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Central Targeting of Trigeminal Primary Afferent Nerve Terminals via Intracisternal Injection of RTX and Its Effect on P...

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Title: Central Targeting of Trigeminal Primary Afferent Nerve Terminals via Intracisternal Injection of RTX and Its Effect on Pain Behavior
Physical Description: 1 online resource (35 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: pain, rtx, trigeminal, trpv1
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: The TRPV1 selective neurotoxin resiniferatoxin (RTX) was evaluated for use in treating orofacial pain. RTX (250 ng) or vehicle was administered intracisternally in rats to lesion TRPV1 expressing neurons in the nucleus caudalis. Nociception was quantified 7 d following the treatment using an operant facial nociception assay in which the rats place their faces against a thermal stimulus (48?C) while obtaining a reward. Stimulus and reward contacts were monitored. The central lesions were verified by immunohistochemistry. RTX produced an analgesic effect to inflammatory pain as indicated by significantly greater (P < 0.05) reward licking events (1490 + or - 360) and reward pain ratio (14.9 + or - 2.5) when compared to vehicle-treated rats (Licks: 396 + or - 86; Pain Ratio: 0.9 + or - 0.1). RTX eliminated TRPV1 labeling in the nucleus caudalis but not in the trigeminal ganglion. These data indicate that RTX lesions may be used to selectively treat orofacial pain.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Neubert, John K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

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Classification: lcc - LD1780 2008
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Permanent Link: http://ufdc.ufl.edu/UFE0022187/00001

Material Information

Title: Central Targeting of Trigeminal Primary Afferent Nerve Terminals via Intracisternal Injection of RTX and Its Effect on Pain Behavior
Physical Description: 1 online resource (35 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: pain, rtx, trigeminal, trpv1
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The TRPV1 selective neurotoxin resiniferatoxin (RTX) was evaluated for use in treating orofacial pain. RTX (250 ng) or vehicle was administered intracisternally in rats to lesion TRPV1 expressing neurons in the nucleus caudalis. Nociception was quantified 7 d following the treatment using an operant facial nociception assay in which the rats place their faces against a thermal stimulus (48?C) while obtaining a reward. Stimulus and reward contacts were monitored. The central lesions were verified by immunohistochemistry. RTX produced an analgesic effect to inflammatory pain as indicated by significantly greater (P < 0.05) reward licking events (1490 + or - 360) and reward pain ratio (14.9 + or - 2.5) when compared to vehicle-treated rats (Licks: 396 + or - 86; Pain Ratio: 0.9 + or - 0.1). RTX eliminated TRPV1 labeling in the nucleus caudalis but not in the trigeminal ganglion. These data indicate that RTX lesions may be used to selectively treat orofacial pain.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Neubert, John K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022187:00001


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1 CENTRAL TARGETING OF TRIGEMINAL PRIMARY AFFERENT NERVE TERMINALS VIA INTRACISTERNAL INJECTION OF RTX AND ITS EFFECT ON PAIN BEHAVIOR By MELANIE M. WEXEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Melanie M. Wexel

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3 To my Mom, for inspiring me to continue education

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4 ACKNOWLEDGMENTS I thank m y committee chair (John Neubert, DDS, PhD) and my committee members (Robert Caudle, PhD and Calegero Dolce, DDS, PhD). I would al so like to thank Heather Rossi, Alan Jenkins, Jean Kaufman, and Wendi Malphurs for assisting with the laboratory experiments. Also, I would like to acknow ledge the NIH grant #R21 DE16704-01A1 and the Southern Association of Orthodontics gr ant for financial support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES.........................................................................................................................6 LIST OF ABBREVIATIONS.......................................................................................................... 7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INTRODUCTION....................................................................................................................9 Background...............................................................................................................................9 Molecular Targets of Pain......................................................................................................10 Pharmacology................................................................................................................... ......12 Significance................................................................................................................... .........14 Hypothesis and Specific Aims................................................................................................ 15 2 METHODS.............................................................................................................................16 Injections..................................................................................................................... ............16 Pain Induction.........................................................................................................................17 Behavioral Measures............................................................................................................ ..17 Operant Thermal Facial Assessment............................................................................... 17 Mechanical Sensitivity.................................................................................................... 18 Capsaicin Eye Wipe Sensitivity...................................................................................... 18 Immunohistochemistry........................................................................................................... 18 Statistical Analysis........................................................................................................... .......19 3 RESULTS...............................................................................................................................21 Operant Thermal Facial Assessment......................................................................................21 Mechanical Sensitiv ity......................................................................................................... ...21 Capsaicin Eye Wipe Sensitivity.............................................................................................. 22 Immunohistochemistry........................................................................................................... 22 4 DISCUSSION.........................................................................................................................26 5 CONCLUSIONS.................................................................................................................... 31 LIST OF REFERENCES...............................................................................................................32 BIOGRAPHICAL SKETCH.........................................................................................................35

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6 LIST OF FIGURES Figure page 3-1. RTX inhibits inflam m atory orofacial pain............................................................................ 23 3-2. Mechanical sensitivity assessment ....................................................................................... 24 3-3. Capsaicin eye wipe response.............................................................................................. ...24 3-4. Effect of intracisternal RTX on TRPV1 expr essing cells in the trigeminal ganglia............. 25 3-5. Effect of intracisternal RTX on TRPV1 staining in the brainstem ........................................ 25 4-1. Central delivery of RTX ................................................................................................. ......30

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7 LIST OF ABBREVIATIONS ICM Intracisternal injection into the cisterna magna TG Trigeminal Ganglion RTX Resiniferatoxin is an agonis t of TRPV1. It is an ultrapotent analog of capsaicin that has been identified and used exte nsively in pain research. TMD Temporomandibular disorders. Painful c onditions of the temporomandibular joint and muscles of mastication. TNC Trigeminal Nucleus Caudalis TRP Transient receptor potential. TRP channels have been identified as important modulators of thermal sensations. TRPV1 Transient receptor potential channel, vanilloid subfamily member 1. Formerly known as the capsaicin receptor or VR1.

