Orofacial Mechanical Sensitivity in Rats

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Title:
Orofacial Mechanical Sensitivity in Rats
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english
Creator:
Bass, Daniel C
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Dental Sciences, Dentistry
Committee Chair:
Neubert, John K
Committee Members:
Dolce, Calogero
Caudle, Robert M

Subjects

Subjects / Keywords:
mechanical -- operant -- orofacial -- rat
Dentistry -- Dissertations, Academic -- UF
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Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
Objectives: The goal was to assess the behavioral responses to mechanical facial stimulation under normal (baseline), analgesic and hyperalgesic conditions. Methods: Animals were trained to place their face against looped nickel titanium wires (0.008”, 0.010”, 0.012”, 0.014”, and 0.016”) providing access to positive reinforcement in a reward conflict paradigm. Rats were tested under naïve, analgesic (morphine 1 and 3 mg/kg, SNC-80 5 and 10 mg/kg, s.c.) and algesic (capsaicin, 0.075%, s.c.) conditions, under conditions of gas anesthesia (isoflurane, 2.5%), and with isoflurane, capsaicin, and morphine 3mg/kg together. We evaluated the reward stimulus:contact ratio (mean±s.e.m.) as our pain outcome measure. Results: Wire diameters of 0.014” and 0.016” produced significantly lower stimulus:contact ratios compared to wires with a diameter of 0.010” under naïve conditions. Morphine treatment at 1mg/kg did not result in a significant increase from naïve values using the 0.010” diameter (t-test, p=0.1087) wire, nor did it show an increase with the 0.016” (p=0.5006) diameter wire. Morphine (3mg/kg) response tested on 0.014” showed a significant increase in the success ratio for morphine treatment (p= 0.0073) as compared to the vehicle group. Facial injection of capsaicin induced a significant decrease (p=0.0180) in the success ratio for wire diameter 0.010". Morphine 3mg/kg reversed the nociceptive effect of the capsaicin injection (p=0.0370). SNC 80 (10mg/kg) showed a significant increase in the success ratio (p= 0.0161) while 5mg/kg caused no significant difference from vehicle injection. Conclusion: Radially arranged looped nickel titanium wires provide an effective mechanism for operant evaluation of facial pain.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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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.
Statement of Responsibility:
by Daniel C Bass.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Neubert, John K.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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1 OROFACIAL MECHANICAL SENSITIVITY IN RATS By DANIEL BASS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Daniel Bass

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3 To my wife

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4 ACKNOWLEDGEMENTS I thank Jesus for giving me peace, and m y wife for always supporting me. I thank Drs. Neubert and Nolan for their wealth of knowledge and advice I thank Vitaly for his t echnical assistance in making the testing plates and I thank my research committee for their guidance I thank my parents for encouraging me to always be excellent.

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5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF FIGURES ................................ ................................ ................................ .......... 6 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 Research Problem ................................ ................................ ................................ .. 10 Literature Review ................................ ................................ ................................ .... 10 Background ................................ ................................ ................................ ...... 11 Pain assessment in rodents ................................ ................................ ....... 12 Preclinical orofacial pain models ................................ ................................ 14 Animal pain assessment and drug testing ................................ ................. 15 Summary ................................ ................................ ................................ .......... 16 2 MATERIALS AND METHODS ................................ ................................ ................ 18 Animals ................................ ................................ ................................ ................... 18 Mechanical Operant Testing System ................................ ................................ ...... 18 Mechanical Stimulus Response Study ................................ ............................. 20 Analgesia response study ................................ ................................ .......... 20 Inflammation response study ................................ ................................ ..... 21 Statistical analysis ................................ ................................ ...................... 21 3 RESULTS ................................ ................................ ................................ ............... 22 Mechanical Stimulus Response Study ................................ ................................ .... 22 Analgesia Response Study ................................ ................................ .............. 22 Inflammation Response Study ................................ ................................ .......... 22 4 DISCUSSION ................................ ................................ ................................ ......... 23 Mechanical Stimulus Response Study ................................ ................................ .... 23 Analgesia Response Study ................................ ................................ .............. 23 Inflammation Response Study ................................ ................................ .......... 25 5 CONCLUSION ................................ ................................ ................................ ........ 27 LIST OF REFERENCES ................................ ................................ ............................... 35 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 40

