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1.  The Dolognawmeter: A Novel Instrument and Assay to Quantify Nociception in Rodent Models of Orofacial Pain 
Journal of neuroscience methods  2010;187(2):207-215.
Rodent pain models play an important role in understanding the mechanisms of nociception and have accelerated the search for new treatment approaches for pain. Creating an objective metric for orofacial nociception in these models presents significant technical obstacles. No animal assay accurately measures pain-induced orofacial dysfunction that is directly comparable to human orofacial dysfunction. We developed and validated a high throughput, objective, operant, nociceptive animal assay, and an instrument to perform the assay termed the dolognawmeter, for evaluation of conditions known to elicit orofacial pain in humans. Using the device our assay quantifies gnawing function in the mouse. We quantified a behavioral index of nociception and demonstrated blockade of nociception in three models of orofacial pain: (1) TMJ inflammation, (2) masticatory myositis, and (3) head and neck cancer. This assay will be useful in the study of nociceptive mediators involved in the development and progression of orofacial pain conditions and it will also provide a unique tool for development and assessment of new therapeutic approaches.
doi:10.1016/j.jneumeth.2010.01.012
PMCID: PMC2832714  PMID: 20096303
orofacial pain; oral cancer; cancer pain; myositis; TMJ pain; nociceptive assay
2.  Operant behavioral responses to orofacial cold stimuli in rats with chronic constrictive trigeminal nerve injury: effects of menthol and capsazepine 
Molecular Pain  2013;9:28.
Both spinal and trigeminal somatosensory systems use the TRPM8 channel as a principal transducer for detecting cold stimuli. It is currently unclear whether this cold transducer may play a role in trigeminal neuropathic pain manifesting cold allodynia and hyperalgesia. In the present study, trigeminal neuropathy was induced by chronic constrictive nerve injury of the infraorbital nerve (ION-CCI). Behavioral responses to cold stimuli in orofacial regions were assessed by the newly developed orofacial operant test in the ION-CCI rats. We tested menthol and capsazepine, two compounds that can activate and inhibit TRPM8 respectively, on orofacial operant responses to cold stimuli in ION-CCI rats. Testing animals performed operant tasks by voluntarily contacting their orofacial regions to a cold stimulation module in order to access sweetened milk as a reward, and contact time and number of the operant behaviors were automatically recorded. Total contact time was significantly reduced at the cooling temperatures of 17°C and 12°C in ION-CCI group in comparison with sham group, indicating the presence of cold allodynia and hyperalgesia in ION-CCI rats. When menthol was administered to ION-CCI rats, total contact time was further reduced and total contact number increased at the cooling temperatures. In contrast, after administration of capsazepine to ION-CCI rats, total contact time was significantly increased at the cooling temperatures. The behavioral outcomes support the idea that TRPM8 plays a role in cold allodynia and hyperalgesia following chronic trigeminal nerve injury.
doi:10.1186/1744-8069-9-28
PMCID: PMC3750444  PMID: 23767981
Trigeminal neuropathic pain; TRPM8 channel; Cold allodynia and hyperalgesia; Orofacial operant behavior test; Menthol; Capsazepine
3.  Orofacial pain management: current perspectives 
Journal of Pain Research  2014;7:99-115.
Some of the most prevalent and debilitating pain conditions arise from the structures innervated by the trigeminal system (head, face, masticatory musculature, temporomandibular joint and associated structures). Orofacial pain (OFP) can arise from different regions and etiologies. Temporomandibular disorders (TMD) are the most prevalent orofacial pain conditions for which patients seek treatment. Temporomandibular disorders include a number of clinical problems that involve the masticatory musculature, the temporomandibular joint (TMJ) or both. Trigeminal neuropathic pain conditions can arise from injury secondary to dental procedures, infection, neoplasias, or disease or dysfunction of the peripheral and/or central nervous system. Neurovascular disorders, such as primary headaches, can present as chronic orofacial pain, such as in the case of facial migraine, where the pain is localized in the second and third division of the trigeminal nerve. Together, these disorders of the trigeminal system impact the quality of life of the sufferer dramatically. A multidisciplinary pain management approach should be considered for the optimal treatment of orofacial pain disorders including both non-pharmacological and pharmacological modalities.
doi:10.2147/JPR.S37593
PMCID: PMC3937250  PMID: 24591846
pain; orofacial; neuropathic; TMD; trigeminal; headache
4.  Metabotropic glutamate receptor 5 contributes to inflammatory tongue pain via extracellular signal-regulated kinase signaling in the trigeminal spinal subnucleus caudalis and upper cervical spinal cord 
Background
In the orofacial region, limited information is available concerning pathological tongue pain, such as inflammatory pain or neuropathic pain occurring in the tongue. Here, we tried for the first time to establish a novel animal model of inflammatory tongue pain in rats and to investigate the roles of metabotropic glutamate receptor 5 (mGluR5)-extracellular signal-regulated kinase (ERK) signaling in this process.
Methods
Complete Freund’s adjuvant (CFA) was submucosally injected into the tongue to induce the inflammatory pain phenotype that was confirmed by behavioral testing. Expression of phosphorylated ERK (pERK) and mGluR5 in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were detected with immunohistochemical staining and Western blotting. pERK inhibitor, a selective mGluR5 antagonist or agonist was continuously administered for 7 days via an intrathecal (i.t.) route. Local inflammatory responses were verified by tongue histology.