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CENTRAL TARGETING OF TRIGEMINAL P RIMARY AFFERENT NERVE TERMINALS VIA INTRACISTERNAL INJECTION OF RTX AND ITS EFFECT ON PAIN BEHAVIOR By Melanie M. Wexel May 2008 Chair: John Neubert Major: Dental Sciences The TRPV1 selective neurotoxin resiniferatoxin (RTX) was evaluated for use in treating orofacial pain. RTX (250 ng) or vehicle was administered intracisternally in rats to lesion TRPV1 expressing neurons in the nucleus caudalis. Nociception was quantified 7 d following the treatment using an operant facial nociception assay in which the rats place their faces against a thermal stimulus (48C) while obtaining a re ward. Stimulus and reward contacts were monitored. The central lesions were verifi ed by immunohistochemistry. RTX produced an analgesic effect to inflammatory pain as i ndicated by significantly greater (P<0.05) reward licking events (1490 360) and reward pain ratio (14.9 2.5) when compared to vehicle-treated rats (Licks: 396 86; Pain Ratio: 0.9 0.1). RTX eliminated TRPV1 labeling in the nucleus caudalis but not in the trigeminal ganglion. These data indicate that RTX lesions may be used to selectively treat orofacial pain.

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9 CHAPTER 1 INTRODUCTION Background Uncontrolled pain is a global problem of epide m ic proportions. The cost of chronic pain is estimated to be about $80 billion per year, with as much as 40% associated with orofacial pain (Israel and Scrivani 2000). Or ofacial pain disorders includi ng temporomandibular disorders (TMD), trigeminal neuralgia, headaches (e.g., mi graine, tension-type), and myofascial pain compose a significant proportion of this widespread phenomenon. Although these disorders affect an estimated 20% of the U.S. population, there is a significant di screpancy in the amount of research focusing on the facial region and tr igeminal system (Lipton et al. 1993). Further research and understanding of the trigeminal pain pathway mechanisms are needed for the development of novel facial pain therapies. Opioids are currently the main form of treatment for moderate to severe pain. Unfortunately, these drugs have dose-limiting ce ntral side effects in cluding sedation and respiratory depression. These side effects combined with their unpr edictable response can vastly undermine the quality of life for patients being treated for chr onic pain conditions. Since few effective analgesic drugs have emer ged in the last 100 years, the sear ch for better pain treatments remains a priority. With the discovery of the cap saicin receptor (TRPV1) there is promise of uncovering the exact molecular mechanisms involved in pain transduction. Capsaicin is the main pungent ingredient of hot chili peppers that causes the activation of many nociceptive neurons (Szallasi and Blumberg 1990). TRPV1 is well known to have a role in pain modulation, both as an activator and inhibitor of transduction. However, the exact mechanisms that are involved with TRPV1-mediated pain inhibition are largely unknow n. For example, the role of desensitization

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10 versus neuronal death following treatment with va nilloids to produce pain relief is currently being debated. Understanding thes e mechanisms is crucial for further development of innovative molecular pain management therapies. Agonists of TRPV1 including resiniferatoxin (R TX), an ultrapotent analog of capsaicin, have been identified and used extensively in pain research. RTX sp ecifically deletes TRPV1 receptors and therefore can be used to investig ate the role of TRPV1 on the elements of pain biology. This research not only aims to investig ate the therapeutic capabil ities of RTX but also to use RTX as a molecular neurosurgical tool to furt her understand pain mechanisms with the removal of the TRPV1 receptor. Molecular Targets of Pain Heat and va nilloid agents such as capsaicin evoke similar burning sensations, which led to the discovery that they have a common mol ecular pathway. It has been postulated for many years that nerve endings detect temperature a nd physical changes by ion channels. Recently, a number of transient receptor potential (TRP) channels have been id entified as important modulators of thermal sensations. There are si x TRP channels that have been cloned and characterized as thermoreceptors. This family has been shown to mediate extracellular calcium influx in response to low intracellular levels (C lapham 1996). Thermoreceptors can be activated by heat (35-45C) or cold (17-35 C) and thermonociceptors are ac tivated at noxious temperatures below 15C and above 43C (Caterina et al. 1997 ; LaMotte and Campbell 1978; Tominaga et al. 1998). These extreme temperatures can burn or freeze the skin and produce high frequency firing of A and C nociceptive fibers (C aterina et al. 1997; McKemy et al. 2002; Peier et al. 2002). TRPV1 is the mammalian heat thermonocicep tor that we will focus on in this study., Hot chili peppers were believed to evoke burning pain through capsaicin-induced ion currents in sensory neurons. Investigations in the late 1980s led to the conclusion that the