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6 LIST OF FIGURES Figure page 2 1 Picture of the plate that is attached to the testing box. 42mm diameteropening in the plate with a 14mm diameter opening between the ................................ ................................ ...... 28 2 2 Picture showing the relationship of the milk dropper just outside of the looped wire arrangement. Animals must insert their faces through the wire arrangement in order to obtain the sweetened milk rewar d. ............................... 29 2 3 event. Facial contacts are also recorded as the face touches the grounded Niti wires. ................................ ................................ ................................ ............ 29 3 1 Graph showing the success ratios of nave testing with the different Niti wire p<0.001). ................................ ................................ ................................ ............ 30 3 2 a. wire size. ................................ ................................ ................................ ............ 30 3 2 b Graph showing success ratios of morphine (1mg/kg, p=0.5006) at 0.016 wires. ................................ ................................ ................................ .................. 31 3 3 wires. ................................ ................................ ................................ .................. 31 3 4 Graph showing success ratios of nave vs nave having been under ................................ ............. 32 3 5a Graph showing success ratios of SNC ...... 32 3 5b Graph showing success ratios of SNC (p= 0.0161). ................................ ................................ ................................ ........ 33 3 6 Graph showing success ratio of capsaicin under isoflur (p= 0.0180). ................................ ................................ ................................ ........ 33 3 7 Graph showing success ratios of morphine (3mg/kg, p= 0.0370) and PBS,both having isoflurane anesthesia and capsaicin injection ......................... 34

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7 LIST OF ABBREVIATIONS N I T I Nickel Titanium wires OFP Orofacial Pain PBS Phosphate Buffered Saline S.C. Sub Cutaneous

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillme nt of the Requirements for the Degree of Master of Science OROFACIAL MECHANICAL SENSITIVITY IN RATS By Daniel Bass May 2012 Chair: John Neubert Major : Dental Sciences Orthodontics Objectives: The goal was to assess the behavioral responses to mechanica l facial stimulation under normal (baseline), analgesic and hyperalgesic conditions. Methods: Animals were trained to place their face against looped nickel titanium wires ement in a reward conflict paradigm. Rats were tested under nave, analgesic (morphine 1 and 3 mg/kg, SNC 80 5 and 10 mg/kg, s.c .) and algesic (capsaicin, 0.075%, s.c.) conditions under conditions of gas anesthesia (isoflurane, 2.5%) and with isoflurane, capsaicin, and morphine 3mg/kg together. We evaluated the reward stimulus:contact ratio (means.e.m.) as our pain outcome measure. stimulus:contact ratios compared to wires with a conditions. Morphine treatment at 1mg/kg did not result in a significant increase from test, p=0.1087) wire, nor did it show an hine (3mg/kg) response tested 0.0073) as compared to the vehicle group. Facial injection of capsaicin induced a

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9 significant decrease (p=0.0180) in the success ratio for wire diameter 0.010". Morphine 3mg/kg reversed the nociceptive effect of the capsaicin injection (p= 0.0370) SNC 80 (10mg/kg) showed a significant increase in the success ratio (p= 0.0161) while 5mg/kg caused no significant difference from vehicle injecti on. Conclusion: Radially arranged looped nickel titanium wires provide an effective mechanism for oper ant evaluation of facial pain.