Results
Submucosal injection of CFA into the tongue produced a long-lasting mechanical allodynia and heat hyperalgesia at the inflamed site, concomitant with an increase in the pERK immunoreactivity in the Vc and C1-C2. The distribution of pERK-IR cells was laminar specific, ipsilaterally dominant, somatotopically relevant, and rostrocaudally restricted. Western blot analysis also showed an enhanced activation of ERK in the Vc and C1-C2 following CFA injection. Continuous i.t. administration of the pERK inhibitor and a selective mGluR5 antagonist significantly depressed the mechanical allodynia and heat hyperalgesia in the CFA-injected tongue. In addition, the number of pERK-IR cells in ipsilateral Vc and C1-C2 was also decreased by both drugs. Moreover, continuous i.t. administration of a selective mGluR5 agonist induced mechanical allodynia in naive rats.
Conclusions
The present study constructed a new animal model of inflammatory tongue pain in rodents, and demonstrated pivotal roles of the mGluR5-pERK signaling in the development of mechanical and heat hypersensitivity that evolved in the inflamed tongue. This tongue-inflamed model might be useful for future studies to further elucidate molecular and cellular mechanisms of pathological tongue pain such as burning mouth syndrome.
doi:10.1186/1742-2094-9-258
PMCID: PMC3543209  PMID: 23181395
Metabotropic glutamate receptor 5; Extracellular signal-regulated kinase; Tongue pain; Inflammation; Trigeminal subnucleus caudalis; Upper cervical spinal cord
5.  Orofacial neuropathic pain mouse model induced by Trigeminal Inflammatory Compression (TIC) of the infraorbital nerve 
Molecular Brain  2012;5:44.
Background
Trigeminal neuropathic pain attacks can be excruciating for patients, even after being lightly touched. Although there are rodent trigeminal nerve research models to study orofacial pain, few models have been applied to studies in mice. A mouse trigeminal inflammatory compression (TIC) model is introduced here which successfully and reliably promotes vibrissal whisker pad hypersensitivity.
Results
The chronic orofacial neuropathic pain model is induced after surgical placement of chromic gut suture in the infraorbital nerve fissure in the maxillary bone. Slight compression and chemical effects of the chromic gut suture on the portion of the infraorbital nerve contacted cause mild nerve trauma. Nerve edema is observed in the contacting infraorbital nerve bundle as well as macrophage infiltration in the trigeminal ganglia. Centrally in the spinal trigeminal nucleus, increased immunoreactivity for an activated microglial marker is evident (OX42, postoperative day 70). Mechanical thresholds of the affected whisker pad are significantly decreased on day 3 after chromic gut suture placement, persisting at least 10 weeks. The mechanical allodynia is reversed by suppression of microglial activation. Cold allodynia was detected at 4 weeks.
Conclusions
A simple, effective, and reproducible chronic mouse model mimicking clinical orofacial neuropathic pain (Type 2) is induced by placing chromic gut suture between the infraorbital nerve and the maxillary bone. The method produces mild inflammatory compression with significant continuous mechanical allodynia persisting at least 10 weeks and cold allodynia measureable at 4 weeks.
doi:10.1186/1756-6606-5-44
PMCID: PMC3563613  PMID: 23270529
Orofacial neuropathic pain; Infraorbital nerve; Inflammation; Nerve compression; Chromic gut suture; Mechanical allodynia; Trigeminal ganglia; Trigeminal nucleus; Mice; Hypersensitivy; Tic douloureux
6.  Assessment of chronic trigeminal neuropathic pain by the orofacial operant test in rats 
Behavioural brain research  2012;234(1):82-90.
Classical behavioral tests in animal models of trigeminal neuropathic pain measure reflexive responses that are not necessarily measures of pain. To overcome the problem, we created a chronic constrictive nerve injury rat model of pain (CCI) by ligation of the infraorbital nerve (ION), and applied the orofacial operant test to assess behavioral responses to mechanical and cold stimulation in these rats. Animals were trained to voluntarily contact their facial region to a mechanical or a cold stimulation module in order to access sweetened milk as a positive reward. ION-CCI rats displayed aversive behaviors to innocuous mechanical stimuli, as indicated by a significant decrease in both contact time and the numbers of long contact events in comparison with sham group. For cold stimulation, ION-CCI rats displayed aversive behaviors to both innocuous (17 °C) and noxious cold temperatures (12 °C and 5 °C), as indicated by a significant decrease in both contact time and the numbers of long contact events at the cooling temperatures. The decreases of the contact time and numbers in ION-CCI rats were partially abolished by morphine. Our orofacial operant test demonstrates mechanical allodynia, cold allodynia, and hyperalgesia in rats with chronic trigeminal nerve injury. The neuropathic pain in ION-CCI rats was partially alleviated by morphine. Thus, orofacial operant test provides a desirable behavioral assessment method for preclinical studies of chronic trigeminal neuropathic pain.