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11 capsaicin receptor was a non-specific cation channe l with limited selectivity for calcium (Wood et al. 1988). The first evidence to support the hypothesi s that ion channels were responsible for the burning response mechanism was the identifica tion of heat gated ion channels in certain primary afferent neurons (Cesare and McNa ughton 1996). In 1997, the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptor, formerly known as VR1, was cloned and characterized as a major molecular component for detecting both chemical and thermal pain (Caterina et al. 1997). Gene expression for TRPV1 has been found to o ccur predominantly in small diameter cells of the unmyelinated c-fibers of trigeminal and dor sal root sensory ganglia (Caterina et al. 1997). TRPV1 is also expressed all along the primary a fferent neurons from the peripheral distribution in the skin and deep tissues of the face to the presynaptic terminals (Cat erina et al. 1997; Denda et al. 2001). TRPV1 is a ligand gated channel that is selective for cations with a very high relative permeability to divalent cations, including calcium. Although, TRP family receptors also respond to many other stim uli (e.g. temperature, protons, lipids, phorbols, phosphorylation, and pressure), TRPV1 is the only channel activated by capsaicin/vanilloids (Gunthorpe et al. 2002). TRPV1 can be thought of as an integrator of noxious stimuli. Agonists (capsaicin and RTX) and potentiators (protons) act by reducing the temperat ure threshold for activation (Gunthorpe et al. 2002; Szallasi and Blumberg 1996; Tominaga et al. 1998). The mechanism of action of TRPV1 is largely unknown, but it appears th at binding or direct thermal activation of the TRPV1 receptor causes a conformational change that allows the influx of cations into the cell. This influx creates an action potential th at begins the cascade of nerve transmission that ultimately results in central pe rception of pain as described above (Caterina et al. 1997).

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12 Pharmacology TRPV1 receptors have a num ber of agoni sts including capsaicin, the main pungent ingredient of hot chili peppers and RTX, an ultrapotent analog of capsaicin derived from the euphorbia rubber plant (Szallasi and Blumberg 1990). Capsaicin and RTX can both activate and inhibit the signaling from nociceptive neurons Inhibition of nociception by capsaicin is primarily produced by desensitiza tion of the neuron, while RTX can specifically delete TRPV1 expressing neurons (Caterina et al. 1997; Caudle et al. 2003; Karai et al. 2004). When capsaicin binds to TRPV1, activation of the channel allows the influx of sodium which results in depolarization a nd subsequent pain. Then, the influx of calcium leads to acute desensitization (Caterina et al 1997; Szabo et al. 1999; Szallasi and Blumberg 1996). RTX is very potent for binding the TRPV1 receptor (Acs et al. 1996) and in contrast to capsaicin, the high affinity binding of RTX keeps the channel open. This allows for excess calcium influx in conjunction with release of intrace llular stores, and subsequent cytotoxicity (Caterina et al. 1997; Szallasi et al. 1999). The ability of RTX to selectively eliminate ce lls expressing the TRPV1 receptor allows for its use both as a therapeuti c prospect, and a tool to study the role of vanilloid receptors in pain modulation. In vivo studies have investigated various met hods of targeting the dorsal root ganglia and trigeminal nerve both peripherally and centrally with RTX. In 1999, Szabo et al. compared the effects of RTX injected epidura lly and subcutaneously in rats. Epidural administration was found to be selective for the spinal cord re gion, while the subcutaneous approach gave a generalized analgesic effect. The epidural approach was found to produce profound, long-lasting segmental analgesia to C-fiber mediated pain as judged by temperatur e withdrawal latency (Szabo et al. 1999). The elimina tion of peripheral nerve endings of the hindpaw showed a long term, reversible attenuation of nociceptive tr ansmission based on increased thermal hindpaw

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13 withdrawal latency (Neubert et al. 2003). Kissin et al. completely prevented pain hypersensitivity caused by the carrageenan infl ammatory process and plantar incisions by prophalactically applying RTX percutaneously to the sciatic and saphenous nerves (Kissin et al. 2002; Kissin et al. 2005b). This group also s howed a decrease in carrageenan induced edema and pain with RTX injections in the knee join t (Kissin et al. 2005a). In 2004, Karai et al. injected RTX into hindpaws of rats and the lumbar cerebrospinal fluid of dogs and was able to demonstrate efficacy in both species. Of si gnificance, the pain response was blocked while keeping the sensations of touch, proprio ception, motor function and mechanosensitive nociception (Karai et al. 2004). There is precedence to using RTX within the tr igeminal system. Karai et al. applied RTX into the trigeminal ganglia (TG) via a stereotaxic approach th rough the brain, while Neubert et al. accessed the trigeminal ganglia via the infrao rbital foramen (Karai et al. 2004; Neubert et al. 2005a). Both studies demonstrated that RTX can specifically delete TRPV 1 within the orofacial region, as evidenced by a loss of capsaicin eye wipe response. In this behavioral assay, a 0.1% solution of capsaicin is applied to the cornea of the eye and the number of eye wipes with the paws is counted for 1 minute. Capsaicin normally elicits extreme burning pain when applied in this fashion, producing over 50 wipes/min on average. Animals treated with RTX in the trigeminal ganglia have a complete elimination of this response. Immunohistochemistry data also revealed that RTX blocked inflammation induced spinal c-fos induction, a marker for nociceptive activity induced w ithin the second order neurons (Neubert et al. 2003). Reduction of orofacial pain responses have al so been achieved by usi ng specific toxins via an intracisternal injection appro ach of substance P conjugated to saporin (SP-SAP) (Simons et al. 2002). While this drug specifically targeted th e superficial medullary neurons expressing the

