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Christie RA Leite-Panissi,1 Bruna B De Paula,2 John K Neubert,3 Robert M Caudle2 1Department of Psychology, School of Philosophy, Science and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil; 2Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, FL, USA; 3Department of Orthodontics, University of Florida, Gainesville, FL, USACorrespondence: Robert M Caudle, Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, FL, USA. PO Box 100416. 1395 Center Drive, Gainesville, FL, 32610, USA, Tel +1 352-273-6767, Fax +1 352-392-7609, Email caudle@ufl.eduBackground: Temporomandibular joint (TMJ)-associated inflammation contributes to the pain reported by patients with temporomandibular disorders (TMD). It is common for patients diagnosed with TMD to report pain in the masticatory muscles and temporomandibular joints, headache, and jaw movement disturbances. Although TMD can have different origins, including trauma and malocclusion disorder, anxiety/depression substantially impacts the development and maintenance of TMD. In general, rodent studies on orofacial pain mechanisms involve the use of tests originally developed for other body regions, which were adapted to the orofacial area. To overcome limitations and expand knowledge in orofacial pain, our group validated and characterized an operant assessment paradigm in rats with both hot and cold stimuli as well mechanical stimuli. Nevertheless, persistent inflammation of the TMJ has not been evaluated with this operant orofacial pain assessment device (OPAD).Methods: We characterized the thermal orofacial sensitivity for cold, neutral, and hot stimuli during the development of TMD using the OPAD behavior test. In addition, we evaluated the role of transient receptor potential vanilloid 1 (TRPV1) expressing nociceptors in rats with persistent TMJ inflammation. The experiments were performed in male and female rats with TMJ inflammation induced by carrageenan (CARR). Additionally, resiniferatoxin (RTX) was administered into the TMJs prior CARR to lesion TRPV1-expressing neurons to evaluate the role of TRPV1-expressing neurons.Results: We evidenced an increase in the number of facial contacts and changes in the number of reward licks per stimulus on neutral (37°C) and cold (21°C) temperatures. However, at the hot temperature (42°C), the inflammation did not induce changes in the OPAD test. The prior administration of RTX in the TMJ prevented the allodynia and thermal hyperalgesia induced by CARR.Conclusion: We showed that TRPV-expressing neurons are involved in the sensitivity to carrageenan-induced pain in male and female rats evaluated in the OPAD.Keywords: temporomandibular disorder, acute pain, TRPV1, operant assay, orofacial pain

Inter-relationships between pain sensitivity, drug reward, and drug misuse are of considerable interest given that many analgesics exhibit misuse potential. Here we studied rats as they underwent a series of pain- and reward-related tests: cutaneous thermal reflex pain, induction and extinction of conditioned place preference to oxycodone (0.56 mg/kg), and finally the impact of neuropathic pain on reflex pain and reinstatement of conditioned place preference. Oxycodone induced a significant conditioned place preference that extinguished throughout repeated testing. Correlations identified of particular interest included an association between reflex pain and oxycodone-induced behavioral sensitization, and between rates of behavioral sensitization and extinction of conditioned place preference. Multidimensional scaling analysis followed by k-clustering identified three clusters: (1) reflex pain, rate of behavioral sensitization and rate of extinction of conditioned place preference (2) basal locomotion, locomotor habituation, acute oxycodone-stimulated locomotion and rate of change in reflex pain during repeated testing, and (3) magnitude of conditioned place preference. Nerve constriction injury markedly enhanced reflex pain but did not reinstate conditioned place preference. These results suggest that high rates of behavioral sensitization predicts faster rates of extinction of oxycodone seeking/reward, and suggest that cutaneous thermal reflex pain may be predictive of both., (© 2023. The Author(s).)

Trigeminal neuralgia is the most common neuropathic pain involving the craniofacial region. Due to the complex pathophysiology, it is therapeutically difficult to manage. Noradrenaline plays an essential role in the modulation of arousal, attention, cognitive function, stress, and pain. The locus coeruleus, the largest source of noradrenaline in the brain, is involved in the sensory and emotional processing of pain. This review summarizes the knowledge about the involvement of noradrenaline in acute and chronic trigeminal pain conditions and how the activity of the locus coeruleus noradrenergic neurons changes in response to acute and chronic pain conditions and how these changes might be involved in pain-related comorbidities including anxiety, depression, and sleep disturbance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Inc.)