doi:10.1016/j.bbr.2012.06.020
PMCID: PMC3418659  PMID: 22743005
Trigeminal neuropathic pain; Mechanical allodynia; Cold allodynia; Behavioral assessment; Operant behavior
7.  Spinal trigeminal neurons demonstrate an increase in responses to dural electrical stimulation in the orofacial formalin test 
Primary headaches are often associated with pain in the maxillofacial region commonly classified under the term “orofacial pain” (OFP). In turn, long-lasting OFP can trigger and perpetuate headache as an independent entity, which is able to persist after the resolution of the main disorder. A close association between OFP and headache complicates their cause and effect definition and leads to misdiagnosis. The precise mechanisms underlying this phenomenon are poorly understood, partly because of the deficiency of research-related findings. We combined the animal models of OFP and headache—the orofacial formalin test and the model of trigeminovascular nociception—to investigate the neurophysiological mechanisms underlying their comorbidity. In anesthetized rats, the ongoing activity of single convergent neurons in the spinal trigeminal nucleus was recorded in parallel to their responses to the electrical stimulation of the dura mater before and after the injection of formalin into their cutaneous receptive fields. Subcutaneous formalin resulted not only in the biphasic increase in the ongoing activity, but also in an enhancement of neuronal responses to dural electrical stimulation, which had similar time profile. These results demonstrated that under tonic pain in the orofacial region a nociceptive signaling from the dura mater to convergent trigeminal neurons is significantly enhanced apparently because of the development of central sensitization; this may contribute to the comorbidity of OFP and headache.
doi:10.1007/s10194-011-0404-7
PMCID: PMC3253149  PMID: 22116533
Formalin test; Trigeminal; Sensitization; Orofacial; Headache
8.  Spinal trigeminal neurons demonstrate an increase in responses to dural electrical stimulation in the orofacial formalin test 
Primary headaches are often associated with pain in the maxillofacial region commonly classified under the term “orofacial pain” (OFP). In turn, long-lasting OFP can trigger and perpetuate headache as an independent entity, which is able to persist after the resolution of the main disorder. A close association between OFP and headache complicates their cause and effect definition and leads to misdiagnosis. The precise mechanisms underlying this phenomenon are poorly understood, partly because of the deficiency of research-related findings. We combined the animal models of OFP and headache—the orofacial formalin test and the model of trigeminovascular nociception—to investigate the neurophysiological mechanisms underlying their comorbidity. In anesthetized rats, the ongoing activity of single convergent neurons in the spinal trigeminal nucleus was recorded in parallel to their responses to the electrical stimulation of the dura mater before and after the injection of formalin into their cutaneous receptive fields. Subcutaneous formalin resulted not only in the biphasic increase in the ongoing activity, but also in an enhancement of neuronal responses to dural electrical stimulation, which had similar time profile. These results demonstrated that under tonic pain in the orofacial region a nociceptive signaling from the dura mater to convergent trigeminal neurons is significantly enhanced apparently because of the development of central sensitization; this may contribute to the comorbidity of OFP and headache.
doi:10.1007/s10194-011-0404-7
PMCID: PMC3253149  PMID: 22116533
Formalin test; Trigeminal; Sensitization; Orofacial; Headache
9.  Antinociceptive Effects of H3 (R-Methylhistamine) and GABAB (Baclofen)-Receptor Ligands in an Orofacial Model of Pain in Rats 
Neurotoxicity Research  2013;24(2):258-264.
The present study explored the antinociceptive effects of H3 (R-alpha-methylhistamine) and GABAB (baclofen) receptor ligands in an orofacial model of pain in rats. Orofacial pain was induced by subcutaneous injection of formalin (50 μl, 5 %) in the upper lip region, and the number of jumps and time spent face rubbing was recorded for 40 min. Formalin produced a marked biphasic pain response; first phase, 0–10 min (jumps), and second phase, 15–40 min, (rubbing). Baclofen (50 μg) injected into the rat wiskerpad 5 min before formalin administration suppressed both phases of pain whereas R-alpha-methylhistamine (12.5 μg) abolished the first phase only. Brains were taken immediately after behavioral testing was completed. HPLC/ED analysis showed that 5-hydroxytryptamine (5-HT) turnover was increased in hippocampus, thalamus, and brain stem of all formalin groups, excepting the baclofen group in which the balance of 5-HT metabolism was restored to control values. These findings demonstrate that GABAB receptors represent peripheral targets for analgesia. Consequently, locally administered baclofen may be a useful approach in treating inflammatory trigeminal pain.
doi:10.1007/s12640-013-9385-4
PMCID: PMC3691488  PMID: 23463522
H3 receptor; GABAB receptor; Orofacial pain; Formalin test; Rats
10.  Spontaneous behavioral responses in the orofacial region: A model of trigeminal pain in mouse 
Headache  2012;53(1):137-151.
OBJECTIVES
To develop a translational mouse model for the study and measurement of non-evoked pain in the orofacial region by establishing markers of nociceptive-specific grooming behaviors in the mouse.
BACKGROUND
Some of the most prevalent and debilitating conditions involve pain in the trigeminal distribution. Although there are current therapies for these pain conditions, for many patients they are far from optimal. Understanding the pathophysiology of pain disorders arising from structures innervated by the trigeminal nerve is still limited and most animal behavioral models focus on the measurement of evoked pain. In patients, spontaneous (non-evoked) pain responses provide a more accurate representation of the pain experience than do responses that are evoked by an artificial stimulus. Therefore, the development of animal models that measure spontaneous nociceptive behaviors may provide a significant translational tool for a better understanding of pain neurobiology.
