong et al. 2014).  Extracellular action potentials of masseter ganglion neurons were recorded with a parylene-coated tungsten microelectrode (0.10”, 2MΩ, A-M Systems Inc., Carlsborg, WA, USA).  Mechanoreceptors innervating the masseter muscle were identified by their response to mechanical stimulation applied to the muscle with a fine-tipped cotton swab.  The mechanoreceptive field of the afferent fiber was confirmed to be in the muscle if the fiber did not respond to mechanical stimulation of the overlying skin and the jaw but did respond to insertion of a catheter needle (to deliver NMDA) into the masseter muscle.  The skin over the mechanoreceptive field was marked with permanent marker.  The distance between the initial NGF injection site and the mechanoreceptive field of the fiber was measured at the end of the recording session.  76   Figure 3.1: Examples of extracellular electrophysiological recordings. (A) To confirm that the masseter muscle afferent fiber projected to the caudal brainstem, antidromic action potential (*) generated by a stimulating electrode in the caudal brainstem was collided with the orthodromic action potential generated by stimulation of the muscle.  The CV of this masseter muscle fiber from a male rat is 10.5 m/s (B) An example of MT measurement is shown.  An electronic von Frey hair was used to apply force to the masseter muscle (lower trace) until an action potential was generated in the masseter muscle afferent fiber (upper trace).  The arrow indicates the MT for this fiber.  The MT of this masseter muscle fiber from a female rat is 34.5 g.   Antidromic collision was performed to confirm projection of the masseter muscle fiber to the caudal brain stem.  A stimulating electrode (parylene-coated tungsten microelectrode, 0.10”, 2MΩ, A-M Systems Inc., Carlsborg, WA, USA) was lowered into the ipsilateral caudal brainstem and a constant-current electrical stimulus (100 µs biphasic pulse, 10-90 µA, 0.5 Hz) was applied to evoke antidromic action potentials.  Orthodromic action potentials were evoked 77  by mechanical stimulation of the masseter muscle. Collision was demonstrated by disappearance of the antidromic spike (Figure 3.1A).  The straight line distance between the stimulating and recording electrodes was divided by latency of the antidromic action potential to estimate conduction velocity (CV).  Mechanical activation threshold (MT) was assessed with an electronic von Frey hair (model 1601C, Life Science, Woodland Hills, CA, USA). Mechanical stimuli were applied to the receptive field of the fiber at 1 min intervals for 10 min to obtain a baseline threshold (minimum force required to evoke afferent discharge, Figure 3.1B).  Force was increased gradually until a response was observed. After baseline recording, a 26-gauge needle connected to a 25 μl Hamilton syringe (Hamilton, Reno, NV, USA) via a polyethylene tube that contained NMDA was inserted into the mechanoreceptive field of the masseter muscle afferent fiber.  A ten-minute recording period followed to record any baseline spontaneous discharge.  After this baseline measurement, a single injection of NMDA (500 mM, 10 µl, pH 7.4) was administered to assess whether NGF alters response of masseter afferents to chemical stimulation. This was followed by a ten-minute recording period to assess NMDA-evoked discharge.  MT was then reassessed at the end of this period for 10 minutes in the same manner as described for baseline measurement.  It was occasionally possible to record from more than one muscle afferent fiber in the same animal.  This occurred in male rats for the vehicle group (12 afferent fibers from 10 animals) 78  and in female rats in both the vehicle (12 afferent fibers from 11 animals) and NGF group (12 afferent fibers from 10 animals).    3.3.5. Plasma Estrogen Concentration Blood samples were collected and centrifuged for 10 min at 1000xG at room temperature to remove blood cells.  Plasma was stored at - 20°C until analysis.  17β-estradiol concentration (pg/ml) was measured with an enzyme-linked immunosorbent assay (ELISA) kit (40-056-205004, Genway).  Samples were assayed in duplicate.  3.3.6. Data analysis A Students t-test sample size estimation suggested that 12 afferent fibers per group would allow the detection of a 50% difference in mechanical activation threshold between the vehicle and NGF treatment groups after NMDA injection (α=0.05, power=0.80).  Spearman’s correlation was used to determine the relationship between CV and MT.  MTs in the same animal before and after NMDA injection were compared using a paired t-test.  Relative mechanical activation threshold (Rel MT) was calculated as Post-NMDA MT/Baseline MT x 100.  Mechanical activation thresholds and Rel MT s were compared between the vehicle and NGF treatment groups by using a Students t-test.  Two-way ANOVA on Rel MT with treatment and conduction velocity as factors was performed on fast and slow Aδ fibers with post hoc Holm-Sidak multiple comparisons.  Differences in the proportion of afferent fibers with spontaneous 79  discharges before and after NMDA injection were compared between NGF and control treatment by using the Fisher’s exact test.  The frequency of discharge (spikes/min) after NGF and control treatments were compared the Mann-Whitney rank sum test. A probability level of less than 0.05 was considered significant for all tests. Error bars represent SEM.  3.4. Results 3.4.1. Mechanical activation threshold Before NMDA injection, no significant difference was observed in the mean baseline mechanical activation thresholds between the vehicle (n=12) and NGF groups (n=12) in male (vehicle, 30.5 ± 9.3 g; NGF, 32.3 ± 8.2 g) or female rats (vehicle, 34.2 ± 9.6g; NGF, 41.9 ± 11.1 g).  The baseline mechanical activation thresholds were plotted against conduction velocities of the afferent fibers (Figure 3.2).  A significant inverse correlation between conduction velocity and mechanical activation threshold was observed in the vehicle group of the male rats (coefficient = -0.776) but not in the female rats, which is consistent with a previous study (Mann et al. 2006).  However, a significant inverse correlation (coefficient = -0.685) between conduction velocity and mechanical activation threshold was found in the NGF-treated female rats.  In Figure 3.2, the lines represent masseter mechanical withdrawal thresholds determined from an earlier behavioral study in male and female rats at 3 days after intramuscular injections of vehicle or NGF into the masseter muscle (Wong et al. 2014).  The injection procedure (vehicle, NGF concentration and injection volume) in that study was 80  identical to the one in this study. The electronic von Frey hair used to determine the mechanical withdrawal thresholds was also from the same manufacturer (IITC) as the one employed here.  Afferent fibers with mechanical activation thresholds above the respective line (vehicle or NGF treatment) were considered to be putative mechano-nociceptors.  The number of masseter afferent fibers with a mechanical activation threshold above the withdrawal threshold was found to be 3/12 in the vehicle group and 5/12 in the NGF group in the male rats, while a bigger difference was found between the vehicle (3/12) and NGF groups (7/12) in the female rats.  None of the differences reached statistical significance (Fisher’s exact test, p>0.05). 81   Figure 3.2: The scatter plot shows the relationship between baseline MT and CV of masseter mechanoreceptors (n = 12/treatment group) three days after NGF or vehicle injection in male (A) and female (B) rats. A significant inverse correlation was observed in the vehicle group of the male rats (Spearman’s correlation coefficient = -0.776)) but not in the female rats.  A significant inverse correlation was found after NGF injection in the female rats (Spearman’s correlation coefficient = -0.685).  The dotted lines represent behavioral mechanical thresholds at 3 days after intramuscular vehicle injection into the masseter muscle as determined from an earlier study (male = 38.1g; female = 30.2g) while the solid lines represent behavioral mechanical thresholds at 3 days after NGF injection (male = 34.5g; female = 26.4g) (Wong et al. 2014). 82  NMDA injection significantly reduced mechanical activation threshold in all treatment groups when compared to the baseline MT in male (vehicle, 25.4 ± 8g; NGF, 21 ± 7.1 g) and female rats (vehicle, 27.7 ± 7.9 g; NGF, 30.7 ± 10.3 g).  In the male rats, NMDA-induced mechanical sensitization was significantly greater in the NGF group than the vehicle group (Figure 3.3 A & B).  In female rats, NMDA-induced mechanical sensitization was increased in the NGF group, however, the difference was not significant (p>0.05).  The relationship between CV and Rel MT is presented in Figure 3.4 A & B.  The scatter plot shows that in female rats, NGF appeared to enhance NMDA-induced mechanical sensitization primarily for Aδ fibers with slower conduction velocities.  Further analysis was carried out by separating the two populations based on conduction velocity (Figure 3.5 A & B).  They were separated at the midpoint of the Aδ fiber’s conduction velocity range into slow Aδ fiber (2-7 m/s) and fast Aδ fiber (>7-12 m/s) groups .  Two-way ANOVA on Rel MT with treatment and conduction velocity as factors was performed with post hoc Holm-Sidak multiple comparisons.  In the male rats, there was a significant effect of treatment, but no significant effect of conduction velocity or interaction between treatment and conduction velocity.  