The prescription opioid oxycodone is widely used for the treatment of pain in humans. Oxycodone misuse is more common among people with an anxiety disorder than those without one. Therefore, oxycodone might be misused for its anxiolytic properties. We investigated if oxycodone affects anxiety-like behavior in adult male and female rats. The rats were treated with oxycodone (0.178, 0.32, 0.56, or 1 mg/kg), and anxiety-like behavior was investigated in the elevated plus-maze test. Immediately after the elevated plus-maze test, a small open field test was conducted to determine the effects of oxycodone on locomotor activity. In the elevated plus-maze test, oxycodone increased the percentage of time spent on the open arms, the percentage of open arm entries, time on the open arms, open arm entries, and the distance traveled. The males treated with vehicle had a lower percentage of open arm entries than the females treated with vehicle, and oxycodone treatment led to a greater increase in the percentage of open arm entries in the males than females. Furthermore, the females spent more time on the open arms, made more open arm entries, spent less time in the closed arms, and traveled a greater distance than the males. In the small open field test, treatment with oxycodone did not affect locomotor activity or rearing. Sex differences were observed; the females traveled a greater distance and displayed more rearing than the males. In conclusion, oxycodone decreases anxiety-like behavior in rats, and oxycodone has a greater anxiolytic-like effect in males than females., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube. In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures. Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

The chemotherapeutic agent oxaliplatin is commonly used to treat colorectal cancer. Although effective as a chemotherapeutic, it frequently produces painful peripheral neuropathies. These neuropathies can be divided into an acute sensitivity to cool temperatures in the mouth and face, and chronic neuropathic pain in the limbs and possible numbness. The chronic neuropathy also includes sensitivity to cool temperatures. Neurons that detect cool temperatures are reported to utilize Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Therefore, we investigated the effects of oxaliplatin on facial nociception to cool temperatures (18°C) in mice and on TRPM8 expressing trigeminal ganglion (TRG) neurons. Paclitaxel, a chemotherapeutic that is used to treat breast cancer, was included for comparison because it produces neuropathies, but acute cool temperature sensitivity in the oral cavity or face is not typically reported. Behavioral testing of facial sensitivity to 18°C indicated no hypersensitivity either acutely or chronically following either chemotherapeutic agent. However, whole cell voltage clamp experiments in TRPM8 expressing TRG neurons indicated that both oxaliplatin and paclitaxel increased Hyperpolarization-Activated Cyclic Nucleotide-Gated channel (HCN), voltage gated sodium channel (Na v ), and menthol evoked TRPM8 currents. Voltage gated potassium channel (K v ) currents were not altered. Histological examination of TRPM8 fibers in the skin of the whisker pads demonstrated that the TRPM8 expressing axons and possible Merkel cell-neurite complexes were damaged by oxaliplatin. These findings indicate that oxaliplatin induces a rapid degeneration of TRG neuron axons that express TRPM8, which prevents evoked activation of the sensitized neurons and likely leads to reduced sensitivity to touch and cool temperatures. The changes in HCN, Na v , and TRPM8 currents suggest that spontaneous firing of action potentials may be increased in the deafferented neurons within the ganglion, possibly producing spontaneously induced cooling or nociceptive sensations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caudle and Neubert.)

Rodent models of human disease can be valuable for understanding the mechanisms of a disease and for identifying novel therapies. However, it is critical that these models be vetted prior to committing resources to developing novel therapeutics. Failure to confirm the model can lead to significant losses in time and resources. One model used for migraine headache is to administer nitroglycerin to rodents. Nitroglycerin is known to produce migraine-like pain in humans and is presumed to do the same in rodents. It is not known, however, if the mechanism for nitroglycerin headaches involves the same pathological processes as migraine. In the absence of known mechanisms, it becomes imperative that the model not only translates into successful clinical trials but also successfully reverse translates by demonstrating efficacy of current therapeutics. In this study female rats were given nitroglycerin and nociception was evaluated in OPADs. Estrous was not monitored. Based on the ED 50 of nitroglycerin a dose of 10 mg/kg was used for experiments. Sumatriptan, caffeine, buprenorphine and morphine were administered to evaluate the reverse translatability of the model. We found that nitroglycerin did not produce mechanical allodynia in the face of the rats, which is reported to be a consequence of migraine in humans. Nitroglycerin reduced the animals' participation in the assay. The reduced activity was verified using an assay to measure exploratory behavior. Furthermore, the effects of nitroglycerin were not reversed or prevented by agents that are effective acute therapies for migraine. Two interesting findings from this study, however, were that morphine and nitroglycerin interact to increase the rats' tolerance of mechanical stimuli on their faces, and they work in concert to slow down the central motor pattern generator for licking on the reward bottle. These interactions suggest that nitroglycerin generated nitric oxide and mu opioid receptors interact with the same neuronal circuits in an additive manner. The interaction of nitroglycerin and morphine on sensory and motor circuits deserves additional examination. In conclusion, based on the results of this study the use of nitroglycerin at these doses in naïve female rats is not recommended as a model for migraine headaches., Competing Interests: JN and RC are co-founders of Velocity Laboratories, LLC, which provided funding for this project. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Caudle, Caudle, Flenor and Neubert.)