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10 CHAPTER 1 INTRODUCTION Research Problem Operant models for assessing mechanical orofacial pain in rodents are not wel l characterized. If we can validate a mechanical operant model for behavioral assessment in rodents, the result will have a significant impact on the way new analgesics are screened and evaluated. Making this validated model available to the scientific com munity will help to screen drugs more efficiently, speeding the process of getting new pain therapies on the market for patients who need relief. Literature Review Orofacial pain disorders have been well described in humans; however, evaluation of orofaci al pain in animals has been difficult 1 There are few animal models in existence that can replicate the human pain experience, especially for the facial region. This lack of testing devices is a problem because it slows drug development and evaluation. Ne ubert et al. have developed a novel thermal operant behavioral assay for characterization of orofacial pain sensitivity 1 They have also demonstrated the utility and innovativeness of the operant assay, using a variety of pain conditions (e.g., inflammatio n, heat, cold), pain states (e.g., allodynia), environmental conditions, and pharmacological agents 1 5 The development of this assay was a major advance in OFP translational studies because it provided the first thermal operant model to quantitatively eva luate pain within the trigeminal system. The benefit of this model compared to reflex based models is that it more closely represents the human pain experience through

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11 emotional and motivational inclusion, incorporation of higher level cognitive processing in evaluating pain. A significant problem for many patients with orofacial pain is that they experience mechanical pain, including touch evoked allodynia, a condition characterized by pain associated with normally non painful stimuli. Mechanical allodyni a affects many patients with burn and trauma, as well as specific disorders such as trigeminal neuralgia. For example, patients with trigeminal neuralgia could find simple mechanical stimuli such as tooth brushing or face washing to be unbearable. Or anoth er example, putting on a shirt is normally not a painful experience; however, after having a severe sunburn, putting on a shirt can be extremely painful. In dentistry, patients undergoing active orthodontic treatment can have facial pain to normal stimuli due to the sensitivity of the teeth. All of these problems mentioned involve mechanical sensitivity. Although the thermal operant behavioral assay has been thoroughly validated, an operant model for assessing mechanical orofacial pain in rodents is not wel l characterized. Previous work in our lab has assessed punctate sensation in a mechanical facial operant assay 6 .The current study uses a mechanical facial operant assay to assess pressure sensation in facial pain. It is important to distinguish between the two different mechanical stimuli because they represent different sensations and may involve different pain pathways. Background The development of novel analgesics is a lengthy process involving a number of different stages, including animal modeling, measurement, and assessment of efficacy. Animal models are intended to replicate human pain conditions, including acute inflammatory pain, chronic arthritic pain, and neuropathic pain conditions. Given that the human pain condition can be induced in the ro dent, the next hurdle is to utilize a reliable

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12 method for assessing the pain. Drug testing is the final step, whereby new pharmacological agents are then evaluated against standard analgesics using these models. Pain assessment in rodents The pain produce d in animal models has been evaluated by assessing: 1) simple reflexes, 2) unlearned or innate behaviors, and 3) learned or operant behaviors 7 No single assessment can evaluate all aspects of pain, with each assessment having advantages and disadvantages. Simple reflexes, such as paw withdrawal and tail flick, do not require learning and can occur automatically 8 During the reflex response, sensory signaling to the spinal cord along sensory neurons initiates a reflex arc that triggers a signal directly back to the involved muscles. The advantages of simple reflex assessments are that they are easy to perform and the results can be related to similar human studies. However, the simple reflex is not directly assessing the amount of pain, it measures the segmental connection between the sensory, intermediate, and motor nerve cell s to deliver a response 7 Reflex response testing has been shown in both spinalized and decerebrated animals. One study demonstrated that after mid thoracic spinalization, both thermal and mechanical stimuli were able to elicit tail flick and hindlimb with drawal reflexes 9 Another study showed that chronic decerebrate animals with aspiration of all the cranial contents rostral to the mesencephalon have intact spinal reflexes in response to mild noxious thermal and mechanical stimuli 1 0 While reflex respons es depend on segmental processing, unlearned or innate behaviors such as paw licking, face rubbing, limb guarding, vocalization, grooming, escape responses, che w ing/biting, or a combination of these behaviors are mediated by