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14 neurokinin 1 receptor (NK-1), this intracisternal a pproach may also be used to target the central terminals of trigeminal primary afferent fibers Surprisingly, this region of the brainstem is relatively unprotected and is eas ily accessible via a percutaneous approach through the atlantooccipital membrane overlying the cisterna magna. Th e cisterna magna is an ideal site to target TRPV1 in the brainstem to evaluate the effect s of RTX on orofacial pa in. Although there is evidence that application of RTX to the peripheral nerve terminals produces a loss of TRPV1 expressing cells in the trigeminal ganglia; the e ffects on TRPV1 cells in the trigeminal ganglia with a intracisternal injections are unknown (Ne ubert et al. 2003). This is one of the outcomes that we investigated. One of the limiting factors for studying pain in the face relates to the limited number of valid behavioral outcome measures. Assessmen t of mechanical sens itivity using von Frey filaments to elicit a head withdrawal response is the typical orofacial pain outcome measure. However, this approach has numerous limitations, in cluding that the animals must be restrained. Our group has devised an alternative to typical reflex and unlearned behavioral measures by using an operant thermal assessment system (Neu bert et al. 2005a). Briefly, this system measures orofacial pain behavior by providing positive rewards for tolerance of thermal nociceptive stimulation. A food fasted animal is given the choice of placing its face on a hot (e.g., 48C) thermode in order to obtain a reward of sweetened condensed milk. (Neubert et al. 2005a). This method readily lends itself to testin g of trigeminally-mediate d pain and was one of the primary outcome measures of this study. This system is described in detail in the materials and methods. Significance Currently, the m ost commonly used drugs and treatment approaches vastly undermine the patients quality of life and many times remain inadequate. There is a need for greater

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15 understanding of the trigeminal pathways of pain in order to develop novel pain management techniques. This study hopes to investigate pain mechanisms with in the trigeminal system that will also evaluate new therapeutic approaches to treating chronic pain. Hypothesis and Specific Aims We hypothesized that deletion of trigem inal TRPV1 expressing primary afferent pain fibers via targeting of the central terminals will si gnificantly affect pain in the orofacial region. We tested this hypothesis with a number of specific aims. Aim 1 was to evaluate the effects of central RTX-treatment on orofacial pain by behavi orally characterizing thermal and mechanical sensitivity following central administration of RTX or vehicle and induction of orofacial inflammation. Aim 2 was to histologically characterize the changes in the TRPV1 expressing cells in the trigeminal ganglion and evaluate the relative amount of TRPV1+ fibers in the nucleus caudalis following central administration of RTX or vehicle.

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16 CHAPTER 2 METHODS Male Sprague Dawley rats (200-300g, N = 30) were ligh tly anesthetized (isoflurane, 12.5%, inhalation) 1 day prior to testing, and the face was shaved using clippers, followed by application of depilatory cream Excess cream was removed with a moistened paper towel to minimize skin irritation. Rats were food fasted (1215 hrs) prior to each testing session but were provided with standard food chow immediatel y following each session and on non-testing days. Animals were brought into the beha vioral procedure room 1 hr prio r to testing at the same time each day and allowed to acclimate to the temperature and ambient noise of the room. Water and food was made available ad libitum when animals were not in a testing session. Animal weight was recorded weekly. Animal testing procedures and general handling complied with the ethical guidelines and standards established by the Inst itutional Animal Care & Use Committee at the University of Florida, and all procedures complied with the Guide for Care and Use of Laboratory Animals (Council 1996). Injections We targeted TRPV1 receptors on th e central te rminal of the trigeminal primary afferent neurons by intracisternal injection into the ci sterna magna. Animals were anesthetized (2.5% isoflurane, USP, inhalation) and the posterior skull region overlying the cisterna magna was disinfected with betadine. A 27 gauge, inch needle attached to a 0.3ml plastic syringe was then directed so as to touch the occiput. Note that contacting the bony surface provides distinct tactile feedback. The tip was sequentially m oved caudally until the needle punctured the dura overlying the cisterna magna. Once the needle wa s in place, aspiration was performed to check for cerebrospinal fluid, and then RTX (250 ng) or vehicle (0.25% Tween 80 in phosphate buffered saline, 0.05% ascorbic acid) was delivered.

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17 Pain Induction Carrageen an, an inflammatory agent derived from seaweed, was used to induce inflammation and subsequent heat sensitivity (Ng and Ong 2001). A volume of 200 l of a 40 mg/ml, 8mg total solution was administered by subcutaneous injection into the face 1 week following intracisternal RTX or vehicle injectio ns. Animals were tested 3 hrs post-carrageenan injection at 48 C. Behavioral Measures The anim als were trained for two weeks prior to injections to allow them to acclimate to the reward-aversion testing boxes. Baseline thermal (37 C, 48C), mechanical (Von Frey), and cap eye wipe responses were recorded on all rats prior to injections. Operant Thermal Facial Assessment Orofacial therm al sensitivity was assessed using a reward-conflict operant paradigm, as described previously (Neubert et al. 2005a). Briefly, unrestrai ned animals were placed into reward-aversion testing boxes. The animals were given a choice to endure a painful thermal stimulus (48 C + inflammation) in order to receive a reward consisting of sweetened condensed milk solution. Sensors were placed on both the re ward bottle and stimulus thermode and data was recorded automatically as a licking or facial contact event whenever the animal contacted either part. The threshold for detecti on of facial contacts and licking contacts was set at 1.0 V and an event was registered when the si gnal went above threshold and ended when the signal drops below threshold. For orofacial thermal sensitivity, six outcome measures were evaluated: (1) reward intake; (2) total number of licking events; (3) total number facial contacts; (4) cumulative facial contact duration; (5) ratio of reward/facial contacts ; (6) duration per contact for the facial contacts. Data analyses were completed using custom-written routines (generously