METHODS
C57BL/6 mice received either an injection of 0.9% Saline solution or complete Freund’s adjuvant (CFA) into the right masseter muscle. Animals were video recorded and then analyzed by an observer blind to the experiment group. The duration of different facial grooming patterns performed in the area of injection were measured. After 2 hrs, mice were euthanized, perfused and the brainstem was removed. Fos protein expression in the trigeminal nucleus caudalis was quantified using immunohistochemistry to investigate nociceptive-specific neuronal activation. A separate group of animals was treated with morphine sulfate, to determine the nociceptive-specific nature of their behaviors.
RESULTS
We characterized and quantified 3 distinct patterns of acute grooming behaviors: fore-paw rubbing, lower lip skin/cheek rubbing against enclosure floor and hind paw scratching. These behaviors occurred with a reproducible frequency and time course, and were inhibited by the analgesic morphine. CFA-injected animals also showed Fos labeling consistent with neuronal activation in nociceptive-specific pathways of the trigeminal nucleus after two hours.
CONCLUSIONS
These behaviors and their correlated cellular responses represent a model of trigeminal pain that can be used to better understand basic mechanisms of orofacial pain and identify new therapeutic approaches to this common and challenging condition.
doi:10.1111/j.1526-4610.2012.02226.x
PMCID: PMC3664930  PMID: 22830495
Trigeminal; mouse; orofacial; spontaneous nociception; pain
11.  Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior 
Molecular Pain  2008;4:43.
Background
Rodent models of orofacial pain typically use methods adapted from manipulations to hind paw; however, limitations of these models include animal restraint and subjective assessments of behavior by the experimenter. In contrast to these methods, assessment of operant responses to painful stimuli has been shown to overcome these limitations and expand the breadth of interpretation of the behavioral responses. In the current study, we used an operant model based on a reward-conflict paradigm to assess nociceptive responses in three strains of mice (SKH1-Hrhr, C57BL/6J, TRPV1 knockout). We previously validated this operant model in rats and hypothesized in this study that wild-type mice would demonstrate a similar thermal stimulus-dependent response and similar operant pain behaviors. Additionally, we evaluated the effects on operant behaviors of mice manipulated genetically (e.g., TRPV1 k.o.) or pharmacologically with resiniferatoxin (RTX), a lesioning agent for TRPV1-expressing neurons. During the reward-conflict task, mice accessed a sweetened milk reward solution by voluntarily position their face against a neutral or heated thermode (37–55°C).
Results
As the temperature of the thermal stimulus became noxiously hot, reward licking events in SKH1-Hrhr and C57BL/6J mice declined while licking events in TRPV1 k.o. mice were insensitive to noxious heat within the activation range of TRPV1 (37–52°C). All three strains displayed nocifensive behaviors at 55°C, as indicated by a significant decrease in reward licking events. Induction of neurogenic inflammation by topical application of capsaicin reduced licking events in SKH1-Hrhr mice, and morphine rescued this response. Again, these results parallel what we previously documented using rats in this operant system. Following intracisternal treatment with RTX, C57BL/6J mice demonstrated a block of noxious heat at both 48 and 55°C. RTX-treated TRPV1 k.o. mice and all vehicle-treated mice displayed similar reward licking events as compared to the pre-treatment baseline levels. Both TRPV1 k.o. and RTX-treated C57BL/6J had complete abolishment of eye-wipe responses following corneal application of capsaicin.
Conclusion
Taken together, these results indicate the benefits of using the operant test system to investigate pain sensitivity in mice. This ability provides an essential step in the development of new treatments for patients suffering from orofacial pain disorders.
doi:10.1186/1744-8069-4-43
PMCID: PMC2584042  PMID: 18828909
12.  Partial infraorbital nerve ligation as a model of trigeminal nerve injury in the mouse: Behavioral, Neural and Glial reactions 
Trigeminal nerve damage often leads to chronic pain syndromes including trigeminal neuralgia, a severely debilitating chronic orofacial pain syndrome. Options for treatment of neuropathic pain are limited in effectiveness and new approaches based on a better understanding of the underlying pathologies are required. Partial ligation has been shown to effectively mimic many of the qualities of human neuropathic pain syndromes. We have devised a mouse model of trigeminal neuralgia using a partial ligation of the infraorbital nerve (pIONL) that induces persistent pain behaviors and morphological changes in the brainstem. We found that the pIONL effectively induced mechanical allodynia lasting for over 3 weeks. Cell proliferation (BrdU), activation of astrocytes and microglia in the ipsilateral caudal medulla, and persistent satellite cell reaction in the ipsilateral ganglion were observed. Neurochemical markers CGRP, substance P were decreased in medullary dorsal horn ipsilateral to the injury side, whereas substance P receptor NK1 expression was increased after 8 days. Nerve injury marker ATF3 was markedly increased in ipsilateral trigeminal ganglion neurons at 8 days after pIONL. The data indicate that partial trigeminal injury in mice produces many persistent anatomical changes in neuropathic pain, as well as mechanical allodynia.
Perspective
The study describes the development of a new mouse model of trigeminal neuropathic pain. Our goal is to devise better treatments of trigeminal pain, and this will be facilitated by characterization of the underlying cellular and molecular neuropathological mechanisms in genetically designed mice.
doi:10.1016/j.jpain.2008.06.006
PMCID: PMC2632609  PMID: 18708302
infraorbital nerve; caudal medulla; allodynia; neuron; glia
13.  Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1–C2 neurons in trigeminal neuropathic pain 
Neuroscience letters  2011;491(1):8-12.