Post hoc testing indicated a significant difference between vehicle and NGF in the fast conduction velocity group.   In the female rats, there was a significant interaction between treatment and conduction velocity, but there was no significant effect of treatment or conduction velocity.  Post hoc testing indicated a significant difference between slow and fast conduction velocity groups in the NGF groups. 83    Figure 3.3: Relative mean mechanical activation threshold (Rel MT) of masseter mechanoreceptors (n = 12), 10 min after intramuscular NMDA injection in male (A) and female (B) rats. Rats were treated with NGF or vehicle three days prior to NMDA injection.  Rel MT = Post injection MT/Baseline MT x 100. *:  p<0.05, Students t-test.      84                A Male                         B Female   Figure 3.4: The scatter plot shows the relationship between Rel MT and CV of masseter mechanoreceptors in male (A) and female (B) rats. Rats were treated with NGF or vehicle three days prior to NMDA injection.    85                                  A Male                                  B Female  Figure 3.5: Comparison between the Rel MT of slow Aδ fibers (CV: 2-7 m/s) and fast Aδ fibers (CV: >7-12 m/s) after vehicle or NGF treatment in male (A) and female (B) rats. Only Aδ fibers (CV: 2-12 m/s) were included in the analysis. Male (slow: vehicle n=3; NGF n=4, fast: vehicle n=5; NGF n=7). Female (slow: vehicle n=5; NGF n=6, fast: vehicle n=5; NGF n=5). *: p< 0.05 two-way ANOVA and post hoc Holm-Sidak multiple comparisons test.   Plasma estrogen concentration of the female rats was determined since systemic estrogen has been shown to modulate the sensitivity of masseter 86  afferent fibers to NMDA (Dong et al. 2007).  The plasma concentration of estrogen was not different between the vehicle (63.3 ± 14.5 pg/ml) and the NGF (70.8 ± 20 pg/ml) groups (Students t-test, p>0.05).  No significant correlation was observed between plasma estrogen concentration and baseline MT or Rel MT (Figure 3.6).    Figure 3.6: The scatter plots show the relationship between plasma estrogen concentration and baseline MT (A) or Rel MT (B) of masseter mechanoreceptors 87  at 10 min following NMDA injection in female rats.  No significant correlations were identified (Spearman’s correlation coefficient, p>0.05).    3.4.2. Spontaneous and NMDA-evoked discharge The number of spontaneous discharges and NMDA-evoked discharges were evaluated over a ten-minute period before and after NMDA injection.  No difference in the proportion of afferent fibers with spontaneous discharges before and after NMDA injection was found between the vehicle and NGF treatment groups in either sex (Table 3.1).  NGF treatment also had no effect on the frequency of spontaneous discharges before and after NMDA injection (Table 3.2). Table 3.1: The proportion of masseter afferent fibers with spontaneous discharge before and after NMDA injection.  No significant difference was observed between vehicle and NGF groups before and after NMDA injection in either sexes by Fisher’s exact test (p > 0.05).  Table 3.2: The mean frequency of  spontaneous discharge (spikes/min) in masseter afferent fibers before and after NMDA injection. The rate of spontaneous discharge was calculated from a ten-minute period before or after NMDA injection. No significant difference was observed between vehicle and NGF groups before and after NMDA injection in either sexes by Mann Whitney rank sum test (p > 0.05).  88  3.4.3. Distance Since the masseter fibers were randomly identified through mechanical stimulation of the masseter muscle, one possible confounding factor could be the distance between the initial NGF injection and the mechanoreceptive field of the recorded fiber (Figure 3.7).  To determine whether this distance affected the results, the distance between the initial NGF injection and the subsequent NMDA injection for the NGF treatment group is plotted against Baseline MT and Rel MT (Figure 3.8).  No correlation was observed, demonstrating that the distance between NGF injection and the mechanoreceptive field of the fiber did not affect the subsequent mechanical sensitization.      Figure 3.7: The picture illustrates an example of the distance between the initial NGF injection site (X) and the subsequent NMDA injection site (■) in the rat masseter muscle.            89                           A Baseline MT                              B Rel MT    Figure 3.8: The scatter plots show the relationship between this distance (mm) and the baseline MT (A) or Rel MT (B) of masseter mechanoreceptors at 10 min following NMDA injection in male and female rats.  No significant correlations were identified (Spearman’s correlation coefficient, p>0.05).90  3.5. Discussion  NGF is an important mediator of pain and elevated levels have been found in many chronic pain conditions (Anand 1995).  Intramuscular injection of NGF into the masseter muscle induced a long lasting local mechanical sensitization in humans, which is mimicked by equivalent injections into the rat masseter muscles, suggesting that this could be a valuable animal model for investigating the mechanism of NGF-induced muscle mechanical sensitization (Svensson et al. 2003; Svensson et al. 2008a; Wong et al. 2014).  In rats, NGF-induced masseter muscle mechanical sensitization was associated with an increased expression of NR2B subunit containing NMDA receptors in masseter ganglion neurons in both male and female rats and was partly attenuated by an intramuscular injection of the NMDA receptor antagonist, APV, three days after NGF injection, in the male rats (Wong et al. 2014).  In the present study, we examined whether NGF alters the response of individual masseter mechanoreceptors to peripheral NMDA receptor activation.  NGF increased the level of mechanical sensitization in masseter muscle afferent fibers after local injection of NMDA into the masseter muscle, suggesting the effect of NGF occurs in the peripheral endings of muscle afferents.  Taken together, these results suggest that a peripheral mechanism whereby NGF induces local mechanical sensitization by increasing the expression of NMDA receptors at the peripheral endings of masseter mechanoreceptors, underlies the sensitization produced by NGF injections in rats at 3 days after NGF injection.   91   Intramuscular injection of NMDA has been shown to evoke discharges in masseter muscle mechanoreceptors and increasing the expression of NMDA receptors by estrogen treatment has been shown to increase the magnitude of this discharge (Dong et al. 2007; McRoberts et al. 2007).  Since we previously found NGF increases expression of NMDA receptors in masseter muscle ganglion neurons, we would predict that a greater number of NMDA receptors would result in an increase in NMDA-evoked discharges (Wong et al. 2014).  However, that was not the case, as NGF had no effect on the proportion of fibers with discharges or the frequency of the discharges after NMDA injection.  Interestingly, this result agrees with an earlier human pain experiment where pre-injection of NGF had no effect on the intensity of glutamate-evoked masseter muscle pain in healthy humans (Svensson et al. 2008b).  Further, intramuscular injections of NGF into the masseter muscles of healthy men and women resulted in a prolonged (7-14 day) localized mechanical sensitization without reports of spontaneous pain (Svensson et al. 2003; Svensson et al. 2008a).  Here we found that NGF increased NMDA-induced mechanical sensitization but had no effect on the frequency of spontaneous discharges.  These results suggest that NGF does not cause spontaneous pain but may modulate mechanical sensitivity in putative masseter nociceptors by increasing the expression of peripheral NMDA receptors.    92  Previous human and rat behavioral experiments found that NGF reduced mechanical thresholds after intramuscular injection into the masseter muscle (Svensson et al. 2003; Svensson et al. 2008a; Wong et al. 2014).  In this study, however, the mean mechanical threshold of masseter afferent fibers did not change after NGF injection.  This may be due to the sample size not being large enough to demonstrate this effect since single fibers were randomly identified, encompassing a large range in mechanical thresholds.  In the vehicle-injected groups, subsequent NMDA injections induced a decrease in mechanical threshold that was comparable to glutamate-induced mechanical sensitization observed in previous studies (Cairns et al. 2002a; Cairns et al. 2003b).  No difference was found between the male and female rats, which is also consistent with mechanical sensitization induced by glutamate (Cairns et al. 2002a).  NMDA induced a greater mechanical sensitization in NGF-treated masseter muscles than the vehicle-treated group in the male rats.  This is in contrast to an earlier human pain study where pre-injection with NGF did not induce greater glutamate-induced mechanical sensitization of the masseter muscle (Svensson et al. 2008b).  However, in that study glutamate was given one day after NGF treatment compared to three days later in the current study.  This may be significant since a prior study in the rat found that significant increases in NMDA receptor expression began 3 days after NGF injection (Wong et al. 2014).  Our results suggest that NGF increased NMDA-evoked mechanical sensitization by increasing the expression of NMDA receptors in the peripheral endings of masseter nociceptors.  