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13 supraspinal or suprasegmental p rocessing 1 1 17 Some methods for assessing these behaviors include the hot plate test 17 the Hargreaves test 13 and Von Frey filament 34 testing which allow a variety of the behaviors to be monitored. Cold allodynia was also demonstrated in a neuropathic pa in model that measured innate behaviors such as flicking and guarding 1 8 Innate behaviors are useful because they are relatively easy to perform and deliver reliable results; however, they are not able to discriminate allodynia from hyperalgesia. In develo ping more ideal testing conditions, one must consider that the experimenter performing the tests and the testing lab have a significant impact on measuring nociception 1 9,20 Also, restraint stress, which is common in many cases of behavior analysis, can be a confounding factor. For example, the magnitude and duration of analgesia produced by opioid agonists was dose dependently increased in restrained vs. unrestrained rats 2 1,22 Therefore, removing the variable of experimenter manipulation and developing in dependent measures of assessing pain behavior is highly important Operant response assays provide the conditions to allow for independent testing of animals where the animal chooses the amount of nociception they endure 23 In reward/conflict paradigms, th e animal decides how much nociceptive stimuli it will endure and is able to modify its behavior based on several factors involving cortical processing. Additionally, the lack of animal restraint in these models removes the confounding variable of restraint stress inherent to other pain testing techniques. Another significant benefit of these strategies is that the procedure can be automated after the animal is placed in the testing box. The automation allows for a high

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14 throughput of behavioral data to be ob tained because several animals can be tested simultaneously. Preclinical orofacial pain models Orofacial pain models do exist, however, the difficulty in making a good model lies in translating from the hindpaw to the facial region 2 3 29 These few assays i nclude assessment of mechanical sensitivity using von Frey filaments 3 0,32 and thermal sensitivity 3 1 using unlearned behaviors such as grooming 3 1,32 or via indirect measures such as food intake 3 3 The problems with these assays are stress and anticipation of the stimulus, as the animal is either aware of the stimulus or is restrained. Even when the animal is unrestrained, it may be able to see the stimulus coming and experience an anticipatory effect. Recently, Neubert et al. have utilized an operant desi gn to assay orofacial pain in rodents 1,2 They used a reward/conflict paradigm to assess thermal hyperalgesia/allodynia in a novel assay in which animals endured painful stimuli to receive a positive reward 1,2 In this assay, animals were fasted and traine d to voluntarily place their face against a stimulus thermode at different temperatures, which provided access to positive reinforcement sweetened condensed milk. The outcomes measured include number of licking contacts on the dropper with the milk reward number of contacts on the thermode bars, and duration of facial contact on the thermode bars. As the temperature increased, facial thermode contacts increased and the duration decreased, replacing long periods of contact/drinking with more numerous short drinking attempts. Also, the ratio of reward licks/facial contact events was significantly decreased as the temperature increased 1 When an inflammatory response was induced, significant thermal allodynic and hyperalgesic

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15 effects were seen. These exaggera ted responses were blocked with morphine pre treatment 2 Animal pain assessment and drug testing Developing a standard for testing is important to allow pharmacological agents to properly characterize nociceptive pathways. Uniform animal testing is needed 3 4 because different measures of pain, such as reflex and operant, can produce different results using similar nociceptive stimuli. The differing results make interpretation of drug for controlling clinical pain, has been used in many animal pain models and drugs, it is important to understand that different behavioral assays can give d ifferent outcomes to the same dose of drug. For example, while high doses of morphine (> 3mg/kg) 3 5 38 can inhibit a reflex based outcome, these doses can also impare motor and motivational responses 3 9 42 and can compromise interpretation of results, as an imals can become immobilized or unresponsive 4 3 It has been shown that low doses (<1 mg/kg) of morphine can decrease the sensitivity to painful stimuli and enhance unlearned behaviors when tested at the same stimulus temperatures 45 These results show that operant assays allow for assessing drugs within their clinically relevant dose range, while minimizing negative side effects. has only been since the 20 th century that major advancements in the knowledge of how opiates like morphine exert their powerful and selective effects 4 4 It was discovered in the 1960s and 70s that different opioid receptors existed. Since then, much work has been done to characterize these receptors and to create agonist drugs that would