To clarify the involvement of GluR2 and GluR3 subunits of AMPA receptor in orofacial neuropathic pain, we studied changes in nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation followed by infraorbital nerve (ION)-partial transection model applied to GluR2 or GluR3 delta7 knock-in (KI) mice. In these animals, last seven amino acids of GluR2 or GluR3 subunit, the binding sites of interacting protein, are deleted in vivo. Head-withdrawal threshold to mechanical stimulation of the whisker pad skin ipsilateral to ION-partial transection was significantly reduced at 1, 3, 5, 7, 11 and 14 days after transection compared with that before transection in wild-type mice. In the GluR2 and GluR3 delta7 KI mice, the head-withdrawal threshold did not change following ION-partial transection. The number of pERK-LI cells examined in Vc and C1–C2 in wild-type mice after the non-noxious stimulation was larger than that of GluR2 and GluR3 delta7 KI mice.
The present findings suggest that GluR2 and GluR3 subunits of AMPA receptor play roles in the trigeminal nerve injury-mediated enhancement of Vc and C1–C2 neuronal excitability, and hyperalgesia.
doi:10.1016/j.neulet.2010.12.060
PMCID: PMC3130337  PMID: 21215292
Infraorbital nerve injury; Phosphorylation of extracellular; signal-regulated kinase; AMPA receptor; Neuropathic pain
14.  Influences of adult-onset diabetes on orofacial pain and related health behaviors 
Journal of public health dentistry  2010;70(2):10.1111/j.1752-7325.2009.00147.x.
OBJECTIVES
This study tested the hypothesis that persons with orofacial pain and comorbid adult-onset diabetes will experience greater functional and emotional impact than persons experiencing orofacial pain without diabetes.
METHODS
A random-digit dialing sampling procedure was used for a disproportionate probability sample of 10,341 persons who were screened for orofacial pain in the past 6 months and diabetes. This paper reports on 1,767 individuals reporting toothache pain and 877 reporting painful oral sores. A structured telephone interview assessed diabetes history, orofacial pain characteristics, oral health care behaviors, and emotional and functional impacts of orofacial pain.
RESULTS
The 6-month point prevalence was 16.8% for toothache pain, 8.9% for painful oral sores, and 9.6% for adult-onset diabetes. Individuals with comorbid orofacial pain and adult-onset diabetes differed significantly on many of the pain characteristics and health behaviors compared to non-diabetic sufferers of orofacial pain. Diabetics were more likely than non-diabetics to have pain every day, to suffer negative emotions associated with pain, to experience disruption of daily activities and sleep, to make an emergency room visit for orofacial pain, and to report the current need for a pain-related health care visit.
CONCLUSIONS
Although diabetes is well known to be associated with neuropathic pain, these results indicate that the experience of nociceptive pain is exacerbated by diabetes. Findings have significance for the subjective experience of oral pain, dental care outcomes and health-related quality of life associated with oral health outcomes among individuals with diabetes.
doi:10.1111/j.1752-7325.2009.00147.x
PMCID: PMC3813016  PMID: 19765201
orofacial pain; diabetes; toothache; self-care; self-medication
15.  Age and Skeletal Sites Affect BMP-2 Responsiveness of Human Bone Marrow Stromal Cells 
Connective tissue research  2009;50(4):270-277.
Bone marrow stromal cells (BMSCs) contain osteoprogenitors responsive to stimulation by osteogenic growth factors like bone morphogenetic proteins (BMPs). When used as grafts, BMSCs can be harvested from different skeletal sites such as axial, appendicular and orofacial bones, but the lower therapeutic efficacy of BMPs on BMSCs-responsiveness in humans compared to animal models may be partly due to effects of skeletal site and age of donor. We previously reported superior differentiation capacity and osteogenic properties of orofacial BMSCs relative to iliac crest BMSCs in same individuals. This study tested the hypothesis that recombinant human BMP-2 (rhBMP-2) stimulates human BMSCs differently based on age and skeletal site of harvest. Adult maxilla, mandible and iliac crest BMSCs from same individuals and pediatric iliac crest BMSCs were comparatively assessed for BMP-2 responsiveness under serum-containing and serum-free insulin-supplemented culture conditions. Adult orofacial BMSCs were more BMP-2-responsive than iliac crest BMSCs based on higher gene transcripts of alkaline phosphatase, osteopontin and osteogenic transcription factors MSX-2 and Osterix in serum-free insulin-containing medium. Pediatric iliac crest BMSCs were more responsive to rhBMP-2 than adult iliac crest BMSCs based on higher expression of alkaline phosphatase and osteopontin in serum-containing medium. Unlike orofacial BMSCs, MSX-2 and Osterix transcripts were similarly expressed by adult and pediatric iliac crest BMSCs in response to rhBMP-2. These data demonstrate that age and skeletal site-specific differences exist in BMSC osteogenic responsiveness to BMP-2 stimulation and suggest that MSX-2 and Osterix may be potential regulatory transcription factors in BMP-mediated osteogenesis of adult orofacial cells.
doi:10.1080/03008200902846262
PMCID: PMC2905683  PMID: 19637063
Bone morphogenic proteins; age; skeletal site; stem cells
16.  Activation of cyclin-dependent kinase 5 mediates orofacial mechanical hyperalgesia 
Molecular Pain  2013;9:66.