However, we cannot exclude the possibility that NGF may 93  also altered the functional properties of NMDA receptors as this has been demonstrated in previous studies performed in cultured neurons (Bai and Kusiak 1997; Jarvis et al. 1997; Di-Luca et al. 2001).  In this study, a sex-related difference was observed in the level of NMDA-evoked mechanical sensitization following NGF injection, with the male rats exhibiting a greater decrease in afferent mechanical threshold than the females.  Further examination found that NGF induced a greater NMDA-induced mechanical sensitization in the slow A fibers than fast A fibers in the female rats, but not male rats.  This result suggests that NGF may have a different sensitizing effect on putative mechano-nociceptors in the female rats compared to the male rats.  This effect was not related to plasma concentrations of estrogen as no correlation was observed between plasma estrogen concentration and the level of sensitization, although previously it has been found that estrogen may increase masseter muscle sensitivity to NMDA by increasing peripheral NMDA receptor expression (Dong et al. 2007; McRoberts et al. 2007).  Sex-related differences in masseter muscle nociception have been documented previously in human and animal experiments.  For example, healthy women exhibited greater sensitization following NGF injection into the masseter muscle than healthy men (Svensson et al. 2003; Svensson et al. 2008a).  A higher dose of ketamine, an NMDA receptor antagonist, was required to attenuate glutamate-evoked masseter pain in women than men, suggesting a similar sex-related difference in peripheral NMDA receptor expression (Cairns et al. 2006; Castrillon 94  et al. 2012).  We previously found that NGF induced a greater mechanical sensitization in female rats than in male rats, which was associated with a greater increase in NMDA receptor expression by masseter ganglion neurons in the female rats (Wong et al. 2014).  These results suggest there is a marked difference in the effect of NGF on peripheral NMDA receptor expression between the sexes.     While our results suggest that NGF induces muscle mechanical sensitization via increasing the expression of NMDA receptors in the peripheral endings of masseter mechanoreceptors, other studies suggest that presynaptic NMDA receptors on the central terminals of primary afferents may also induce hyperexcitability during injury via facilitation of the release of glutamate and substance P in the dorsal horn (Liu et al. 1997; Yan et al. 2013).  However, conflicting results have been observed where intrathecal NMDA did not evoke SP release in one in vivo study (Nazarian et al. 2008) and NMDA was found to decrease excitatory postsynaptic potentials (EPSPs) recorded in spinal cord slices in another (Bardoni et al. 2004).  The interpretation of these results is not straightforward as activation of presynaptic glutamate receptors may shunt the action potential propagation and reduce neurotransmitter release, a mechanism similar to the proposed role of type A gamma-aminobutyric acid (GABAA) receptors in primary afferent depolarization (Bardoni 2013). Further experiments are needed to determine what the consequence of increasing central presynaptic NMDA receptors would be on synaptic transmission. 95  It is also likely that sex-related differences in NGF-induced mechanical sensitization of the masseter result from a mechanism other than increased peripheral NMDA receptor expression.  For example, we previously reported that intramuscular injection of NGF increased the expression of the neuropeptides SP and CGRP by masseter ganglion neurons that co-expressed NMDA receptors, but only in female rats (Wong et al. 2014).  However, peripheral NMDA receptor induced mechanical sensitization of masticatory muscle afferent fibers in female rats is not inhibited by local injections of antagonists for SP or CGRP receptors (Gazerani et al. 2010). The greater expression of neuropeptides by masseter muscle afferent fibers in female rats after NGF treatment could result in an increased central release of SP/CGRP, which may increase the excitability of neurons in the trigeminal subnucleus caudalis (Coste et al. 2008; Meng et al. 2009).  This central mechanism may contribute more to NGF-induced mechanical sensitization in female rats than in male rats (Latremoliere and Woolf 2009).   Thus, a combination of peripheral and central sensitization may explain the observed sex-related difference in the effect of intramuscularly injected NGF on masseter muscle mechanical threshold.   In this study we found NGF increases the mechanical sensitivity of masseter muscle nociceptors to NMDA receptor activation.  These results suggest that NGF, an important mediator of pain, may induce mechanical allodynia, in part, by increasing the expression of NMDA receptors in the peripheral endings of muscle nociceptors.  In an earlier study, NGF increased the 96  median soma size of masseter ganglion neurons expressing NMDA receptors, which suggests that peripheral NMDA receptors may be part of the phenotypic change that occurs in muscle nociceptors during injury and inflammation (Wong et al. 2014).  These results may be relevant to humans, as NMDA receptors have been found in human masseter muscle afferent fibers (Wong et al. 2014).  Our results merit further study into the role of peripheral NMDA receptors in craniofacial muscle pain development. 97  Chapter 4 Conclusion 4.1. Peripheral NMDA Receptors in NGF-Induced Mechanical Sensitization The overall objective of this study was to determine whether peripheral NMDA receptors contribute to the development of NGF-induced sensitization in masseter muscles.  To answer this question, a multifaceted approach was followed: 1) behavorial studies were performed to establish whether NGF induces a prolonged sensitization and whether it can be blocked by the local administration of a NMDA antagonist.  2) Immunohistochemical studies were performed to assess expression of NMDA receptor in trigeminal ganglion neurons innervating the masseter muscle and whether increased expression is associated with the observed NGF-induced sensitization behaviorally.  3) In vivo electrophysiological recording was performed to investigate the effect of NGF on the properties of individual masseter afferents and whether NGF changes their response to a NMDA receptor agonist.  Finally, experiments were completed in male and female rats and to assess for any sex-related differences.  My results suggested that NGF-induced mechanical sensitization might be mediated, in part, through an effect of peripheral NMDA receptors on peripheral mechanisms of pain perception.  In my experiments, NGF induced a prolonged local mechanical sensitization in rats, which was associated with an increase in NMDA receptor expression in masseter ganglion neurons.  A phenotypic change also occurred with an increase in the soma size of masseter ganglion neurons expressing the 98  NR2B subunit in both sexes.  In the male rats, local administration of APV partly attenuated the NGF-induced sensitization.  Single fiber recording also demonstrated that NGF increased NMDA-induced mechanical sensitization in masseter mechanoreceptors.  These results showed that in the male rats, NGF induced local mechanical sensitization in the muscle, in part, through increasing the expression in NMDA receptors in the peripheral endings of muscle afferent fibers.  This peripheral mechanism could result in sensitization of the afferent fibers by endogenous glutamate present in the muscle.  In the female rats, however, differences were observed suggesting peripheral and central mechanisms may be involved.  NGF induced mechanical sensitization was longer lasting in the female rats.  An increase in neuropeptide expression (CGRP/SP) accompanied NMDA receptor upregulation in masseter ganglion neurons in the female rats but not in the male rats.  In contrast to the male rats, APV did not attenuate NGF-induced sensitization in the female rats.  Single fiber recording also revealed no effects by NGF on NMDA-induced mechanical sensitization in female masseter muscle afferent fibres; however; further analysis found NGF had a preferential effect on mechano-nociceptors with slow conduction velocity; an effect not observed in the male rats.  These results suggest that the greater effect of NGF in the female rats may be due to the preferential effect of NGF on mechano-nociceptors, mediated by an increased expression of NMDA receptors in their peripheral endings.  In my immunohistochemical studies, I measured the frequency of neurons expressing 99  the NMDA receptors, which did not allow for the quantification of the amount of increase in expression within individual neurons.  Therefore, it is possible that there is a variation in the NMDA receptor upregulation among different fiber populations.  This process could be enhanced by an increase in central input by masseter muscle afferent fibers accompanied by an increased release of neuropeptides in their central endings.  My results suggest peripheral and central mechanisms may be involved in the sex-related difference in NGF-induced sensitization.   4.1.1. Alternate explanations An alternate explanation for the NGF-induced sensitization is that increased expression of presynaptic NMDA receptor in the central terminals facilitates the release of glutamate and SP, as discussed in Chapter 3.  However, this proposed mechanism has been controversial due to inconsistent results and more research is needed to investigate whether this occurs following NGF injection.  