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16 activate analgesia while minimizing negative side effects. The classic opioid drug morphine, which targets the receptor, has been shown to exhibit a good anti nociceptive profile in many animal models; however, morphin e also exhibits many negative side effects, including constipation, respiratory depression, and loss of motor coordination 4 5 opioids have been shown to have fewer side effects after systemic administration in rats, although their analgesic efficiency h as been reported to be inferior to opioids 45 SNC opioid agonist that has potential to be a good analgesic drug and needs to be characterized in operant testing. Summary behavioral model are to remove confounding factors, such as restraint and anticipation, and have the ability to observe behavior indicative of pain intensity. Neubert et al. have developed such an operant test to examine pain behavior with a thermal stimu lus 1 After validating the operant assay, it has been used to study thermal allodynia and hyperalgesia 2 cold allodynia 4 3 the effect environment plays on thermal sensitivity 5 thermal sensitivity 4 6 the effect of lesioning neurons on pain 4 7 and animal thermal preference 4 The operant test validated in 2005 has made a large impact on how thermal pain is studied. However, a similar mechanical operant test still needs to be developed to study pressure sensitivity. To address t his current deficit, we propose an innovative, automated, software controlled operant behavioral approach for assessing mechanical pressure induced orofacial pain. This approach takes advantage of the operant model, using higher level processing to more co nvincingly evaluate human pain conditions in rodents, and applies its benefits to testing and treating mechanical allodynia. Additionally, the speed with which drugs can be

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17 evaluated is greater using an automated operant system as compared with reflexive t esting with von Frey filaments, translating into quicker development of novel drugs at a reduced cost.

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18 CHAPTER 2 MATERIALS AND METHOD S Animals Experiments were performed on adult male Sprague Dawley rats (200 470g). Animals were housed two per cage, und er a 12 hour light/dark cycle, in a temperature and humidity controlled environment. Animals were fasted for 15 18 hrs prior to each testing day and were provided with standard food chow and water ad libitum following each session. All testing was done bet ween 9:00am and 1:00pm. All experimental protocols conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Mechanical O perant Testing System The testing device consists of a testing cage (20.3 cm W x 20.3 cm D x 16.2 cm H) with acrylic walls constructed with an opening on the back wall (4 x 6 cm). On the back of the cage, a hard circular plastic plate was secured over the opening that has embedded in it eight looped nickel titanium wires arranged in a radial fashion to act as the mechanical stimulus. We chose nickel titanium (NiTi) wires due to their high elasticity and ability to deliver the same force reliably, and the ra dial design because it allowed us to have contacts all around the face. When the animals are placed in the clear acrylic box, they have access to a standard rodent watering bottle that contains a diluted (1:2 with water) sweetened condensed milk solution ( Nestle, room temp) by placing their head through the opening between the wire arrangement. The hard plastic plates have a 42mm diameter circular hole cut out of the center. Eight NiTi wires of the same diameter are cut at 10cm in length and folded over on