Background
Cyclin-dependent kinase 5 (Cdk5) is a unique member of the serine/threonine kinase family. This kinase plays an important role in neuronal development, and deregulation of its activity leads to neurodegenerative disorders. Cdk5 also serves an important function in the regulation of nociceptive signaling. Our previous studies revealed that the expression of Cdk5 and its activator, p35, is upregulated in nociceptive neurons during peripheral inflammation. The aim of the present study was to characterize the involvement of Cdk5 in orofacial pain. Since mechanical hyperalgesia is the distinctive sign of many orofacial pain conditions, we adapted an existing orofacial stimulation test to assess the behavioral responses to mechanical stimulation in the trigeminal region of the transgenic mice with either reduced or increased Cdk5 activity.
Results
Mice overexpressing or lacking p35, an activator of Cdk5, showed altered phenotype in response to noxious mechanical stimulation in the trigeminal area. Mice with increased Cdk5 activity displayed aversive behavior to mechanical stimulation as indicated by a significant decrease in reward licking events and licking time. The number of reward licking/facial contact events was significantly decreased in these mice as the mechanical intensity increased. By contrast, mice deficient in Cdk5 activity displayed mechanical hypoalgesia.
Conclusions
Collectively, our findings demonstrate for the first time the important role of Cdk5 in orofacial mechanical nociception. Modulation of Cdk5 activity in primary sensory neurons makes it an attractive potential target for the development of novel analgesics that could be used to treat multiple orofacial pain conditions.
doi:10.1186/1744-8069-9-66
PMCID: PMC3882292  PMID: 24359609
Cdk5; p35; Trigeminal ganglia; Orofacial pain; Mouse model
17.  Protective Effect of Leaves of Murraya koenigii on Reserpine-Induced Orofacial Dyskinesia 
Murraya koenigii L. (Rutaceae), commonly known as curry leaf tree, closely associated with south India where the word “curry” originates from the Tamil “kari” for spiced sauces. Curry leaves are a rich source of carbazole alkaloids which possess various biological activities such as antitumor, antioxidant and anti-inflammatory. Curry leaf has a potential role in the treatment of diabetes. Reserpine-induced orofacial dyskinesia in rats is an animal model of tardive dyskinesia that has been linked with free radical generation and oxidative stress. In this study, neuroprotective potential and in-vivo antioxidant status of methanol extract of the leaves of Murraya koenigii (MEMK) in reserpine-induced orofacial dyskinesia are investigated. Reserpine was used to induce orofacial dyskinesia. The effect of MEMK on locomotion and catalepsy was studied using Open-field apparatus and Bar-test, respectively. The effect of MEMK on the levels of protective anti-oxidant enzymes i.e. superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSH) and inhibited lipid peroxidation (LPO) in forebrain region were investigated in reserpine-treated animals. Results demonstrated that the MEMK significantly inhibited the reserpine-induced vacuous chewing movements (VCM), tongue protrusion (TP), orofacial burst (OB) and catalepsy. MEMK significantly increased the number of squares traversed and rearing in open field apparatus. Treatment with MEMK significantly restored the levels of protective anti-oxidant enzymes i.e. SOD, CAT, GSH and inhibited LPO in forebrain region when compared with reserpine. It also inhibited haloperidol-induced catalepsy. The present study concludes that the oxidative stress might play an important role in reserpine-induced abnormal oral movements, and Murraya koenigii may have great potential in the treatment of neuroleptic-induced orofacial dyskinesia
PMCID: PMC3832166  PMID: 24250488
Vacuous chewing movements; Tongue protrusions; Free radicals; Murraya koenigii
18.  Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury 
PLoS ONE  2014;9(3):e92264.
Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good therapeutic tool to attenuate programmed cell death, including apoptosis and autophagy, consequent to CCI-induced peripheral nerve injury.
doi:10.1371/journal.pone.0092264
PMCID: PMC3958488  PMID: 24642655
19.  Astroglia in Medullary Dorsal Horn (Trigeminal Spinal Subnucleus Caudalis) Are Involved in Trigeminal Neuropathic Pain Mechanisms 
The aim of this study was to investigate whether astroglia in the medullary dorsal horn (trigeminal spinal subnucleus caudalis; Vc) may be involved in orofacial neuropathic pain following trigeminal nerve injury. The effects of intrathecal administration of the astroglial aconitase inhibitor sodium fluoroacetate (FA) were tested on Vc astroglial hyperactivity [as revealed by glial fibrillary acid protein (GFAP) labeling], nocifensive behavior, Vc extracellular signal-regulated kinase phosphorylation (pERK), and Vc neuronal activity in inferior alveolar nerve-transected (IANX) rats. Compared with sham-control rats, a significant increase occurred in GFAP-positive cells in ipsilateral Vc at postoperative day 7 in IANX rats, which was prevented following FA administration. FA significantly increased the reduced head withdrawal latency to high-intensity heat stimulation of the maxillary whisker pad skin in IANX rats, although it did not significantly affect the reduced escape threshold to low-intensity mechanical stimulation of the whisker skin in IANX rats. FA also significantly reduced the increased number of pERK-like immunoreactive cells in Vc and the enhanced Vc nociceptive neuronal responses following high-intensity skin stimulation that were documented in IANX rats, and glutamine administration restored the enhanced responses. These various findings provide the first documentation that astroglia is involved in the enhanced nociceptive responses of functionally identified Vc nociceptive neurons and in the associated orofacial hyperalgesia following trigeminal nerve injury.