For example, neurokinin 1 (NK1) receptor internalization can be used to determine whether NGF increases SP release in the dorsal horn (Liu et al. 1997).  Glutamate release from the central terminals of muscle afferent fibers can be assessed by measuring excitatory postsynaptic currents (EPSCs) in the pair-pulse protocol in in vitro slice preparations (Yan et al. 2013).  These experiments would help to determine whether NGF has an effect on neurotransmitter release in the central endings of masseter muscle afferents.  100  Another possibility is that NGF may have an effect through increasing the functional properties of NMDA receptors in the peripheral endings of masseter afferents.  In one experiment, intramuscular injection of NGF induced mechanical sensitization in masseter mechanoreceptors starting at thirty minutes after injection (Svensson et al. 2010).  In my behavioral experiments, NGF induced sensitization began three hours after injection but expression of NMDA receptors did not increase until three days following injection.  Previous in vitro cell culture studies also found that retrograde transport of NGF does not begin until one hour after NGF application (Ure and Campenot 1997).  Hence other mechanisms may be involved in the rapid effects of NGF.  For example, NGF has been found to rapidly increase NMDA receptor mediated currents in rat hippocampal neurons (Jarvis et al. 1997; Kovalchuk et al. 2004).  Also, activation of trkA receptor by NGF in spinal neurons resulted in phosphorylation of tyrosine residues of NR2B subunits (Di-Luca et al. 2001).  Tyrosine phosphorylation has been shown to increase NMDA receptor mediated currents (Yu et al. 1997).  Effects on the functional properties of peripheral NMDA receptors may mediate, in part, the early phase of the NGF-induced sensitization.  To determine whether NGF increases peripheral NMDA receptor phosphorylation, experiments using Western blot analysis could be carried out to compare the relative levels of phosphorylated NR2B subunits in vehicle and NGF-injected masseter muscles.   In addition to the peripheral NMDA receptors, NGF may have effects on other peripheral targets.  For example, it has been speculated that NGF may 101  facilitate the sprouting of sympathetic fibers in the skin, which has been observed in some inflammatory and neuropathic models in the rat (Osikowicz et al. 2013).  Interaction between sympathetic and sensory afferents has been proposed to contribute to sensory abnormalities including pain after nerve injury (Bongenhielm et al. 1999).  Sprouting of sympathetic baskets in the DRG has been reported after spinal nerve injury (Chung et al. 1997); however, the same did not occur in the trigeminal ganglion, suggesting potential anatomical differences between trigeminal and dorsal root ganglia (Bongenhielm et al. 1999; Benoliel et al. 2001).  Further studies are needed to investigate whether sympathetic sprouting plays a part in craniofacial muscles following NGF injection.   As mentioned in Chapter 2, NGF may also have effects on other peripheral receptors.  In particular, NGF has been shown to potentiate the responses of nociceptive sensory neurons to capsaicin (Shu and Mendell 2001; Chao et al. 2006).  Co-localization of trkA and TRPV1 channels, the receptor activated by capsacin as well as by heat and low pH, has been reported previously (Chao 2003).  In addition, NGF induced mechanical hyperalgesia in lateral gastrocnemius muscle in wild type mice but not in TRPV1-knockout mice (Ota et al. 2013).  In another study performed in the rats, a TRPV1 antagonist partially attenuated NGF-induced heat and mechanical hyperalgesia after NGF injection into the paws (Mills et al. 2013).  These results suggest that TRPV1 102  receptors may also be a part of the downstream mechanism in NGF-induced sensitization.    4.2. Animal model of myofascial pain in TMD The limitations of using animal models as proxies for human clinical pain have been debated in the literature (Langley et al. 2008; Mogil et al. 2010).  Pain is a subjective phenomenon that involves multiple areas of the nervous system (Craig et al. 1995; Wall 1995).  Since we might never be able to know how an animal feels, we could only infer pain based on behaviors in any animal models (Mogil et al. 2010).  Ideally, the pain-like behaviors will mimic human pain behaviors.  In this study, I investigated whether intramuscular injections of NGF into the rat masseter muscle could reproduce the effect of NGF observed in human pain experiments, which mimics the myofascial pain found in TMD patients.  My results showed that intramuscular injection of NGF into the rat masseter muscle induces a mechanical sensitization that has similar features to NGF-induced mechanical sensitization of the masseter muscle in humans.  First, it induces a prolonged reduction in masseter muscle mechanical threshold, which is also found in human pain experiments.  A reduction in mechanical threshold is a characteristic seen in TMD patients with myofascial pain (Gomes et al. 2008; Cairns 2010; Basi et al. 2012).  Secondly, no overt spontaneous pain-like behaviors or corresponding increase in spontaneous discharges from masseter muscle nociceptors was observed after NGF injection in behavioral and electrophysiological experiments, respectively.  This corresponds to the fact that 103  in human pain studies, subjects did not report spontaneous pain after NGF injection.  Pain was only reported during strenuous activities such as chewing and yawning, which parallels symptoms of myofascial pain in TMD patients (Svensson et al. 2003).  Thirdly, the same sex-related difference in human NGF experiments was observed, with NGF having a greater behavioral effect in female rats than in male rats.  This result may be translatable to TMD as a greater incidence of this condition is observed in women than men.  Taken together, these results suggest that intramuscular injection of NGF into rat masseter muscle maybe used as a proxy of NGF-induced mechanical sensitization in human and may shed light on the mechanism of myofascial pain in TMD patients.  Although elevated levels of NGF have been associated with many chronic pain conditions and antibodies against NGF have been demonstrated to be efficacious in treating certain pain conditions, the association between NGF and TMD has yet to be demonstrated (Anand 1995; Lane et al. 2010; Katz et al. 2011).  In a recent study, the NGF level in the masseter muscle in TMD patients with TMJ and masseter muscle myofascial pain was measured (Basi et al. 2012).  No significant correlation between NGF level and ongoing pain intensity measured with the visual analogue scale was observed.  This analysis may be flawed, as NGF injections have been shown previously to not induce spontaneous pain in human pain studies (Svensson et al. 2003; Svensson et al. 2008a).  Additionally, no significant difference in NGF level in the masseter 104  muscle was observed between painful TMD patients and pain-free control group.  However, this result is complicated by the inclusion of pain-free TMD patients with disc displacement in the control group in conjunction with non-TMD patients.  Since NGF is known to be released upon tissue injury and trauma, this may be a confounding factor in the analysis (Pezet and McMahon 2006; Watson et al. 2008).  More research is needed to elucidate the relationship between the level of NGF and the presence of myofascial pain in the masseter muscle of TMD patients.  4.3 Other potential limitations   Isoflurane, an inhalation anesthetic, was used to anesthetize the animals to allow for the administration of NGF and APV in the behavioral experiments and to permit the in vivo electrophysiological recordings.  Inhalation anesthetics induce loss of consciousness, with additional actions including analgesia, amnesia and muscle relaxation (Chau 2010).  The exact mechanism of the inhalation anesthetics has yet to be determined, however, NMDA receptors have been proposed as a potential site of action (Chau 2010; Torri 2010).  Therefore, the effect of isoflurane on peripheral NMDA receptors could be a confounding factor in this study and may result in an increase in the muscle mechanical activation threshold measured in the behavioral and electrophysiological studies.  Parallel vehicle control groups and blinding were applied to minimize this potential confounder in these studies.  105   The specificity of the antibody to the NR2B subunit is critical to the immunohistochemical experiments in this study.  To confirm this, we performed removal of the primary antibody as described in Chapter 2.  In the literature, specific binding of this antibody (BD Transduction Laboratories) to recombinant NR2B subunit has been demonstrated (Robinson et al. 2005).    4.4. Future studies My studies suggest that NGF can decrease the mechanical activation threshold of masseter mechano-nociceptors by increasing the expression of NMDA receptors in peripheral endings.  Since it has been found that primary afferent fibers may also release glutamate upon stimulation, lowering the activation threshold may result in a positive feedback cycle of glutamate-NMDA receptor sensitization in masseter muscle nociceptors (Kirk 1974; Carlton 2001; Lam et al. 2005).  Furthermore, NGF has been shown to induce glutamate release from cultured neurons and trigeminal satellite glial cells (Numakawa et al. 2003; Raiteri et al. 2003; Laursen et al. 2014).  