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19 themselves with stainless steel crimpable stops cinched over the two wire ends. The wires are secured in place, leaving a 14mm diameter opening for the rats to push through in getting the reward. After the wires are secured, copper is wrapped around the p late, contacting all the niti wires (Figure 2 1). The free end of the copper wire connected to the plate has a female adapter that connects to a male adapter on a copper wire that is plugged into the multi channel data acquisition module (WinDaq Lite Data Acq D148U, DATAQ Instruments, Inc). The reward bottle was positioned just outside of the opening through the wire arrangement (Figure 2 2). The metal spout on the watering bottle was connected to a DC power supply and, in series, to the multi channel data acquisition module. When the animal drank from the bottle, the tongue contacted the metal spout on the water bottle, completing an electrical circuit (Figure 2 3). The closed circuit was registered in the was established from the metal wire to the animal by grounding the floor with an to determine if the animal was discouraged by the mechanical stimulus. The investigator monitored online data acquisition to ensure that each recorded licking event from the first circuit corresponded to a recorded facial contact on the wires (the second circuit). This ensured that the animal was not acce ssing the reward without contacting the wires, thus eliminating false positive recordings of licks. A complete testing session lasts for twenty minutes. For each behavioral experiment, we evaluated the following operant outcome measures: total number of licking events, total number of facial contacts, and the lick/facial contact ratio. We

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20 classify the lick/facial contact ratio as our pain indices and lower values are representative of a painful behavior 1 Mechanical Stimulus Response Study Rats (N=8) wer e first trained to the operant task by using non nociceptive plates testing period (20min), we determined that they were trained to do the task of accessing the milk. We then evaluated the effects of increasing wire diameter on the operant of three testin g sessions/week to minimize excessive fasting of the animals in any given week. All the animals were fasted for 15 16 hours and at the same time to control for level of appetite. Analgesia response study Animals were injected with morphine (1mg/kg, and 3mg /kg, s.c.), and SNC 80, a delta opioid receptor agonist (5mg/kg, and 10mg/kg, s.c.). Each testing session where a drug was used had half of the animals injected with the drug and the other half injected with phosphate buffered saline (PBS, 0.9%, s.c.) as a control for the effects of the injection. The analgesic drugs and PBS were administered between the scapulae 30 original animals were tested They were then tested with SNC80 (5mg/kg and 10mg/kg, s. c.) and morphine (3mg/kg)

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21 Inflammation response study The original eight animals were anesthetized with isoflurane for delivery of a capsaicin injection. Each rat was injected (s.c. ) with 20ul of either capsaicin (0.075%) or PBS bilate rally over the center of the body of the superficial masseter. Half of the rats tested received the capsaicin and the other half received PBS 30 minutes prior to The new set of 12 rats was also tested with capsaicin anesthetized with isoflurane and received the same capsaicin injections mentioned above, while half of the animals received morphine (3mg/kg, s.c.) and the other half PBS (s.c.). Statistical analysis Statistical analysis inclu ded an unpaired t test to compare between two different treatment groups. Additionally, comparison of multiple wire types was performed using a one hoc comparisons. All statistical evaluations were made using GraphPad Prism (v 4.02, GraphPad Software, San Diego, CA). Significance was set at p< 0.05.

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22 CHAPTER 3 RESULTS Mechanical Stimulus Response Study ratios of 2.80.6 and 1.80.4, respectiv ely (ANOVA, p<0.001) as compared with a 1). This data indicates that the larger diameter wires produced an aversive response. Analgesia Response Study Morphine (1mg/kg) 2 a and b) wires.Morphine (1mg/kg) did not produce a significant increase in the success ratio on 3 3) wires. There w as a significant increase in the success ratio for morphine treatment wires showed a significant increase in the success ratio compared to nave on the same wire size ( p= 0.0016) (Figure 3 4). SNC 80 at 5mg/kg did not show a significant increase in the success ratio compared to PBS (Figure 3 5a). SNC 80 at 10mg/kg did show a significant increase in the success ratio compared to PBS, p= 0.0161 (Figure 3 5b). Inflammation Response Study When both capsaicin and saline animals were anesthetized with isoflurane, (Figure 3 6). Morphi ne (3mg/kg, s.c.) was significant in reducing the pain response 7).