doi:10.1523/JNEUROSCI.3365-09.2009
PMCID: PMC2804401  PMID: 19741123
20.  Endoneurial pathology of the needlestick-nerve-injury model of Complex Regional Pain Syndrome, including rats with and without pain behaviors 
Current rodent models of neuropathic pain produce pain hypersensitivity in almost all lesioned animals and not all identified experimental effects are pain specific. 18G needlestick-nerve-injury (NNI) to one tibial nerve of outbred Sprague-Dawley rats models the phenotype of Complex Regional Pain Syndrome (CRPS), a post-traumatic neuropathic pain syndrome, leaving roughly half of NNI rats with hyperalgesia. We compared endoneurial data from these divergent endophenotypes searching for pathological changes specifically associated with pain-behaviors. Tibial, sural, and common sciatic nerves from 12 NNI rats plus 10 nerves from sham-operated controls were removed 14 days post-surgery for morphometric analysis. PGP9.5+ unmyelinated-fibers were quantitated in plantar hindpaw skin. Distal tibial nerves of NNI rats had endoneurial edema, 30% fewer axons, twice as many mast cells, and thicker blood-vessel walls than uninjured tibial nerves. However the only significant difference between nerves from hyperalgesic versus non-hyperalgesic NNI rats was greater endoneurial edema in hyperalgesic rats (p < 0.01). We also discovered significant axonal losses in uninjured ipsilateral sural nerves of NNI rats, demonstrating spread of neuropathy to nearby nerves formerly thought spared. Tibial and sural nerves contralateral to NNI had significant changes in endoneurial bloodvessels. Similar pathological changes have been identified in CRPS-I patients. The current findings suggest that severity of endoneurial vasculopathy and inflammation may correlate better with neuropathic pain behaviors than degree of axonal loss. Spread of pathological changes to nearby ipsilateral and contralesional nerves might potentially contribute to extraterritorial pain in CRPS.
doi:10.1016/j.ejpain.2011.05.004
PMCID: PMC3176948  PMID: 21676634
Neuralgia; reflex sympathetic dystrophy; hyperalgesia; allodynia; dystonia; morphometry; mast cells
21.  Satellite glial cell P2Y12 receptor in the trigeminal ganglion is involved in lingual neuropathic pain mechanisms in rats 
Molecular Pain  2012;8:23.
Background
It has been reported that the P2Y12 receptor (P2Y12R) is involved in satellite glial cells (SGCs) activation, indicating that P2Y12R expressed in SGCs may play functional roles in orofacial neuropathic pain mechanisms. However, the involvement of P2Y12R in orofacial neuropathic pain mechanisms is still unknown. We therefore studied the reflex to noxious mechanical or heat stimulation of the tongue, P2Y12R and glial fibrillary acidic protein (GFAP) immunohistochemistries in the trigeminal ganglion (TG) in a rat model of unilateral lingual nerve crush (LNC) to evaluate role of P2Y12R in SGC in lingual neuropathic pain.
Results
The head-withdrawal reflex thresholds to mechanical and heat stimulation of the lateral tongue were significantly decreased in LNC-rats compared to sham-rats. These nocifensive effects were apparent on day 1 after LNC and lasted for 17 days. On days 3, 9, 15 and 21 after LNC, the mean relative number of TG neurons encircled with GFAP-immunoreactive (IR) cells significantly increased in the ophthalmic, maxillary and mandibular branch regions of TG. On day 3 after LNC, P2Y12R expression occurred in GFAP-IR cells but not neuronal nuclei (NeuN)-IR cells (i.e. neurons) in TG. After 3 days of successive administration of the P2Y12R antagonist MRS2395 into TG in LNC-rats, the mean relative number of TG neurons encircled with GFAP-IR cells was significantly decreased coincident with a significant reversal of the lowered head-withdrawal reflex thresholds to mechanical and heat stimulation of the tongue compared to vehicle-injected rats. Furthermore, after 3 days of successive administration of the P2YR agonist 2-MeSADP into the TG in naïve rats, the mean relative number of TG neurons encircled with GFAP-IR cells was significantly increased and head-withdrawal reflex thresholds to mechanical and heat stimulation of the tongue were significantly decreased in a dose-dependent manner compared to vehicle-injected rats.
Conclusions
The present findings provide the first evidence that the activation of P2Y12R in SGCs of TG following lingual nerve injury is involved in the enhancement of TG neuron activity and nocifensive reflex behavior, resulting in neuropathic pain in the tongue.
doi:10.1186/1744-8069-8-23
PMCID: PMC3386019  PMID: 22458630
Neuron-Glia interactions; Lingual nerve injury; Mechanical allodynia; Heat hyperalgesia; Purinergic receptor
22.  Differential involvement of trigeminal transition zone and laminated subnucleus caudalis in orofacial deep and cutaneous hyperalgesia: the effects of interleukin-10 and glial inhibitors 
Molecular Pain  2009;5:75.
Background
In addition to caudal subnucleus caudalis (Vc) of the spinal trigeminal complex, recent studies indicate that the subnuclei interpolaris/caudalis (Vi/Vc) transition zone plays a unique role in processing deep orofacial nociceptive input. Studies also suggest that glia and inflammatory cytokines contribute to the development of persistent pain. By systematically comparing the effects of microinjection of the antiinflammatory cytokine interleukin (IL)-10 and two glial inhibitors, fluorocitrate and minocycline, we tested the hypothesis that there was a differential involvement of Vi/Vc and caudal Vc structures in deep and cutaneous orofacial pain.