Therefore, it would be of interest to determine whether intramuscular injection of NGF could result in a prolonged increase in interstitial concentration of glutamate in the masseter muscle.  Microdialysis could be used to collect interstitial fluids from the muscle and glutamate concentration can be quantified with ELIZA kit.  Additionally, I have speculated that NGF may have a preferential effect on peptidergic sensory fibers in the female rats; therefore, it would follow to determine whether neuropeptide release is enhanced in the central and peripheral endings of muscle afferents 106  after NGF treatment.  NK1 receptor internalization can be used to measure SP release in the dorsal horn while neuropeptides in the muscle can be measured in conjunction with glutamate concentration using microdialysis as described above.   Functional effects of neuropeptides can also be evaluated by measuring blood flow in the muscle using a Doppler monitor and changes in temperature can be measured with a thermal probe.  Extracellular single unit electrophysiological recordings on second-order neurons in the trigeminal spinal nucleus caudalis can also be performed to determine whether central sensitization occurs after NGF injection, especially in the female rats.  For example, the mechanical threshold, frequency of spontaneous discharges and responses after mechanical stimulation can be measured and compared between animals treated with intramuscular injections of NGF or vehicle into the masseter muscle.  Antagonists to NMDA receptor (e.g. APV, ketamine) and neuropeptides (e.g. NK1 receptor antagonist) can also be used intrathecally to assess whether this sensitization is mediated by these receptors.  This thesis project is based on human pain experiments where injection of NGF into the masseter muscle induces local mechanical sensitization that mimics the myofascial pain of TMD patients.  The next step would be to assess whether the results from this study can be translated back to human subjects and especially, TMD patients with myofascial pain.  In particular I have found 107  evidence suggesting that NMDA receptors and SP are expressed in peripheral nerves innervating the masseter muscle of healthy individuals with similar frequency of expression as in the rats.  We recognized that the small sample size and large variability in the data make it difficult to draw firm conclusions including comparison between sexes, therefore, it would be beneficial to expand the sample size.    Masseter muscle tissue samples from TMD patients with myofascial pain could also be collected and analyzed for comparison with baseline levels in healthy subjects.  The intensity of myofascial pain and pressure pain threshold (PPT) in the muscle can be evaluated to determine if there is any correlation with peripheral NMDA receptor expression in masseter afferents.  Efficacy studies can also be performed to investigate whether APV is effective in alleviating masseter muscle pain in TMD patients.  Previous studies have found ketamine to be efficacious in reducing glutamate-induced pain in the masseter muscle in healthy subjects (Cairns et al. 2006; Castrillon et al. 2012).  In one study performed in TMD patients, ketamine was not reported to be effective overall for myofascial TMD, but did improve pain by >50% in more than half of the patients (Siegel and Sapru 2010).  However, ketamine crosses the blood-brain barrier and it would be interesting to see whether a peripheral restricted NMDA antagonist such as APV can reduce pain in TMD patients.  The results from this study would be important in determining whether peripheral NMDA receptors play a role in the development of myofascial pain in TMD.  108  4.5. Biomarkers of pain One limitation in current pain management is the lack of ability to quantify and measure pain in an objective manner (Chang 2013).  Patients suffering from painful conditions are often asked to assess the level of their pain using subjective rating scales (Chang 2013).  However, these subjective ratings are variable because pain perception is highly individualized and can be influenced by many physiological and psychosocial factors (Mogil et al. 2010; Chang 2013; DeVon et al. 2014).  For example, studies have shown that pain and sensory perception can be affected by circadian cycle (Odrcich et al. 2006).  Additionally, some patients may lack the ability to verbalize the presence of pain due to aphasia or altered mentation (DeVon et al. 2014).  Because of these reasons, an objective and quantifiable pain measurement or biomarker would be beneficial to the diagnosis of pain, assessment of pain severity and the evaluation of efficacy of treatments (Marchi et al. 2009; Chang 2013).   Human neuroimaging techniques such as positron emission tomography (PET), proton magnetic resonance spectroscopy (MRS) and functional magnetic resonance imaging (fMRI) have been investigated as neuroimaging biomarker for chronic pain (Chang 2013).  However, this approach is limited by the specificity and sensitivity of these methods and current technologies are unable to distinguish the precise location of pain function in the brain (Mogil et al. 2010).  This is problematic as it is recognized that pain experience is highly complex and can involve many components including affective and cognitive factors (Wall 109  1995).  Another potential limitation is that most neuroimaging techniques measure indirect proxies of neuronal activity, for example, fMRI measure the blood oxygen level in the brain.    Pro-inflammatory substances such as tumor necrosis factor alpha (TNF) and cytokines have also been investigated as peripheral markers of pain (Marchi et al. 2009; DeVon et al. 2014).  However, the results have been variable which may be due to the differences among study design, variations in symptoms and the fact that although these substances are important in the development of inflammation, many of them do not induce pain directly (Marchi et al. 2009; DeVon et al. 2014).  Further studies are needed to find appropriate biomarkers of pain.  In this study, the expression of NMDA receptors in primary afferent neurons was found to be a part of the mechanism of NGF induced mechanical sensitization of the masseter muscle, highlighting the potential of using the frequency of expression of this receptor in peripheral fibers as a biomarker for the myofascial pain in TMD as well as in other musculoskeletal pain conditions where elevated tissue levels of NGF have been reported.   4.6. Significance A growing body of data has suggested that NGF may play a significant role in chronic muscle pain development; however, its downstream mechanism 110  remains unclear.  The results from this study may help us gain new insights into the neurobiological mechanism of NGF-induced muscle pain and may lead to potential new targets for the treatment of chronic muscle pain conditions, such as TMD and chronic low back pain.  These patients suffer from significant pain and impairment that does not respond well to existing pharmaceutical agents.  Chronic muscle pain affects a significant segment of the population, for example, an estimated 10% of Americans currently suffer symptoms of TMD.  Recent clinical trials have found anti-NGF antibodies to be efficacious in treating osteoarthritic pain and chronic low back pain (Cattaneo 2010).  However, several studies have been suspended due to cases of joint damage (Garber 2011).  This is not surprising because of the diverse range of functions of neurotrophins.  Our results suggested that NGF induces mechanical sensitization in part through a mechanism that involves peripheral NMDA receptor activation, supporting further investigations into the potential of this receptor as a target for analgesia. This might be an attractive strategy since a more selective blockade of a downstream target of NGF might result in fewer side effects than the general blockade by the anti-NGF antibodies.   111  References  Abe, T., S. Matsumura, et al. (2005). "Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain." Eur J Neurosci 22: 1445-1454.   Alfredson, H., S. Forsgren, et al. (2001). "Glutamate NMDAR1 receptors localised to nerves in human Achilles tendons. Implications for treatment?" Knee Surg Sports Traumatol Arthrosc 9: 123-126.   Alfredson, H. and R. Lorentzon (2002). "Chronic tendon pain: no signs of chemical inflammation but high concentrations of the neurotransmitter glutamate. Implications for treatment?" Curr Drug Targets 3: 43-54.   Ali, A. M. and M. M. Sharawy (1994). "Histopathological changes in rabbit craniomandibular joint associated with experimentally induced anterior disk displacement (ADD)." J Oral Pathol Med 23: 364-374.   Almarza, A. J., C. K. Hagandora, et al. (2011). "Animal models of temporomandibular joint disorders: implications for tissue engineering approaches." Annals of Biomedical Engineering 39: 2479-2490.   Anand, P. (1995). "Nerve growth factor regulates nociception in human health and disease." British Journal of Anaesthesia 75: 201-208.   Apfel, S. C., J. C. Arezzo, et al. (1994). "Nerve growth factor administration protects against experimental diabetic sensory neurography." Brain Res 634: 7-12.   Apfel, S. C., J. A. Kessler, et al. (1998). "Recombinant human nerve growth factor in the treatment of diabetic polyneurography, NGF study group." Neurology 51: 695-702.   Apfel, S. C., S. Schwartz, et al. (2000). "Efficacy and safety of recombinant human nerve growth factor in patients with diabetic polyneurography: A randomized controlled trial. rhNGF Clinical Investigator Group." JAMA 284: 2215-2221.   