Objective: To assess changes in orofacial tactile sensitivity and gnawing related to capsaicin-mediated cutaneous, myogenic, and arthrogenic nociception in the rat., Design: After recovery from anesthesia, orofacial tactile sensitivity and gnawing were assessed using operant testing methods following capsaicin application. Twenty female CD-Hairless rats were tested with bilateral capsaicin cream application to the cheek or with isoflurane anesthesia alone. Following several weeks of recovery, animals (n = 20) received either 10 μL unilateral masseter injections of vehicle, or phosphate buffered saline (PBS) to assess injection sensitization. After several weeks, masseter capsaicin (1.0%) injections (10 μL) were assessed compared to vehicle and PBS (n = 13). Weeks later capsaicin TMJ injections were evaluated. Animals (n = 11) received either 10 μL unilateral TMJ injections of capsaicin solution (1%) or vehicle., Results: Capsaicin cream to the skin significantly altered gnawing activity (increased puncture time by 248 s (p = 0.0002)) and tactile sensitivity (decreased tolerated bottle distance by 0.980 cm compared to isoflurane only (p = 0.0001)). Similarly, capsaicin masseter injection increased puncture time (339.6 s, p = 0.07) and decreased tolerated bottle distance (1.04 cm, p = 0.005) compared to vehicle. However, intra-articular capsaicin in the TMJ only modified gnawing (increased puncture time by 133 s), with no changes found in tactile sensitivity compared to vehicle., Conclusion: Application of capsaicin to the skin and masseter had similar behavioral effects; however, intra-articular injections to the TMJ only affected gnawing. These data indicate the behavioral changes in rodent models of myogenic and cutaneous pain may be markedly different than models of arthrogenic pain originating from the TMJ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Agonists of the vanilloid receptor transient vanilloid potential 1 (TRPV1) are emerging as highly efficacious nonopioid analgesics in preclinical studies. These drugs selectively lesion TRPV1+ primary sensory afferents, which are responsible for the transmission of many noxious stimulus modalities. Resiniferatoxin (RTX) is a very potent and selective TRPV1 agonist and is a promising candidate for treating many types of pain. Recent work establishing intrathecal application of RTX for the treatment of pain resulting from advanced cancer has demonstrated profound analgesia in client-owned dogs with osteosarcoma. The present study uses transcriptomics and histochemistry to examine the molecular mechanism of RTX action in rats, in clinical canine subjects, and in 1 human subject with advanced cancer treated for pain using intrathecal RTX. In all 3 species, we observe a strong analgesic action, yet this was accompanied by limited transcriptional alterations at the level of the dorsal root ganglion. Functional and neuroanatomical studies demonstrated that intrathecal RTX largely spares susceptible neuronal perikarya, which remain active peripherally but unable to transmit signals to the spinal cord. The results demonstrate that central chemo-axotomy of the TRPV1+ afferents underlies RTX analgesia and refine the neurobiology underlying effective clinical use of TRPV1 agonists for pain control.

Multiple myeloma (MM) is a hematological cancer with underlying causes associated with increased oxidative stress. Through signaling of their receptor RAGE, advanced glycation endproducts (AGEs) are known to increase oxidative stress associated with malignant transformation. In the present study, we have demonstrated that the levels of these compounds are increased in the saliva of myeloma patients with bone lesions. This data may provide a potential marker for bone lesions in MM and a potential target for the treatment of myeloma by blocking the AGEs or their receptor.