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18 provided by Dr. Charles Widmer, University of Florida) in LabView Express (National Instruments Corporation) and Excel (Microsoft). Mechanical Sensitivity Mechanical sensitivity was evalu ated as a refl exive pain outcome measure. An electronic aesthesiometer (IITC Inc., Woodland Hills, CA) wa s used to assess the threshold of painful response to mechanical stimulation. The rigid ti p attached to the probe was pressed against the skin overlying the superficial masseter until an aversive response wa s displayed and the probe was removed. An aversive response included withdrawal of the head, voc alization, twitching of the back, struggling against restraint, or any co mbination thereof. The sensor displayed the force (g) needed to achieve this aversive response and this value was recorded by the investigator. Three measurements were taken at each test site, alternati ng between the left and right sides. Capsaicin Eye Wipe Sensitivity The capsaicin eye wipe response was assessed before and 1 hour after the cisterna m agna injections of RTX and vehicle. A ca psaicin solution (0.1%, intraocular, 50 l) was placed directly into the cornea and th e number of eye wipes was counte d for one minute (Karai et al. 2004). In preliminary studies, we found that RTX delivered intracisternally completely eliminated the capsaicin eye wipe response. Theref ore, we used this assay to verify that the RTX injections were successful. RTX injected ra ts that did not experi ence elimination of the capsaicin eye wipe response were re-injected. Immunohistochemistry We investigated the expression of T RPV1 in the trigeminal ganglion (TG) and the trigeminal nucleus caudalis (TNC) following RTX or vehicle treatment in the cisterna magna. After animals were euthanized, right and left trigeminal ganglia and brainstem tissues were

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19 immediately harvested and placed into a 10% formalin solution and post-fixed overnight. Samples were paraffin embedded, sectioned (10 m) and mounted on Fisher Plus slides. Following deparaffinization and epitope unmas king with Target Retrieval Solution (S1700, Dako, Carpinteria, CA, 60C overnight), sections were blocked with 10% normal goat serum (S1000, Vector Laboratories, Inc., Burlingame, CA) followed by a peroxidase quenching (Dako Peroxidase Blocking Reagent, S200 1) and incubated overnight at 4C with the TRPV1 primary antibody (1:10,000, rabbit an ti-VR1, Affinity Bioreagents). Antibody detection was performed using the Vectastain Elite ABC Goat anti-Rabbit IgG and Per oxidase Substrate Kits (SK-4700 and SK-4100, Vector Laboratories, Inc., Bu rlingame, CA) and visualized using 3,3diaminobenzidene tetrahydrochloride (DAB, V ector Lab, SK-4100). Control specimens for assessment of non-specific binding were processed in parallel, with the omission of the primary antibody. Histological secti ons were visualized u nder light microscopy. A blinded observer chose two sections from di fferent levels of the TG and TNC for each group (treated vs. non-treated) and the number of TRPV1 immunoreactive and nonimmunoreactive cells within a specific, standardized area were counted by visual inspection (100x magnification); the ratio of TRPV1 to the to tal number of small to medium sized cells was calculated. For brainstem sections, the presen ce of positive staining was graded by an observer blinded to the animal treatments using a 3 poi nt scale: 0=none; 1=li ght staining; 2=heavy staining. Ten sections at random for each treat ment were scored by visual inspection (10x objective). Statistical Analysis Data normality was assessed (Kolmogorov-Smirnov with Lilliefors Significance test) and the appropriate statistical analyses were co mpleted to determine whether the effects of

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20 temperature were significant. An ANOVA was used to evaluate the effects of treatment (none, inflammation/RTX, inflammation/vehicle) on mech anical sensitivity and operant thermal facial assessment outcomes at 48 C (SPSS Inc). An ANOVA was also used for the capsaicin eye wipe testing response to evaluate differences betw een the treatment groups and baseline. When significant differences were found, post-hoc comp arisons were made using the Tukey HSD, using a probability level of 0.05. In the trigeminal ganglia, the ratio of TRPV 1 to the total number of cells in the two treatment groups (RTX, vehicle) was compared usi ng an unpaired t-test. The Kruskal-Wallis test was used for brainstem histological comparisons A significance level of P < 0.05 was used in all instances.

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21 CHAPTER 3 RESULTS Operant Thermal Facial Assessment Previous studies by Neubert et al demonstr ated s ignificant temperature effects on the operant behavior of untreated animals (Neubert et al. 2006; Neubert et al. 2005b). In these studies, a number of outcomes were signi ficantly decreased at temperatures > 45.5C, including reward licking events, reward/attempts and the facial duration/contact ratios. Additionally, carrageenan-induced inflammation produced a signi ficant decrease on these outcome measures (Neubert et al. 2005b). Based on these studies, we chose to use the carrageenan-inflammation model and test animals at stimulus temperatures > 45.5C. Following intracisternal injections, vehicle animals that were tested at 48C showed no significant differe nce from 48C baseline values (data not shown). Note that RTX animals were not tested at 48C prior to carrageenan treatment because in preliminary studies these animals displayed insensitivity at this noxious stimulus temperature and therefore risk ed severe burning of their faces. There was a significant difference between RTX and vehicle groups for all operant thermal facial outcome measures following infla mmation with carrageenan, when tested at 48C (Figure 3-1). Five outcome meas ures were significantly higher in the RTX animals: intake, licking contact events, duration, ra tio licks/contacts, and ratio faci al duration/contacts. Facial contact events were significantly lower for RTX animals. Overall, these data indicate an analgesic effect following intracisternal RTX treatment. Mechanical Sensitivity At baseline, there was no signi fican t difference in mechanical sensitivity between the two groups. After intracisternal treatment, the RTX/ carrageenan group demonstrated a significantly higher threshold (P<0.05) as compared to base line and vehicle/carrageenan groups (Figure 3-2).