Asaki, S., M. Sekikawa, et al. (2006). "Sensory innervation of temporomandibular joint disk." J Orthop Surg (Hong Kong) 14: 3-8.   Averill, S., S. B. McMahon, et al. (1995). "Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons." Eur J Neurosci 7: 1484-1494.   112  Bai, G. and J. W. Kusiak (1997). "Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells." J Biol Chem 272: 5936-5942.   Bal-Kucuk, B., S. Tolunay-Kaya, et al. (2014). "Pharmacotherapeutic agents used in temporomandibular disorders." Oral Diseases 20: 740-743.   Bardoni, R. (2013). "Role of Presynaptic glutamate receptors in pain transmission at the spinal cord level." Current Neuropharmacology 11: 477-483.   Bardoni, R., C. Torsney, et al. (2004). "Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn." J Neurosci 24: 2774-2781.   Basi, D. L., A. M. Velly, et al. (2012). "Human temporomandibular joint and myofascial pain biochemical profiles: a case-control study." J Oral Rehabil 39: 326-337.   Bennett, D. H. (2001). "Neurotrophic factors: important regulators of nociceptive pain." The Neuroscientist 7: 13-17.   Benoliel, R., E. Eliav, et al. (2001). "No sympathetic nerve sprouting in rat trigeminal ganglion following painful and non-painful infraorbital nerve neuropathy." Neurosci Lett 2001: 151-154.   Bereiter, D. A. (2001). "Sex Differences in brainstem neural activation after injury to the TMJ region." Cells Tissues Organs 169: 226-237.   Bereiter, D. A. and D. J. Barker (1975). "Facial receptive fields of trigeminal neurons: increased size following estrogen treatment in female rats." Neuroendocrinology 18: 115-124.   Bongenhielm, U., F. M. Boissonade, et al. (1999). "Sympathetic nerve sprouting fails to occur in the trigeminal ganglion after peripheral nerve injury in the rat." Pain 82: 283-288.   Brooks, S. L., P. L. Westesson, et al. (1992). "Prevalence of osseus changes in the temporomandibular joint of asymptomatic persons without internal derangement." Oral Surg Oral Med Oral Pathol Oral Radiol Endod 73: 118-122.   Bruno, M. A. and A. C. Cuello. (2006). "Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade." PNAS 103: 6735-6740.  113  Burchiel, K. J. (1984). "Spontaneous impulse generation in normal and denervated dorsal root ganglia: sensitivity to alpha-adrenergic stimulation and hypoxia." Experimental Neurology 85: 257-272.   Bush, F. M., S. W. Harkins, et al. (1993). "Analysis of gender effects on pain perception and symptom presentation in temporomandibular pain." Pain 53: 73-80.   Cairns, B. (2007a). Nociceptors in the Orofacial Region (Temporomandibular Joint and Masseter Muscle). Encyclopedia of Pain. R. F. Schmidt and W. D. Willis, Springer Berlin Heidelberg: 1424-1427.   Cairns, B. E. (2007b). "The influence of gender and sex steroids on craniofacial nociception." Headache 47: 319-324.   Cairns, B. E. (2008). Physiological properties of thin-fibers muscle afferents: excitiation and modulatory effects. Fundamentals of musculoskeletal pain. Seattle, IASP Press: 19-32.   Cairns, B. E. (2010). "Pathophysiology of TMD pain – basic mechanisms and their implications in pharmacotherapy." J Oral Rehabil 37: 391-410.   Cairns, B. E., X. Dong, et al. (2007). "Systemic administration of monosodium glutamate elevates intramuscular glutamate levels and sensitizes rat masseter muscle afferent fibers." Pain 132: 33-41.   Cairns, B. E., G. Gambarota, et al. (2003a). "Activation of peripheral excitatory amino acid receptors decreases the duration of local anesthesia." Anesthesiology 98: 521-529.   Cairns, B. E., G. Gambarota, et al. (2002a). "Glutamate-induced sensitization of rat masseter muscle fibers." Neuroscience 109: 389-399.   Cairns, B. E. and P. Gazerani (2009). "Sex-related differences in pain." Maturitas 63(4): 292-296.   Cairns, B. E. and P. Prateepavanich (2009). Role of peripheral mechanisms in spinal pain conditions. Peripheral targets for analgesia: novel approaches to pain management. B. E. Cairms, John Riley & Sons: 21-40.   Cairns, B. E., Y. Sim, et al. (2002b). "Influence of sex on reflex jaw muscle activity evoked from the rat temporomandibular joint." Brain Res 957: 338-344.   Cairns, B. E., P. Svensson, et al. (2006). "Ketamine attenuates glutamate-induced mechanical sensitization of the masseter muscle in human males." Exp Brain Res 169: 467-472. 114    Cairns, B. E., P. Svensson, et al. (2003b). "Activation of peripheral NMDA receptors contributes to human pain and rat afferent discharges evoked by injection of glutamate into the masseter muscle." J Neurophysiol 90: 2098-2105.   Cairns, B. E., K. Wang, et al. (2003c). "The effect of glutamate-evoked masseter muscle pain on the human jaw-stretch reflex differs in men and women." J Orofac Pain 17: 317-325.   Capsoni, S., S. Covaceuszach, et al. (2011). "Taking pain out of NGF: a “painless” NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrphic activity." PLoS ONE 6: e17321.   Carlton, S. M. (2001). "Peripheral excitatory amino acids." Curr Opin Pharmacol 1: 52-56.   Carlton, S. M., G. L. Hargett, et al. (1995). "Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin." Neurosci Lett 197: 25-28.   Cascos-Romero, J., E. Vazquez-Delgado, et al. (2009). "The use of tricyclic antidepressants in the treatment of temporomandibular joint disorders: systemic review of the literature of the last 20 years." Med Oral Patol Oral Cir Bucal 14: E3-E7.   Castrillon, E. E., B. E. Cairns, et al. (2007). "Effect of a peripheral NMDA receptor antagonist on glutamate-evoked masseter muscle pain and mechanical sensitization in women." J Orofac Pain 21: 216-224.   Castrillon, E. E., B. E. Cairns, et al. (2012). "Comparison of glutamate-evoked pain between the temporalis and masseter muscles in men and women." Pain 153(4): 823-829.   Castrillon, E. E., M. Ernberg, et al. (2010). "Interstitial glutamate concentration is elevated in the masseter muscle of myofascial temporomandibular disorder patients." J Orofac Pain 24: 350-360.   Cattaneo, A. (2010). "Tanezumab, a recombinant humanized mAb against nerve growth factor for the treatment of acute and chronic pain." Curr Opin Mol Ther 12: 94-106.   Chang, L. (2013). "Altered glutamatergic metabolism and activated glia: biomarkers for neuropathic pain?" Pain 154: 181-182.   Chao, M. V. (2003). "Neurotrophins and their receptors: a convergence point for many signaling pathways." Nat Rev Neurosci 4: 299-309. 115    Chao, M. V., R. Rajagopal, et al. (2006). "Neurotrophin signalling in health and disease." Clinical Science 110: 167-173.   Chatterton, J. E., M. Awobuluyi, et al. (2002). "Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits." Nature 415: 793-798.   Chau, P.-L. (2010). "New insights into the molecular mechanisms of general anaesthetics." British Journal of Pharmacology 161: 288-307.   Chen, W., W. Walwyn, et al. (2014). "BDNF released during neuropathic pain potentiates NMDA receptors in primary afferent terminals." Eur J Neurosci 39: 1439-1454.   Cheng, J.-K. and R.-R. Ji (2008). "Intracellular signaling in primary sensory neurons and persistent pain." Neurochem Res 33: 1970-1978.   Chung, K., Y. W. Yoon, et al. (1997). "Sprouting sympathetic fibers form synaptic varicosities in the dorsal root ganglion of the rat with neuropathic injury." Brain Res 751: 275-280.   Coste, J., D. L. Voisin, et al. (2008). "Dorsal horn NK1-expressing neurons control windup of downstream trigeminal nociceptive neurons." Pain 137: 340-351.   Cox, L. J., U. Hengst, et al. (2008). "Intr-axonal translation and retrograde transport of CREB mediates neuronal survival." Nature Cell Biology 10: 149-159.   Craig, A. D., E. T. Zhang, et al. (1995). "A reply." Pain 62: 391-392.   da-Silva, S. and F. Wang (2011). "REtrograde neural circuit specification by target-derived neurotrophins and growth factors." Curr Opin Neurobiol 21: 61-67.   deGroot, J., S. Zhou, et al. (2000). "Peripheral glutamate release in the hindpaw following low and high intensity sciatic stimulation." Neuroreport 11: 497-502.   DeSantis, M. and J. W. Duckworth (1982). "Properties of primary afferent neurons from muscles which are spontaneously active after a lesion of their peripheral process." Experimental Neurology 75: 261-274.   DeVon, H. A., M. R. Piano, et al. (2014). "The association of pain with protein inflammatory biomarkers." Nursing Research 63: 51-62.   Devor, M. and Z. Seltzer (1999). Pathophysiology of damages nerves in relation to chronic pain. Textbook of Pain. P. D. Wall and R. Melzack.   116  Di-Luca, M., F. Gardoni, et al. (2001). "NMDA receptor subunits are phosphorylated by activation of neurotrophin receptors in PSD of rat spinal cord." Neuroreport 12: 1301-1305.   Dong, X.-D., M. K. Mann, et al. (2007). "Sex-related differences in NMDA-evoked rat masseter muscle afferent discharges result from estrogen-mediated modulation of peripheral NMDA receptor activity." Neuroscience 146: 822-832.   Dostrovsky, J. O. and B. J. Sessle (2007). Nociceptive processing in the brainstem. Encyclopedia of Pain. R. F. Schmidt and W. D. Willis, Springer Berlin Heidelberg: 1370-1374.   Doubell, T. P., R. J. Mannion, et al. (1999). The dorsal horn: state-dependent sensory processing, plasticity and the generation of pain. Textbook of Pain. P. D. Wall and R. Melzack.   Dworkin, S. F. and L. LeResche (1992). "Research diagnostic criteria for temporomandibular discorders: review, criteria, examinations and specifications, critique." J Craniomandib Disord 6: 301-355.   Ernberg, M., B. Hedenberg-Magnusson, et al. (1999). "The level of serotonin in the superficial masseter muscle in relation to local pain and allodynia." Life Sci 65: 313-325.   