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23 CHAPTER 4 DISCUSSION Mechanical Stimulus Response Study We were successful in validating a new mechanical operant test ing model in rodents evaluating pressure mechanical sensation. We showed that as the force levels upon the face increased with the increasing wire diameter, success ratios decreased. This indicates that the animals sensed the aversive stimuli and decided t hat the reward was increasingly not worth the sensation they would have to endure when the larger wires were in place. r enough resistance. The rats were able to penetrate too much of the hole and may have become distracted with the task at hand. In addition to low resistance, another factor influencing the lower ratio on volved. Because the tickling sensation depends in part on the nerves that transmit pain 4 8 we chose it for our baseline measurements. Analgesia Response Study due to its lowest success measurement in baseline testing; however, this wire seemed to be too rigid to allow for an increased response even with an algesic drugs. For that analgesic drugs against in the new batch of animals.

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24 Our data shows that morphine 1mg/kg did not have a significant increase on the success ratio of indicates that morphine at 1mg/kg was not efficacious at reducing OFP from pressure sensation in our operant model. However, at 3mg/kg morphine produced a significant difference in the succes s ratio (p=0.0073). Although these results are consistent with other projects showing that morphine is dose dependent in relieving mechanical pain 4 9,50 we anticipated that our pressure operant testing model would be similar to the punctate model in the m orphine response. This difference may be accounted for by differing numbers of mu opiod receptors on the afferent pain fibers transmitting the pressure versus punctate mechanical pain. Although isoflurane has been used as an inhaled anesthetic for nearly t hirty years 5 1 we did not anticipate that anesthetizing the rats briefly thirty minutes prior to testing would not have an effect on the results. However, isoflurane was recently shown to significantly suppress spontaneous paw flinches and paw withdrawal i n rats induced by thermal and mechanical stimuli in a dose dependent manner 51 While this study was analyzing the anti nociceptive effects of an intrathecal injection of isoflurane, our study shows that a brief period of inhaled isoflurane thirty minutes p rior to testing had a significant anti nociceptive effect specific to mechanical testing. This was an interesting finding that should be addressed in future studies. Error! Bookmark not defined. After studying SN C 80 at 5 and 10 mg/kg, we found that at 10mg/kg, there was a significant increase in the success ratio and that 5mg/kg caused no significant difference from vehicle injection. Sluka et al. assessed the effects of opioid agonists on mechanical hyperalgesia induced by repeated intramuscular injections of acid and

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25 found that SNC80 dose dependently increased the mechanical withdrawal threshold back toward baseline responses 5 2 The significant increases in threshold did not occur until a higher dosage of 30nmol was reached. Another experiment showed SNC80 produced neither a blockade nor a prolongation of the tail withdrawal response latency even at the highest dose tested (80 mg/kg) 5 3 The supraspinal methods used to assess pain in these tests require higher con centrations of SNC 80 to produce analgesia. We have shown in our operant assay that SNC 80 is dose dependent in relieving pressure mechanical pain and that 10mg/kg is capable of providing significant analgesia without negative side effects. The ability of opioid agonists to block nociception is influenced by many factors, as opioids have different profiles in different pain models 5 4,55 The test used to assess nociception also may have a dramatic effect on opioid potency 55 We have characterized both mu and delta opioid efficacy in the operant assay to be used as baseline data to test new drugs against in the future. Inflammation Response Study Capsaicin significantly reduced the success ratio as compared to saline. This is similar to previous studies that h ave also shown capsaicin reducing mechanical success measurements 2,6,56 Intraplantar injection of capsaicin in rats was shown to increase the frequency of withdrawal to von Frey filaments 58 Facial application of capsaicin cream was shown to reduce the s uccess outcome in a punctate mechanical operant model 6 Our tests have shown a capsaicin injection superficial to the masseter muscles can reduce success ratios in a pressure mechanical operant model, thus this provides a simple, reproducible model of neur ogenic inflammation.