Objectives: To investigate and discuss the effects of cocoa on orofacial pain., Setting and Sample Population: The Department of Orthodontics at the University of Florida (UF). Male and female hairless rats (N=20/group) were tested., Materials and Methods: Rats were tested using the Orofacial Pain Assessment Device (OPAD) before and after changing their food from the standard chow to a cocoa-enriched or control-equivalent diet., Results: Male rats fed the cocoa diet had a significantly higher operant pain index when tested at 37°C as compared to control diet-fed animals. Female rats on the cocoa diet had a significantly higher pain index when tested at 18°C and 44°C, as compared to animals fed the control diet. Capsaicin-induced pain was inhibited, with cocoa-diet male rats having a significantly higher pain index than control-diet male rats and cocoa-diet female rats at both 37°C and 44°C. Cocoa-diet female rats had a significantly higher pain index at 44°C than control-diet females. Mechanical sensitivity was affected following capsaicin cream, with a significantly decreased tolerated bottle distance in both cocoa- and control-diet animals, but there was no difference between cocoa- and control-diet groups., Conclusion: Using the OPAD operant system, we demonstrated that a diet rich in cocoa was effective in inhibiting neurogenic inflammatory pain in rats. This has implications for the use of novel alternative therapies such as diet modification for pain control., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The detection of cool temperatures is thought to be mediated by primary afferent neurons that express the cool temperature sensing protein Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Using mice, this study tested the hypothesis that sex differences in sensitivity to cool temperatures were mediated by differences in neurons that express TRPM8. Ion currents from TRPM8 expressing trigeminal ganglion (TRG) neurons in females demonstrated larger hyperpolarization-activated cyclic nucleotide-gated currents (Ih) than male neurons at both 30° and 18°C. Additionally, female neurons' voltage gated potassium currents (Ik) were suppressed by cooling, whereas male Ik was not significantly affected. At the holding potential tested (-60mV) TRPM8 currents were not visibly activated in either sex by cooling. Modeling the effect of Ih and Ik on membrane potentials demonstrated that at 30° the membrane potential in both sexes is unstable. At 18°, female TRPM8 TRG neurons develop a large oscillating pattern in their membrane potential, whereas male neurons become highly stable. These findings suggest that the differences in Ih and Ik in the TRPM8 TRG neurons of male and female mice likely leads to greater sensitivity of female mice to the cool temperature. This hypothesis was confirmed in an operant reward/conflict assay. Female mice contacted an 18°C surface for approximately half the time that males contacted the cool surface. At 33° and 10°C male and female mice contacted the stimulus for similar amounts of time. These data suggest that sex differences in the functioning of Ih and Ik in TRPM8 expressing primary afferent neurons leads to differences in cool temperature sensitivity.

Post-traumatic headache (PTH) following TBI is a common and often persisting pain disability. PTH is often associated with a multimodal central pain sensitization on the skin surface described as allodynia. However, the particular neurobiology underlying cTBI-induced pain disorders are not known. These studies were performed to assess trigeminal sensory sensitization and to determine if sensitization measured behaviorally correlated with detectable changes in portions of the trigeminal sensory system (TSS), particularly trigeminal nucleus, thalamus, and sensory cortex. Thermal stimulation is particularly well suited to evaluate sensitization and was used in these studies. Recent advances in the use of reward/conflict paradigms permit use of operant measures of behavior, versus reflex-driven response behaviors, for thermal sensitization studies. Thus, to quantitate facial thermal sensitization (allodynia) in the setting of acute TBI, the current study utilized an operant orofacial pain reward/conflict testing paradigm to assess facial thermal sensitivity in uninjured control animals compared with those two weeks after cTBI in a rodent model. Significant reductions in facial contact/lick behaviors were observed in the TBI animals using either cool or warm challenge temperatures compared with behaviors in the normal animals. These facial thermal sensitizations correlated with detectable changes in multiple levels of the TSS. The immunohistochemical (IHC) studies revealed significant alterations in the expression of the serotonin (5-HT), neurokinin 1 receptor (NK1R), norepinephrine (NE), and gamma-aminobutyric acid (GABA) in the caudal trigeminal nucleus, thalamic VPL/VPM nucleus, and sensory cortex of the orofacial pain pathways. There was a strong correlation between increased expression of certain IHC markers and increased behavioral markers for facial sensitization. The authors conclude that TBI-induced changes observed in the TSS are consistent with the expression of generalized facial allodynia following cTBI. To our knowledge, this is the first report of orofacial sensitization correlated with changes in selected neuromodulators/neurotransmitters in the TSS following experimental mild TBI., (Published by Elsevier Ltd.)