Fernandes-de-las-Penas, C., F. Galan del Rio, et al. (2009). "Bilateral widespread mechanical pain sensitivity in women with myofascial temporomandibular disorder: evidence of impairment in central nociceptive processing." J Pain 10: 1170-1178.   Foy, M. R., J. Xu, et al. (1999). "17β-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation." J Neurophysiol 81: 925-929.   Fricton, J. (2007). "Myogenous temporamandibular disorders: diagnostic and management considerations." Dent Clin N Am 51: 61-83.   Furukawa, H., S. K. Singh, et al. (2005). "Subunit arrangement and function in NMDA receptors." Nature 438: 185-192.   Garber, K. (2011). "Fate of novel painkiller mAbs hangs in balance." Nat Biotechnol 29: 173-174.   Gazerani, P., S. Au, et al. (2010). "Botulinum neurotoxin type A (BoNTA) decreases the mechanical sensitivity of nociceptors and inhibits neurogenic vasodilation in a craniofacial muscle targeted for migraine prophylaxis." Pain 151: 606-616.   117  Ginty, D. D. and R. A. Segal (2002). "Retrograde neurotrophin signaling: trk-ing along the axon." Current Opinion in Neurobiology 12: 268-274.   Gomes, M. B., J. P. Guimaraes, et al. (2008). "Palpation and pressure pain threshold: reliability and validity in patients with temporomandibular disorders." Cranio 26: 202-210.   Graff-Radford, S. B. and J. P. Bassiur (2014). "Temporomandibular disorders and headaches." Neuro Clin 32: 525-537.   Guo, W., H. Wang, et al. (2010). "Long lasting pain hypersensitivity following ligation of the tendon of the masseter muscle in rats: a model of myogenic orofacial pain." Molecular Pain 6: 40.   Gupta, S., K. E. McCarson, et al. (2011). "Mechanisms of pain modulation by sex hormones in migraine." Headache 51: 905-922.   Haas, D. A., O. Nakanishi, et al. (1992). "Development of an orofacial model acute inflammation in the rat." Arch Oral Biol 37: 417-422.   Haas, H. S., A. Linecker, et al. (2010). "Peripheral glutamate signaling in head and neck areas." Head & Neck 32: 1554-1572.   Helmy, E., R. Bays, et al. (1988). "Osteoarthrosis of the temporomandibular joint following experimental disc perforation in Macaca fascicularis." J oral Maxillofac Surg 46: 979-990.   Hendry, S. and S. Hsiao (2012). The somatosensory system. Fundamental Neuroscience. L. Squire, D. Berg, F. E. Bloomet al, Academic Press.   Jankowski, M. P. and H. R. Koerber (2010). Neurotrophic factors and nociceptor sensitization. Translational Pain Research: From Mouse to Man. L. Kruger and A. R. Light. Boca Raton, FL, CRC Press.   Jarvis, C. R., J. G. Xiong, et al. (1997). "Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons." J Neurophysiol 78: 2363-2371.   Jasmin, L., J.-P. Vit, et al. (2010). "Can satellite glial cells be therapeutic targets for pain control?" Neuron Glia Biology 6: 63-71.   Jasmin, L., J.-P. Vit, et al. (2009). "Silencing GLAST in the trigeminal ganglion produced mechanical hypersensitivity, Program No. 171.1 " Neuroscience Meeting Planner, Chicago, IL: Society for Neuroscience: Online.   118  Juranek, I. and F. Lembeck (1997). "Afferent C-fibres release substance P and glutamate." Canadian Journal of Physiology and Pharmacology 75: 661-664.   Kalia, L. V., J. R. Gingrich, et al. (2004). "Src in synaptic transmission and plasticity." Oncogene 23: 8007-8016.   Katz, N., D. G. Borenstein, et al. (2011). "Efficacy and safety of tanezumab in the treatment of chronic low back pain." Pain 152: 2248-2258.   Kirk, E. J. (1974). "Impulses in dorsal spinal nerve rootlets in cats and rabbits arising from dorsal root ganglia isolated from the periphery." Journal of Comparative Neurology 2: 165-176.   Kovalchuk, Y., K. Holthoff, et al. (2004). "Neurotrophin action on a rapid timescale." Curr Opin Neurobiol 14: 558-563.   Lam, D. K., B. J. Sessle, et al. (2005). "Neural mechanisms of temporomandibulat joint and masticatory muscle pain: a possible role for peripheral glutamate receptor mechanisms." Pain Res Manage 10: 145-152.   Lane, N. E., T. J. Schnitzer, et al. (2010). "Tanezumab for the treatment of pain from osteoarthritis of the knee." N Engl J Med 363: 1521-1531.   Langley, C. K., Q. Aziz, et al. (2008). "Volunteer studies in pain research - opportunities and challenges to replace animal experiments.  The report and recommendations of a focus on alternatives workshop." Neuroimage 42: 467-473.   Latremoliere, A. and C. J. Woolf (2009). "Central sensitization: a generator of pain hypersensitivity by central neural plasticity." J Pain 10: 895-926.   Laursen, J. C., B. E. Cairns, et al. (2014). "Glutamate dysregulation in the trigeminal ganglion: a novel mechanism for peripheral sensitization of the craniofacial region." Neuroscience 256: 23-35.   LeResche, L. (1997). "Epidemiology of temporomandibular disorders: implications for the investigation of etiologic factors." Crit Rev Oral Biol Med 8: 291-305.   Levine, J. D. and D. B. Reichling (1999). Peripheral mechanismd of inflammatory pain. Textbook of Pain. P. D. Wall and R. Melzack.   Lewin, G. R. and S. B. McMahon (1991). "Physiological properties of primary sensory neurons appropriately and inappropriately innervating skin in the adult rat." J Neurophysiol 66: 1205-1217.   119  Li, J., J. A. McRoberts, et al. (2006). "Experimental colitis modulates the functional properties of NMDA receptors in dorsal root ganglia neurons." Am. J. Physiol. Gastrointest. Liver Physiol 291: G219-G228.   Li, J., J. A. McRoberts, et al. (2004). "Electrophysiological characterization of N-methyl-D-aspartate receptors in rat dorsal root ganglia neurons." Pain 109: 443-452.   Lieberman, A. R. (1976). Sensory ganglia. The Peripheral Nerve. D. N. London. London, Chapman and Hall: 188-278.   Liu, H., P. W. Mantyh, et al. (1997). "NMDA-receptor regulation of substance P release from primary afferent nociceptors." Nature 386: 721-724.   Liu, X. J. and M. W. Salter (2010). "Glutamate receptor phosphorylation and trafficking in pain plasticity in spinal cord dorsal horn." European Journal of Neuroscience 32: 278-289.   Lodge, D., S. N. Davis, et al. (1988). "A comparison between the in vivo and in vitro activity of five potent and competitive NMDA antagonists." Br J Pharmacol 95: 957-965.   Malcangio, M., N. E. Garrett, et al. (1997). "Nerve growth factor treatment increases stimulus-evoked release of sensory neuropeptides in the rat spinal cord." Eur J Neurosci 9: 1101-1103.   Manfredini, D., L. Guarda-Nardini, et al. (2011). "Research diagnostic criteria for temporomandibular disorders: a systemic review of axis I epidemiologic findings." Oral Surg Oral Med Oral Pathol Oral Radiol Endod 112: 453-462.   Mann, M. K., X. D. Dong, et al. (2006). "Influence of intramuscular nerve growth factor injection on the response properties of rat masseter muscle afferent fibers." J Orofac Pain 20: 325-336.   Marchi, A., R. Vellucci, et al. (2009). "Pain biomarkers." Clin Drug Invest 29 Suppl 1: 41-46.   McMahon, S. B. and D. L. H. Bennett (1999). Trophic factors and pain. Textbook of Pain. P. D. Wall and R. Melzack.   McMahon, S. B., G. R. Lewin, et al. (1993). "Central hyperexcitability triggered by noxious inputs." Curr Opin Neurobiol 3: 602-610.   McRoberts, J. A., J. Li, et al. (2007). "Sex-dependent differences in the activity and modulation of N-methyl-d-aspartic acid receptors in rat dorsal root ganglia neurons." Neuroscience 148: 1015-1020. 120    Meloto, C. B., P. O. Serrano, et al. (2011). "Genomics and the new perspectives for temporomandibular disorders." Archives of Oral Biology 56: 1181-1191.   Meng, J., S. V. Ovsepian, et al. (2009). "Activation of TRPV1 mediates calcitonin gene-related peptide release, which excites trigeminal sensory neurons and is attenuated by a retargeted botulinum toxin with anti-nociceptive potential." J Neurosci 29: 4981-4992.   Miller, E. M., E. W. Hoffman, et al. (2011). "Glutamate pharmacology and metabolism in peripheral primary afferents: Physiological and pathophysiological mechanisms." Pharmacology & Therapeutics 130: 283-309.   Mills, C. D., T. Nguyen, et al. (2013). "Characterization of nerve growth factor-induced mechanical and thermal hypersensitivity in rats." Eur J Pain 17: 469-479.   Mogil, J. S., K. D. Davis, et al. (2010). "The necessity of animal models in pain research." Pain 151: 12-17.   Nazarian, A., G. Gu, et al. (2008). "Spinal NMDA receptors and nociception-evoked release of primary afferent substance P." Neuroscience 152: 119-127.   Neugebaur, V. and S. Carlton (2002). "Peripheral metabotropic glutamate receptors as drug targets for pain relief." Expert Opin Ther Targets 6: 349-361.   Neumann, S., T. P. Doubell, et al. (1996). "Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons." Nature 384: 360-364.   Newsholme, E. A. and P. C. Calder (1997). "The proposed role of glutamine in some cells of the immune system and speculative consequences for the whole animal." Nutrition 13: 728-730.   Numakawa, N., H. Nakayama, et al. (2003). "Nerve growth factor-induced glutamate release is via p75 receptor, ceramide, and Ca(2+) from ryanodine receptor in developing cerebellular neurons." J Biol Chem 278: 41259-41269.   Odrcich, M., J. M. Bailey, et al. (2006). "Chronobiological characteristics of painful diabetic neuropathy and postherpetic neuralgia: diurnal pain variation and effects of analgesic therpay." Pain 120: 207-212.   Ohtori, S., K. Takahashi, et al. (2001). "Phenotypic inflammation switch in rats shown by calcitonin gene-related peptide immunoreactive dorsal root ganglion neurons innervating the lumbar facet joints." Spine 26: 1009-1013.   