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26 Morphine 3mg/kg reversed the nociceptive effect of the capsaicin injection. Another study analyzing the effects of morphine injection on the inflammatory response induced by intraplantar capsaicin injection showed dose dependent attenu ation of pain responses with morphine 5 7 They found that morphine induced pain attenuation was dose dependent and significant at 1mg/kg, 3mg/kg, and 10mg/kg, measuring the paw withdrawal threshold to von Frey filaments. Quantifying the effective morphine d ose in each animal model is important to accurately relate to human studies, which have shown the ability of morphine 1mg/kg in humans to reduce capsaicin induced mechanical hypersensitivity 5 8

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27 CHAPTER 5 CONCLUSION Our novel mechanical operant testing ass ay will fill the current void of validated automated in vivo assays for measuring mechanical pressure pain and provide a key to the advancement of translational pain research. This is accomplished because the assay provides behavioral measures that require expression of both the physiological and cortical aspects of pain. As pain spans any number of diseases, ranging from diabetes to cancer, quickly identifying an analgesic to treat the pain would provide a tremendous societal benefit. After hundreds of yea rs using the same analgesic drugs and billions of dollars spent every year on treating pain patients, new methods of assessing pain are requisite if we hope to discover new effective treatments. The successful translation of even one novel therapy that dem onstrates significant analgesic properties without compromising other normal functions would provide an avenue for significant commercial success of our operant assay. Future studies will evaluate wire density, structural arrangement, and differing pain c onditions. We will also further adapt this operant facial testing paradigm for use with mice. This line of research is significant because it allows for investigator independent measures of facial pain, which can lead to better assessments of novel analges ic therapies.

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28 Figure 2 1. Picture of the plate that is attached to the testing box. 42mm diameter opening in the plate with a 14mm diam eter opening between the wires.

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29 Figure 2 2. Picture showing the relationship of the m il k dropper just outside of the looped wire arrangement. Animals must inse rt their faces through the wire arrangement in order to obtain the sweetened milk reward. Figure 2 3. Picture showing the tongue contacting the mil e vent. Facial contacts are also recorded as the face touches the grounded Niti wires

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30 Figure 3 1. Graph showing the success ratios of nave testing with the different Niti wire diameters. Signific ANOVA, p< 0.001). Figure 3 2 a. Graph showing the success ratio of morphine (1mg/kg, p= 0.1087) at

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31 Figure 3 2 b. Graph showing success ratios of morphine (1mg/kg, p=0.5006 wires. Figure 3 3. Graph showing success ratio o f morphin wires.

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32 Figure 3 4. Graph showing success ratios of nave vs nave having been under Figure 3 5a. Graph showing success ratios of SNC 80 (5mg/kg) vs PBS a wires.

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33 Figure 3 5b. Graph showing success ratios of SN C wires (p= 0.0161). Figure 3 (p= 0.0180).

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34 Figure 3 7. Graph showing suc cess ratios of morphine (3mg/kg, p= 0.0370) and PBS both having isoflurane anesthesia and capsaicin injection

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40 BIOGRAPHICAL SKETCH Daniel Bass was raised in Sarasota, Florida where he attended Pine View School for the Gifted from 2 nd grade through high school. After graduating high school, he dawned the Ora nge and Blue colors on a journey towards a Bachelor of Arts in Business Administration He enjoyed the University of Florida so much that after graduating with a bachelor s degree, he decided to stay for another four years to complete dental school. He was surely glad that he stayed and got to witness all of the Gator national championship victories! After graduating dental school and getting married all in the same weekend, he was prepared and excited to stay another three years at the University of Florid a to complete his post doctorate training in the dental specialty of orthodontics. After an amazing eleven years at the University of Florida, he became a husband, father of a beautiful one year old princess, and a certified orthodontist. Dr. Bass open ed an orthodontic practice in Wellington, FL. He was sad to leave the home town of his alma mater, but he is happy to live close to the Atlantic ocean, where he enjoys surfing.