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22 The vehicle treated anim als had a significantly lower threshold (P<0.05) following carrageenan inflammation, as compared to baseline values. Th ese results indicate that carrageenan produced mechanical hyperalgesia that was blocked by intracisternal RTX treatment. Capsaicin Eye Wipe Sensitivity The capsaicin eye wipe response w as completely eliminated in RTX animals and remained intact in vehicle injected animals. There was no significant difference between the eye wipe response of baseline and vehicle animals following capsaicin applic ation. (Figure 3-3) Immunohistochemistry There were no significant diffe rences found in the ratio of TRPV1-expressing cells in the trig eminal ganglia for RTX-treated animals comp ared to vehicle-treated (Figure 3-4). There were significant differences found in the level of TRPV1 staining of the trigeminal nucleus caudalis between the two treatment groups (Fig ure 3-5). The RTX group showed a complete elimination of TRPV1-positive staining in the nucleu s caudalis. All RTX-treated animal sections (N=15) were scored zero for no st aining, while all sections (N=15) from vehicle-treated animals were scored two for heavy staining. Collectively these data indicate that the RTX intracisternal treatment was specific to lesioning of the centr al terminals of the TR PV1-expressing neurons, but did not lesion the entire neuron.

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23 Figure 3-1. RTX inhibits infl ammatory orofacial pain. Ther e was a significant difference (*P<0.05) in all outcomes between RTX and vehicle animals. These measures were taken 3h following induction of orofacial inflammation. Note th at all behavioral outcome measures are expressed as a percent of 48C baseline values.

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24 Von Frey Figure 3-2. Mechanical sensitivity assessment 3h after inflammation using Von Frey filaments. RTX significantly increased the threshold co mpared to both vehicle and baseline (+P<0.05). Vehicle treated animals disp layed a significantly lower threshold (*P<0.05) as compared to baseline. Figure 3-3. Capsaicin eye wipe response. RTX significantly eliminated the number of eye wipes following application of 0.1% cap saicin to the eye (P< 0.05).

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25 Figure 3-4. Effect of Intracister nal RTX on TRPV1 expressing cells in the trigeminal ganglia. Intracisternal (ICM) injection of RTX doe s not significantly re duce the number of TRPV1+ neurons within the trigeminal ganglia. Immunohistochemical analysis demonstrated that RTX (A) and vehicle (B ) treated animals had a similar proportion (C) of TRPV1+ cells 2 weeks following intr acisternal treatment with either RTX or vehicle. Figure 3-5. Effect of intracist ernal RTX on TRPV1 staining in the brainstem. This is a representative histological section of TRPV1+ staining in the brainstem following either intracisternal (ICM) inject ion of vehicle (A) or RTX (B, 250ng, 10 l). Note that there was comlete elimination of TR PV1 staining in the br ainstem for the RTXtreated animal.

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26 CHAPTER 4 DISCUSSION To evaluate the effects o f a therapy in the or ofacial region of animal s, one must consider the use of validated behavioral outcome measures. We used a va riety of tests that included reflex, unlearned, and operant measures to evaluate both mechanical and sensory aspects of pain. A novel operant thermal test assay was used to examine changes in thermal sensitivity after intracisternal targeting of TRPV1 receptors in the brainstem using RTX and subsequent inflammation in the face. Vehicl e/carrageenan animals tested at 48C demonstrated behavior indicative of hyperalgesia whereas the behavi or of RTX/carrageenan animals indicated analgesia. All six of the behavioral outco mes were significantly different for the RTX-treated animals as compared to the vehicle-treated animal s. For the RTX group, five of the outcomes significantly increased (intake, licking contact ev ents, duration, ratio licks/ contacts, ratio facial duration/contacts), which we interpret as an analgesic response. Additionally, the decrease in facial contact events also indicates analgesia b ecause the animal was able to keep its face on the thermode for a longer duration, therefore requiri ng fewer contacts to access the reward. For vehicle-treated animals, there was a significan t decrease in intake, licking contact events, duration, licks/contacts ratio, facial duration/contacts ratio, as comp ared to baseline testing at 48C. This is typical of a hyperalgesic response after carrageenan infl ammation and has been documented previously (Neubert et al. 2005b). However, there was no significant difference between baseline and vehicle facial contact events ( data not shown ). This is not completely surprising, because the number of facial contact events can increase due to the limited duration the animal can tolerate its face on the thermode. Since there is reduced duration with elevated contact attempts the duration/contact ratio was ve ry low in vehicle rats, confirming hyperalgesia.