121  Osikowicz, M., G. Longo, et al. (2013). "Inhibition of endogenous NGF degradation induces mechanical allodynia and thermal hyperalgesia in rats." Mol Pain 9: 37.   Ota, H., K. Katanosaka, et al. (2013). "TRPV1 and TRPV4 play pivotal roles in delayed onset muscle soreness." PLoS ONE 8: e65751.   Paoletti, P. (2011). "Molecular basis of NMDA receptor functional diversity." Eur J Neurosci 33: 1351-1365.   Park, K. A., J. C. Fehrenbacher, et al. (2010). "Signaling pathways that mediate nerve growth factor-induced increase in expression and release of calcitonin gene-related peptide from sensory neurons." Neuroscience 171: 910-923.   Peng, H. Y., G. D. Chen, et al. (2010). "Endogenous ephrinB2 mediates colon-uretha cross-organ sensitization via src kinase-dependent tyrosine phosphorylation of NR2B." Am J Physiol Renal Physiol 298: F109-F117.   Petrenko, A. B., T. Yamakura, et al. (2003). "The Role of N-methyl-D-asparate (NMDA) receptors in pain: a review." Anesth Analg 97: 1108-1116.   Petty, B. G., D. R. Cornblath, et al. (1994). "The effect of systemically administered recombinant human nerve growth factor in healthy human subjects." Annals of Neurology 36: 244-246.   Pezet, S. and S. B. McMahon (2006). "Neurotrophins: mediators and modulators of pain." Annu Rev Neurosci 29: 207-538.   Platt, S. R. (2007). "The role of glutamate in central nervous system health and disease--a review." Vet J 173: 278-286.   Raiteri, L., S. Giovedi, et al. (2003). "Cellular mechanisms of the acute increase of glutamate release induced by nerve growth factor in rat cerebral cortex." Neuropharmacology 44: 390-402.   Raja, S. N., R. A. Meyer, et al. (1999). Peripheral neural mechanisms of nociception. Textbook of Pain. P. D. Wall and R. Melzack.   Rappaport, Z. H. and M. Devor (1994). "Trigeminal neuralgia: the role of self-sustaining discharge in the trigeminal ganglion." Pain 56: 127-138.   Ren, K. (1999). "An imporved method for assessing mechanical allodynia in the rat." Physiol Behav 67: 711-716.   Ren, K. and R. Dubner (2011). "The role of trigeminal interpolaris-caudalis transition zone in persistent orofacial pain." Int Rev Neurobiol 97: 207-225. 122    Robinson, A. J., M. A. Bass, et al. (2005). "Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2." Journal of Biological Chemistry 280: 35329-35336.   Roveroni, R. C., C. A. Parada, et al. (2001). "Development of a behavioal model of TMJ pain in rats: the TMJ formalin test." Pain 94: 185-191.   Salter, M. W. and L. V. Kalia (2004). "Src kinases: a hub for NMDA receptor regulation." Nature reviews 5: 317-328.   Saper, C. B. (2000). Brain Stem, Reflexive behavior, and the cranial nerves. Principles of neural science. E. R. Kandel, J. H. Schwartz and T. M. Jessell, McGraw-Hill.   Schizas, N., Ø. Lian, et al. (2010). "Coexistence of up-regulated NMDA receptor 1 and glutamate on nerves, vessels and transformed tenocytes in tendinopathy." Scand J Med Sci Sports 20: 208-215.   Sessle, B. J. (1999). "The neural basis of temporomandibular joint and masticatory muscle pain." J Orofac Pain 13: 238-245.   Sessle, B. J. (2005). "Peripheral and central mechanisms of orofacial pain and their clinical correlates." Minerva Anestesiol 71: 117-136.   Sessle, B. J. (2009). Role of peripheral mechanisms in craniofacial pain conditions. Peripheral receptor targets for analgesia: novel approaches to pain management. B. E. Cairns, John Wiley & Sons: 3-20.   Shaefer, J. R., N. Holland, et al. (2013). "Pain and temporomandibular disorders: a pharmaco-gender dilemma." Dent Clin N Am 57: 233-262.   Shu, X. and L. M. Mendell (2001). "Acute sensitization by NGF of the response of small-diameter sensory neurons to capsaicin." J Neurophysiol 86: 2931-2938.   Siegel, A. and H. N. Sapru (2010). Anatomical organization of the cranial nerves within the brainstem. Essential Neuroscience. B. Sun. Philadelphia, Lippincott Williams and Wilkins.   Siemionow, M., B. B. Gharb, et al. (2011). "The face as a sensory organ." Plastic and Reconstructive Surgery 127: 652-662.   Slack, S., A. Battaglia, et al. (2008). "EphrinB2 induces tyrosine phosphorylation of NR2B via src-family kinases during inflammatory hyperalgesia." Neuroscience 156: 175-183. 123    Sofroniew, M. V., C. L. Howe, et al. (2001). "Nerve growth factor signaling, neuroprotection, and neural repair." Annu Rev Neurosci 24: 1217-1281.   Stawski, P., H. Janovjak, et al. (2010). "Pharmacology of inotropic glutamate receptors: a structural perspective." Bioorganic & Medicinal Chemistry 18: 7759-7772.   Stohler, C. S. (1997). "Masticatory myalgias." Pain Forum 6: 176-180.   Svennson, P., B. E. Cairns, et al. (2003). "Glutamate-evoked pain and mechanical allodynia in the human masseter muscle." Pain 101: 221-227.   Svensson, P., B. E. Cairns, et al. (2003). "Injection of nerve growth factor into human masseter muscle evokes long lasting mechanical allodynia and hyperalgesia." Pain 104: 241-247.   Svensson, P., E. Castrillon, et al. (2008a). "Nerve growth factor-evoked masseter muscle sensitization and perturbation of jaw motor function in healthy women." J Orofac Pain 22: 340-348.   Svensson, P., K. Wang, et al. (2008b). "Effects of NGF-induced muscle sensitization on proprioception and nociception." Exp Brain Res 189(1): 1-10.   Svensson, P., K. Wang, et al. (2010). "Human nerve growth factor sensitizes masseter muscle nociceptors in female rats." Pain 148: 473-480.   Swift, J. Q., M. T. Roszkowski, et al. (1998). "Effect of intra-articular versus systemic anti-inflammatory drugs in a rabbit model of temporomandibular joint inflammation." J oral Maxillofac Surg 56: 1288-1295.   Tal, M. and M. Devor (1992). "Ectopic discharge in injured nerves: comparison of trigeminal and somatic afferents." Brain Res 579: 148-151.   Tan, P. H., L. C. Yang, et al. (2005). "Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat." Gene Ther 12: 59-66.   Torri, G. (2010). "Inhalation anesthetics: a review." Minerva Anestesiol 75: 215-228.   Ueki, K., D. Takazakura, et al. (2003). "The use of polylactic acid/polyglycolic acid copolymer and gelatin sponge complex containing human recombinant bone morphogenetic protein-2 following condylectomy in rabbits." J Craniomaxillofac Surg 31: 107-114.   124  Ure, D. R. and R. B. Campenot (1997). "Retrograde transport and steady-state distribution of I-125-nerve growth factor in rat sympathetic neurons in compartmented cultures." J Neurosci 17: 1282-1290.   Wall, P. D. (1995). "A pain in the brain and lower parts of the anatomy." Pain 62: 389-391.   Wall, P. D. and M. Devor (1983). "Sensory afferent impulses originate from dorsal root ganglia as well as from the periphery in normal and nerve-injured rats." Pain 17: 321-339.   Wang, H., R. J. Liu, et al. (1997). "Peripheral NMDA receptors contribute to the activation of nociceptors: a c-fos expression study in rats." Neuro Lett 221: 101-104.   Wang, M. W., U. Kumar, et al. (2012). "Expression of NMDA and oestrogen receptors by trigeminal ganglion neurons that innervate the rat temporalis muscle." Chin J Dent Res 15: 89-97.   Watson, J. J., S. J. Allen, et al. (2008). "Targeting nerve growth factor in pain." Biodrugs 22: 349-359.   Welker, J. A., R. M. Henshaw, et al. (2000). "The percutaneous needle biopsy is safe and recommended in the diagnosis of musculoskeletal masses." Cancer 89: 2677-2686.   Westberg, K.-G. and A. Kolta (2011). "The trigeminal circuitrs responsible for chewing." International Review of Neurobiology 97: 77-98.   Westlund, K. N., D. L. McNeill, et al. (1989). "Glutamate immunoreactivity in rat dorsal root axons." Neuroscience Letters 96: 13-17.   Whitten, J. P., B. M. Baron, et al. (1990). "R-4-Oxo-5-phosphonorvaline: a new competitive glutamate antagonist at the NMDA receptor complex." J Med Chem 33: 2961-2963.   Wong, H., I. Kang, et al. (2014). "NGF-induced mechanical sensitization of the masseter muscle is mediated through peripheral NMDA receptors." Neuroscience 269C: 232-244.   Woolf, C. J. (2011). "Central sensitization: implications for the diagnosis and treatment of pain." Pain 152: S2-S15.   Woolf, C. J. and Q. Ma (2007). "Nociceptors--noxious stimulus detectors." Neuron 55: 353-364.   125  Woolley, C. S., N. G. Weiland, et al. (1997). "Estradiol increases the sensitivity of hippocampal CA1 pyramidal cells to NMDA receptor-mediated synaptic input: correlation with dendritic spine density." J Neurosci 17: 1848-1859.   Wu, L.-J. and M. Zhuo (2009). "Targeting the NMDA receptor subunit NR2B for the treatment of neuropathic pain." Neurotherapeutics 6: 693-702.   Yan, X., E. Jiang, et al. (2013). "Endogenous activation of presynaptic NMDA receptors enhances glutamate release from the primary afferents in the spinal dorsal horn in a rat model of neuropathic pain." J Physiol 591: 2001-2019.   Yu, X. M., R. Askalan, et al. (1997). "NMDA channel regulation by channel-associated protein tyrosine kinase src." Science 275: 647-678.   Yun, K. I., C. H. Chae, et al. (2008). "Effect of estrogen on the expression of cytokines of the temporomandibular joint cartilage cells of the mouse." J Oral Maxillofac Surg 65: 871-876.   Zhu, M., J. Wang, et al. (2012). "Upregulation of protein phosphatase 2A and NR3A-pleiotropic effect of simvastatin on ischemic stroke rats." PLoS ONE 7: e51552.   Zweifel, L. S., R. Kuruvilla, et al. (2005). "Functions and mechanisms of retrograde neurotrophin signalling." Nature Reviews 6: 615-625.     

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0135676/manifest

Comment

Related Items