Opioid withdrawal causes a dysphoric state that can lead to complications in pain patients and can propagate use in drug abusers and addicts. Opioid withdrawal changes the activity of neurons in the nucleus accumbens, an area rich in both opioid-binding mu opioid receptors and glutamate-binding NMDA receptors. Because the accumbens is an area important for reward and aversion, plastic changes in this area during withdrawal could alter future behaviors in animals. We discovered an increase in phosphorylation of serine 897 in the NR1 subunit of the NMDA receptor (pNR1) during acute morphine withdrawal. This serine can be phosphorylated by protein kinase A (PKA) and dephosphorylated by calcineurin. We next demonstrated that this increased pNR1 change is associated with an increase in NR1 surface expression. NR1 surface expression and pNR1 levels during acute withdrawal were both reduced by the NMDA receptor antagonist MK-801 (dizocilpine hydrogen maleate) and the PKA inhibitor H-89(N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride hydrate). We also found that pNR1 levels remained high after an extended morphine withdrawal period of 2 months, correlated with reward-seeking behavior for palatable food, and were associated with a decrease in accumbal calcineurin levels. These data suggest that NR1 phosphorylation changes during the acute withdrawal phase can be long lasting and may reflect a permanent change in NMDA receptors in the accumbens. These altered NMDA receptors in the accumbens could play a role in long-lasting behaviors associated with reward and opioid use., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Lidocaine is the most widely utilized intraoral injected dental anesthetic, used for more than 500 million dental injections per year. Local anesthesia is essential for pain-free dentistry, yet intraoral injections are often considered painful and a source of anxiety for many patients. Any new anesthetics that will reduce the stress and anxiety of dental injection are expected to be beneficial. A novel chemical approach to taste modulation is proposed, in which the lidocaine cation is coupled with anionic sweeteners such as saccarinate and acesulfamate. The ionic conjugates synthesized using anion exchange techniques, were much less bitter, demonstrated a high local anesthetic potential in animal studies, and were as safe as the original hydrochloride. Based on the currently robust market for lidocaine it is expected that the resulting anesthetics will be in high demand in clinical practices worldwide.

Background: Detecting behaviors related to orofacial pain in rodent models often relies on subjective investigator grades or methods that place the animal in a stressful environment. In this study, an operant-based behavioral assay is presented for the assessment of orofacial tactile sensitivity in the rat., New Methods: In the testing chamber, rats are provided access to a sweetened condensed milk bottle; however, a 360° array of stainless steel wire loops impedes access. To receive the reward, an animal must engage the wires across the orofacial region. Contact with the bottle triggers a motor, requiring the animal to accept increasing pressure on the face during the test. To evaluate this approach, tolerated bottle distance was measured for 10 hairless Sprague Dawley rats at baseline and 30 min after application of capsaicin cream (0.1%) to the face. The experiment was repeated to evaluate the ability of morphine to reverse this effect., Results: The application of capsaicin cream reduced tolerated bottle distance measures relative to baseline (p<0.05). As long as morphine did not cause reduced participation due to sedation, subcutaneous morphine dosing reduced the effects of capsaicin (p<0.001). Comparison with existing method: For behavioral tests, experimenters often make subjective decisions of an animal's response. Operant methods can reduce these effects by measuring an animal's selection in a reward-conflict decision. Herein, a method to measure orofacial sensitivity is presented using an operant system., Conclusions: This operant device allows for consistent measurement of heightened tactile sensitivity in the orofacial regions of the rat., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aim: To investigate the vasoactive intestinal peptides (VIP) expression in irritable bowel syndrome (IBS) and trinitrobenzene sulfonic acid (TNBS) induced colitis., Methods: The VIP gene expression and protein plasma levels were measured in adult participants (45.8% male) who met Rome III criteria for IBS for longer than 6 mo and in a rat model of colitis as induced by TNBS. Plasma and colons were collected from naïve and inflamed rats. Markers assessing inflammation (i.e., weight changes and myeloperoxidase levels) were assessed on days 2, 7, 14 and 28 and compared to controls. Visceral hypersensitivity of the rats was assessed with colo-rectal distension and mechanical threshold testing on hind paws. IBS patients (n = 12) were age, gender, race, and BMI-matched with healthy controls (n = 12). Peripheral whole blood and plasma from fasting participants was collected and VIP plasma levels were assayed using a VIP peptide-enzyme immunoassay. Human gene expression of VIP was analyzed using a custom PCR array., Results: TNBS induced colitis in the rats was confirmed with weight loss (13.7 ± 3.2 g) and increased myeloperoxidase activity. Visceral hypersensitivity to colo-rectal distension was increased in TNBS treated rats up to 21 d and resolved by day 28. Somatic hypersensitivity was also increased up to 14 d post TNBS induction of colitis. The expression of an inflammatory marker myeloperoxidase was significantly elevated in the intracellular granules of neutrophils in rat models following TNBS treatment compared to naïve rats. This confirmed the induction of inflammation in rats following TNBS treatment. VIP plasma concentration was significantly increased in rats following TNBS treatment as compared to naïve animals (P < 0.05). Likewise, the VIP gene expression from peripheral whole blood was significantly upregulated by 2.91-fold in IBS patients when compared to controls (P < 0.00001; 95%CI). VIP plasma protein was not significantly different when compared with controls (P = 0.193)., Conclusion: Alterations in VIP expression may play a role in IBS. Therefore, a better understanding of the physiology of VIP could lead to new therapeutics.