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27 We assessed mechanical sensitivity using Von Frey filaments, with a head withdrawal being the endpoint to stimulation. This was completed to evaluate whether RTX is primarily affecting thermo-sensing versus mechano-sens ing neurons. We found that the RTX group had a significantly (P<0.05) higher thresh old than both vehicle and ba seline, while the vehicle group demonstrated mechanical hyperalgesia with a si gnificantly (P<0.05) lower threshold than RTX and baseline. This finding suggests that some mechanical nerve transmission is also being affected by the intracisternal RTX injections. This is interesting given our prior studies (Neubert et al. 2003; Neubert et al. 2008a; Neubert et al. 2005a), in whic h RTX was given subcutaneously in the foot, percutaneously in the trigeminal ganglion, and perineurally in the sciatic nerve, demonstrating little effect of RTX on mechanical sensitivity. This difference in mechanical sensitivity between peripherally and centrally admi nistered RTX would sugge st that the site of drug application may have different effects on mechanical responses. We completed a histological su rvey of TRPV1 in various poin ts in the trigeminal sensory pathway, including at the cell body level in the tr igeminal ganglion and at the brainstem level where the primary afferent neurons project centr ally into the nucleus caudalis. In previous studies, perineural appl ication of RTX around the infraorbital nerve (p<0.05) demonstrated a loss of TRPV1-expressing cells in the TG and orofacial pain responses are eliminated (Neubert et al. 2008b). Initially, we hypothesized that the deletion of TRPV1 from the pres ynaptic terminal of TRPV1 expressing neurons would also lead to deletion of their cell bodies within the TG following intracisternal RTX application. On th e contrary, this study showed that centrally delivered RTX causes specific elimination of TRPV1-positive staining in the nucleus caudalis but no differences in the ratio of TG TRPV 1 expressing cells following RTX treatment, suggesting that the central terminal is being clea ved off in a receptor specific fashion (Figure 4-

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28 1). This has interesting implications clinicall y, as central administrati on of RTX may have the advantage over peripheral delivery systems. This is due to its specifi c targeting of the presynaptic TRPV1 fibers with no damage to the TG TRPV1 cells or the peripheral fibers. The peripheral elimination of TG TRPV1 cells and infraorbital nerv e fibers may come with a price as the cell body may also perform other tasks and fu nctions. For example, different growth factors (e.g., nerve growth facto r) and neuropeptides (e.g., substan ce P) are colocalized in cells expressing TRPV1 and elimination of these f actors may have implications on healing. Although ~20% of the U.S. population is affected by a facial pain disorder, there still remains many questions regarding mechanisms of trigeminal pain processing. Current treatment modalities for chronic orofacial pain disorders ca n include the use of pharmaceutical agents such as the opioid receptor agonists (e.g., morphine) th at also cause CNS and respiratory depression. The goal of this project was to explore the use of alternative analgesic agents that lack these untoward side effects. Targeting the TRPV1 receptor with the agonist RTX in rats has led us one step closer to understanding molecular mechanisms of trigeminal pain tr ansduction in humans. This knowledge will provide a foundation for devel opment of novel therapeutic approaches that specifically target the trigeminal pain pathwa y without inducing sedati ve side effects and diminished the quality of life for those dealing with chronic facial pain. Further investigation is necessa ry to evaluate the negative si de effects that may occur due to removal of this receptor. For example, with loss of sensation associat ed with elimination of the TRPV1 receptor, the response to painful stimuli as a protectiv e mechanism against injury in these patients may be compromised. Also, TRPV 1 may have other roles of which we are not aware that could complicate the therapy. For th ese reasons, RTX therapy should initially be

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29 reserved to improve the quality of life for patients with terminal illnesses causing chronic facial pain.

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30 Figure 4-1. Central delivery of RTX A. Baseline animal with peri pheral and central TRPV1 receptors intact B. Delivery of RTX intr acisternally C. Specific deletion of TRPV1 only on the presynaptic fibers of the TNC with no effect on the TG cell bodies.

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31 CHAPTER 5 CONCLUSIONS The use of RTX has confirm ed the role of TR PV1 in trigeminal pain processing. This molecular understanding of the trigeminal pain mechanism is essential for the development of novel pain management techniques. In studies co mpleted in the lab including the body of work in this project, both peripheral and central ad ministration of RTX in the trigeminal region eliminated pain behavior in animals. We concl ude that intracisternal RTX treatment may be an effective means for specific pain control in the orofacial region.

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32 LIST OF REFERENCES Acs, G., Palkovits, M. and Blum berg, P.M., Sp ecific binding of [3H]re siniferatoxin by human and rat preoptic area, locus ceruleus, medi al hypothalamus, reticular formation and ventral thalamus membrane prepar ations, Life Sci, 59 (1996) 1899-908. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D. and Julius, D., The capsaicin receptor: a heat-activated ion cha nnel in the pain pathway, Nature, 389 (1997) 816-24. Caudle, R.M., Karai, L., Mena, N., Cooper, B.Y., Mannes, A.J., Perez, F.M., Iadarola, M.J. and Olah, Z., Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells, Neurotoxicology, 24 (2003) 895-908. Cesare, P. and McNaughton, P., A novel heat-activ ated current in nociceptive neurons and its sensitization by bradykinin, Proc Na tl Acad Sci U S A, 93 (1996) 15435-9. Clapham, D.E., TRP is cracked but is CRAC TRP?, Neuron, 16 (1996) 1069-72. Council, N., In: Guide for the Care and Use of Laboratory Animals., National Academy Press, Washington, DC, 1996. Denda, M., Fuziwara, S., Inoue, K., Denda, S., Ak amatsu, H., Tomitaka, A. and Matsunaga, K., Immunoreactivity of VR1 on epidermal keratin ocyte of human skin, Biochem Biophys Res Commun, 285 (2001) 1250-2. Gunthorpe, M.J., Benham, C.D., Randall, A. and Davis, J.B., The diversity in the vanilloid (TRPV) receptor family of ion channels, Trends Pharmacol Sci, 23 (2002) 183-91. Israel, H.A. and Scrivani, S.J., The interdisciplinar y approach to oral, faci al and head pain, J Am Dent Assoc, 131 (2000) 919-26. Karai, L., Brown, D.C., Mannes, A.J., Connelly, S.T., Brown, J., Gandal, M., Wellisch, O.M., Neubert, J.K., Olah, Z. and Iadarola, M.J., Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control, J Clin Invest, 113 (2004) 1344-52. Kissin, E.Y., Freitas, C.F. and Kissin, I., The effects of intraarticular resiniferatoxin in experimental knee-joint arthritis, Anesth Analg, 101 (2005a) 1433-9. Kissin, I., Bright, C.A. and Bradley, E.L., Jr., Se lective and long-lasting neural blockade with resiniferatoxin prevents inflammatory pa in hypersensitivity, Anesth Analg, 94 (2002) 1253-8, table of contents. Kissin, I., Davison, N. and Bradley, E.L., Jr., Perineural resinifera toxin prevents hyperalgesia in a rat model of postoperative pain, Anesth Analg, 100 (2005b) 774-80.