Results
Deep or cutaneous inflammatory hyperalgesia, assessed with von Frey filaments, was induced in rats by injecting complete Freund's adjuvant (CFA) into the masseter muscle or skin overlying the masseter, respectively. A unilateral injection of CFA into the masseter or skin induced ipsilateral hyperalgesia that started at 30 min, peaked at 1 d and lasted for 1-2 weeks. Secondary hyperalgesia on the contralateral site also developed in masseter-, but not skin-inflamed rats. Focal microinjection of IL-10 (0.006-1 ng), fluorocitrate (1 μg), and minocycline (0.1-1 μg) into the ventral Vi/Vc significantly attenuated masseter hyperalgesia bilaterally but without an effect on hyperalgesia after cutaneous inflammation. Injection of the same doses of these agents into the caudal Vc attenuated ipsilateral hyperalgesia after masseter and skin inflammation, but had no effect on contralateral hyperalgesia after masseter inflammation. Injection of CFA into the masseter produced significant increases in N-methyl-D-aspartate (NMDA) receptor NR1 serine 896 phosphorylation and glial fibrillary acidic protein (GFAP) levels, a marker of reactive astrocytes, in Vi/Vc and caudal Vc. In contrast, cutaneous inflammation only produced similar increases in the Vc.
Conclusion
These results support the hypothesis that the Vi/Vc transition zone is involved in deep orofacial injury and suggest that glial inhibition and interruption of the cytokine cascade after inflammation may provide pain relief.
doi:10.1186/1744-8069-5-75
PMCID: PMC2806354  PMID: 20025765
23.  Chronic Constriction Injury of the Infraorbital Nerve in the Rat using modified syringe needle 
Journal of neuroscience methods  2008;172(1):43-47.
Here we report a method for performing a chronic constriction injury (CCI) of the infraorbital nerve (ION) in the rat as a component of a chronic pain model. The surgical approach to the ION is described together with the use of a modified dental syringe needle that simplifies placing two chromic gut ligatures around the ION. This method makes the surgical procedure easier, the nerve injury more consistent across animals and reduces secondary damage to the ION and surrounding tissue. Pain behavior testing together with immunostaining for markers of nerve injury in the spinal trigeminal nucleus show the suitability of this procedure as a model of orofacial pain.
doi:10.1016/j.jneumeth.2008.04.013
PMCID: PMC2497464  PMID: 18501433
CCI; ION; trigeminal ganglion; technique; neuropathy; chronic pain; animal model; microsurgery
24.  A New Program in pain medicine for medical students: Integrating core curriculum knowledge with emotional and reflective development 
Pain medicine (Malden, Mass.)  2011;12(2):186-195.
Objective
Improvements in clinical pain care have not matched advances in scientific knowledge, and innovations in medical education are needed. Several streams of evidence indicate that pain education needs to address both the affective and cognitive dimensions of pain. Our aim was to design and deliver a new course in pain establishing foundation-level knowledge while comprehensively addressing the emotional development needs in this area.
Setting
118 first year medical students at Johns Hopkins School of Medicine.
Outcome measures
Performance was measured by multiple choice tests of pain knowledge, attendance, reflective pain portfolios and satisfaction measures.
Results
Domains of competence in pain knowledge included central and peripheral pain signaling, pharmacological management of pain with standard analgesic medications, neuromodulating agents and opioids; cancer pain, musculoskeletal pain, nociceptive, inflammatory, neuropathic, geriatric, and pediatric pain. Socio-emotional development (portfolio) work focused on increasing awareness of pain affect in self and others and enhancing the commitment to excellence in pain care. Reflections included observations on a brief pain experience (cold pressor test), the multi-dimensionality of pain, the role of empathy and compassion in medical care, the positive characteristics of pain-care role models, the complex feelings engendered by pain and addiction including frustration and disappointment, and aspirations and commitments in clinical medicine. The students completing feedback expressed high levels of interest in pain medicine as a result of the course.
Discussion
We conclude that a four-day pain course incorporating sessions with pain- specialists, pain medicine knowledge, and design-built elements to strengthen emotional skills is an effective educational approach.
doi:10.1111/j.1526-4637.2010.01050.x
PMCID: PMC3078763  PMID: 21276187
25.  Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome 
BMC Neuroscience  2010;11:71.
Background
Voltage gated sodium channels Nav1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders.
Aims and Objectives
To study Nav1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS), considered a neuropathic orofacial pain disorder.
Methods
Two groups of patients were recruited for this study. One group consisted of patients with dental pulpitis pain (n = 5) and controls (n = 12), and the other patients with BMS (n = 7) and controls (n = 10). BMS patients were diagnosed according to the International Association for the Study of Pain criteria; a pain history was collected, including the visual analogue scale (VAS). Immunohistochemistry with visual intensity and computer image analysis were used to evaluate levels of Nav1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group.
Results
There was a significantly increased visual intensity score for Nav1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Nav1.7 immunoreactive % area in the painful pulp group, but this was not statistically significant. When expressed as a ratio of the neurofilament % area, there was a strong trend for an increase of Nav1.7 in the painful pulp group. Nav1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls.
Conclusion
Nav1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Nav1.7 channel blockers should prioritise dental pulp pain rather than BMS.
doi:10.1186/1471-2202-11-71
PMCID: PMC2890014  PMID: 20529324

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