Background: Transient receptor potential (TRP) cation channels are involved in the perception of hot and cold pain and are targets for pain relief in humans. We hypothesized that agonists of TRPV1 and TRPM8/TRPA1, capsaicin and menthol, would alter nociceptive behaviors in the rat, but their opposite effects on temperature detection would attenuate one another if combined., Methods: Rats were tested on the Orofacial Pain Assessment Device (OPAD, Stoelting Co.) at three temperatures within a 17 min behavioral session (33°C, 21°C, 45°C)., Results: The lick/face ratio (L/F: reward licking events divided by the number of stimulus contacts. Each time there is a licking event a contact is being made.) is a measure of nociception on the OPAD and this was equally reduced at 45°C and 21°C suggesting they are both nociceptive and/or aversive to rats. However, rats consumed (licks) equal amounts at 33°C and 21°C but less at 45°C suggesting that heat is more nociceptive than cold at these temperatures in the orofacial pain model. When menthol and capsaicin were applied alone they both induced nociceptive behaviors like lower L/F ratios and licks. When applied together though, the licks at 21°C were equal to those at 33°C and both were significantly higher than at 45°C., Conclusions: This suggests that the cool temperature is less nociceptive when TRPM8/TRPA1 and TRPV1 are co-activated. These results suggest that co-activation of TRP channels can reduce certain nociceptive behaviors. These data demonstrate that the motivational aspects of nociception can be influenced selectively by TRP channel modulation and that certain aspects of pain can be dissociated and therefore targeted selectively in the clinic.

Despite an immense investment of resources, pain remains at epidemic proportions. Given this, there has been an increased effort toward appraising the process by which new painkillers are developed, focusing specifically on why so few analgesics make it from the benchside to the bedside. The use of behavioral assays and animal modeling for the preclinical stages of analgesic development is being reexamined to determine whether they are truly relevant, meaningful, and predictive. Consequently, there is a strengthening consensus that the traditional reflex-based assays upon which several decades of preclinical pain research has been based are inadequate. Thus, investigators have recently turned to the development of new preclinical assays with improved face, content, and predictive validity. In this regard, operant pain assays show considerable promise, as they are more sensitive, present better validity, and, importantly, better encompass the psychological and affective dimensions of pain that trouble human pain sufferers. Here, we briefly compare and contrast reflex assays with operant assays, and we introduce a particular operant orofacial pain assay used in a variety of experiments to emphasize how operant pain assays can be applied to preclinical studies of pain.

Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 °C and 60 °C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

The current study tested the hypothesis that conditioned pain modulation is mediated by the release of endogenous opioids with a placebo-controlled (sugar pill) study of naltrexone (50 mg) in 33 healthy volunteers over two counter-balanced sessions. Pain modulation consisted of rating of heat pain (palm) during concurrent cold water immersion (foot). Compared to baseline heat pain ratings, concurrent foot immersion lowered pain intensity ratings, which suggests an inhibitory effect, was reduced with naltrexone, suggesting at least partial dependence of inhibition on endogenous opioids. An exploratory analysis revealed that individual differences in catastrophizing moderated the effects of naltrexone; endogenous opioid blockade abolished modulation in subjects lower in catastrophizing while modulation was unaffected by naltrexone among high catastrophizers. The results suggest a role of endogenous opioids in endogenous analgesia, but hint that multiple systems might contribute to conditioned pain modulation, and that these systems might be differentially activated as a function of individual differences in responses to pain.

Obesity has been associated with multiple chronic pain disorders, including migraine. We hypothesized that diet-induced obesity would be associated with a reduced threshold for thermal nociception in the trigeminal system. In this study, we sought to examine the effect of diet-induced obesity on facial pain behavior. Mice of two different strains were fed high-fat or regular diet (RD) and tested using a well-established operant facial pain assay. We found that the effects of diet on behavior in this assay were strain and reward dependent. Obesity-prone C57BL/6J mice fed a high-fat diet (HFD) display lower number of licks of a caloric, palatable reward (33% sweetened condensed milk or 30% sucrose) than control mice. This occurred at all temperatures, in both sexes, and was evident even before the onset of obesity. This diminished reward-seeking behavior was not observed in obesity-resistant SKH1-E (SK) mice. These findings suggest that diet and strain interact to modulate reward-seeking behavior. Furthermore, we observed a difference between diet groups in operant behavior with caloric, palatable rewards, but not with a non-caloric neutral reward (water). Importantly, we found no effect of diet-induced obesity on acute thermal nociception in the absence of inflammation or injury. This indicates that thermal sensation in the face is not affected by obesity-associated peripheral neuropathy as it occurs when studying pain behaviors in the rodent hindpaw. Future studies using this model may reveal whether obesity facilitates the development of chronic pain after injury or inflammation., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

In order to better understand and treat neuropathic pain, scientific study must use methods that can assess pain processing at the cortical level where pain is truly perceived. Operant behavior paradigms can accomplish this. We used an operant task to evaluate changes following chronic constriction injury to the trigeminal nerves. We also relate these behavioral changes to immunohistochemistry of transient receptor potential channels vanilloid 1 and melastatin 8 (TRPV1 and TRPM8) in the trigeminal ganglia. Following nerve injury, successful performance of the operant task was reduced and aversive behaviors were observed with 10 and 37 °C stimulation, indicating cold allodynia and mechanical allodynia respectively. In contrast, while aversive behaviors were observed with 48 °C stimulation, successful performance of the operant task was not substantially hindered following injury. These behavioral changes were accompanied by an increase in TRPV1 positive cells and an increased intensity of TRPM8 staining at 2 weeks post-injury, when cold allodynia is maximal. These findings suggest that the incorporation of operant behavioral assessment in the study of pain may provide insight into the relationship among peripheral changes, motivational drive, and pain. Understanding this relationship will allow us to better treat and prevent chronic neuropathic pain., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The NR1 subunit of the NMDA receptor can be alternatively spliced by the insertion or removal of the N1, C1, C2, or C2' regions. Morphine dependence and withdrawal were previously demonstrated to lower N1 and C2' in the accumbens and lower N1, C1, and C2' in the amygdala (AMY). Withdrawal has also been demonstrated to increase motivational and anxiety/stress behaviors in rats. We tested the hypothesis that NR1 splicing would be associated with these behaviors during an extended withdrawal period of 2 months. Motivation was measured using an operant orofacial assay at non-aversive temperatures (37°C) while anxiety and stress were measured by examining this behavior at aversive temperatures (46°C). Lower C1 and C2 expression levels were observed in the AMY in a subset of the population of withdrawn rats even after 2 months of morphine withdrawal. These subsets were associated with a hypersensitivity to adverse conditions which may reflect long-term alterations in the withdrawn population., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Analgesia is particularly susceptible to placebo responses. Recent studies in humans have provided important insights into the neurobiology underlying placebo-induced analgesia. However, human studies provide incomplete mechanistic explanations of placebo analgesia because of limited capacity to use cellular, molecular, and genetic manipulations. To address this shortcoming, this article describes the development of a rat model of conditioned analgesia in an operant pain assay. Specifically, rats were conditioned to associate a placebo manipulation with the analgesic effect of 1mg/kg morphine (subcutaneously) on facial thermal pain. We found that conditioned (placebo) responding bore 3 of the hallmarks of placebo-induced analgesia: (1) strong interanimal variability in the response, (2) suppression by the opiate antagonist naloxone (5mg/kg subcutaneously), and (3) a positive predictive relationship between the unconditioned analgesic effect and the conditioned (placebo) effect. Because of the operant nature of the assay and the use of only a mild noxious thermal stimulus, we suggest that these results provide evidence of placebo-induced analgesia in a preclinical model that utilizes an affective behavioral end point. This finding may provide opportunities for invasive preclinical studies allowing greater understanding of placebo-induced analgesia, thus paving the way for avenues to harness its benefits., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