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33 LaMotte, R.H. and Campbell, J.N., Comparison of responses of warm and nociceptive C-fiber afferents in monkey with hu man judgments of thermal pain, J Neurophysiol, 41 (1978) 509-28. Lipton, J.A., Ship, J.A. and Larach-Robinson, D ., Estimated prevalence and distribution of reported orofacial pain in the United St ates, J Am Dent Assoc, 124 (1993) 115-21. McKemy, D.D., Neuhausser, W.M. and Julius, D., Identification of a cold receptor reveals a general role for TRP channels in th ermosensation, Nature, 416 (2002) 52-8. Neubert, J.K., Karai, L., Jun, J.H., Kim, H.S., Ol ah, Z. and Iadarola, M. J., Peripherally induced resiniferatoxin analgesi a, Pain, 104 (2003) 219-28. Neubert, J.K., Mannes, A.J., Karai, L.J., Jenkins, A.C., Zawatski, L., Abu-Asab, M. and Iadarola, M.J., Perineural resiniferatoxi n selectively inhibits inflammatory hyperalgesia, Mol Pain, 4 (2008a) 3. Neubert, J.K., Mannes, A.J., Keller, J., Wexel, M., Iadarola, M.J. and Caudle, R.M., Peripheral targeting of the trigeminal ga nglion via the infraorbital fora men as a therapeutic strategy, Brain Res Brain Res Protoc, 15 (2005a) 119-26. Neubert, J.K., Rossi, H.L., Malphurs, W., Vierc k, C.J., Jr. and Caudle, R.M., Differentiation between capsaicin-induced all odynia and hyperalgesia usi ng a thermal operant assay, Behav Brain Res, 170 (2006) 308-15. Neubert, J.K., Rossi, H.L., Mannes, A.J., Jenkins, A.C., Wexel, M. and Iadarola, M.J., Central targeting of trigeminal TRPV 1-expressing neurons for cont rol of orofacial pain, In preparation (2008b). Neubert, J.K., Widmer, C.G., Malphurs, W., Rossi, H.L., Vierck, C.J., Jr. and Caudle, R.M., Use of a novel thermal operant behavioral assa y for characterization of orofacial pain sensitivity, Pain, 116 (2005b) 386-95. Ng, C.H. and Ong, W.Y., Increased expression of gamma-aminobutyr ic acid transporters GAT-1 and GAT-3 in the spinal trigeminal nucleus af ter facial carrageenan injections, Pain, 92 (2001) 29-40. Peier, A.M., Moqrich, A., Hergarden, A.C., Reev e, A.J., Andersson, D.A., Story, G.M., Earley, T.J., Dragoni, I., McIntyre, P., Bevan, S. a nd Patapoutian, A., A TRP channel that senses cold stimuli and menthol, Cell, 108 (2002) 705-15. Simons, C.T., Gogineni, A.G., Iodi Carstens, M. and Carstens, E., Reduced aversion to oral capsaicin following neurotoxic destruction of superficial medullary neurons expressing NK-1 receptors, Brain Res, 945 (2002) 139-43.

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34 Szabo, T., Olah, Z., Iadarola, M.J. and Blumberg, P.M., Epidural re siniferatoxin induced prolonged regional analgesia to pa in, Brain Res, 840 (1999) 92-8. Szallasi, A. and Blumberg, P.M., Specific bindin g of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglion memb ranes, Brain Res, 524 (1990) 106-11. Szallasi, A. and Blumberg, P.M., Vanilloid receptors: new insights enhance potential as a therapeutic target, Pain, 68 (1996) 195-208. Szallasi, A., Blumberg, P.M., Annicelli, L.L., Kr ause, J.E. and Cortright, D.N., The cloned rat vanilloid receptor VR1 mediat es both R-type binding and C-type calcium response in dorsal root ganglion neurons, Mol Pharmacol, 56 (1999) 581-7. Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbert, H., Skinner, K., Raumann, B.E., Basbaum, A.I. and Julius, D., The cl oned capsaicin receptor integrates multiple pain-producing stimuli, Neuron, 21 (1998) 531-43. Wood, J.N., Winter, J., James, I.F., Rang, H.P., Y eats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J Neurosci, 8 (1988) 3208-20.

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35 BIOGRAPHICAL SKETCH Melanie W exel received her Bachelor of Sc ience at James Madison University, in Harrisonburg, Virginia in 2000. In 2004, she comple ted her Doctor of Dental Surgery degree at Virginia Commonwealth University School of Dentistry, in Richmond, Virginia. In 2005, she received a certificate of con tinuing education after a 1-year fellowship in or thodontics at the University of Florida. She began her orthodont ic residency in 2005 at University of Florida College of Dentistry. She is expected to gradua te with a certificate of orthodontics and a Master of Science in May 2008. She plans to practice orthodontics in central Virginia.