The NMDA receptor plays a large role in opioid-induced plastic changes in the nervous system. The expression levels of its NR1 subunit are altered dramatically by morphine but no changes in its alternative splicing have been reported. Changes in the splicing of the N1, C1, C2, and C2' cassettes can alter the pharmacology and regulation of this receptor. Western Blots run on brain tissue from rats made tolerant to morphine revealed altered splicing of the N1 cassettes in the accumbens and amygdala (AMY), and the C1 cassette in the AMY and the dorsal hippocampus (HIPP). After 3days of withdrawal C2'-containing NR1 subunits were down-regulated in each of these areas. These were not due to acute doses of morphine and may represent long-term alterations in drug-induced neuroplasticity. We also examined the effects of morphine tolerance on an operant orofacial nociception assay which forces an animal to endure an aversive heat stimulus in order to receive a sweet milk reward. Morphine decreased pain sensitivity as expected but also increased motivational reward seeking in this task. NMDAR antagonism potentiated this reward seeking behavior suggesting that instead of attenuating tolerance, MK-801 may actually alter the rewarding and/or motivational properties of morphine. When combined, MK-801 and morphine had an additive effect which led to altered splicing in the accumbens, AMY, and the HIPP. In conclusion, NR1 splicing may play a major role in the cognitive behavioral aspects especially in motivational reward-seeking behaviors., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Proteinase activated receptor 2 (PAR-2) is expressed by many neurons in the colon, including primary afferent neurons that co-express transient receptor potential vanilloid 1 (TRPV1). Activation of PAR-2 receptors was previously found to enhance colonic motility, increase secretion and produce hypersensitivity to mechanical stimuli. This study examined the functional role of TRPV1/PAR-2 expressing neurons that innervate the colon by lesioning TRPV1 bearing neurons with the highly selective and potent TRPV1 agonist resiniferatoxin., Methods: Colonic motility in response to PAR-2 activation was evaluated in vitro using isolated segments of descending colon and in vivo using manometry. Colonic mechanical nociceptive thresholds were measured using colorectal distension. Transient receptor potential vanilloid 1 expressing neurons were selectively lesioned with resiniferatoxin., Key Results: In vitro, the PAR-2 agonists, trypsin and SLIGRL did not alter contractions of colon segments when applied alone, however, the agents enhanced acetylcholine stimulated contraction. In vivo, PAR-2 agonists administered intraluminally induced contractions of the colon and produced hypersensitivity to colorectal distention. The PAR-2 agonist enhancement of colonic contraction was eliminated when TRPV1 expressing neurons were lesioned with resiniferatoxin, but the PAR-2 agonist induced hypersensitivity remained in the lesioned animals., Conclusions & Inferences: Our findings indicate that TRPV1/PAR-2 expressing primary afferent neurons mediate an extrinsic motor reflex pathway in the colon. These data, coupled with our previous studies, also indicate that the recently described colospinal afferent neurons are nociceptive, suggesting that these neurons may be useful targets for the pharmacological control of pain in diseases such as irritable bowel syndrome., (© 2011 Blackwell Publishing Ltd.)

Mechanical pain sensitivity is characteristic of many orofacial pain conditions; however, few models exist to quantify this pain. Here we evaluated a novel adaptation of our existing operant system to characterize orofacial pain following mechanical and thermal stimuli. We demonstrate that the operant system is able to detect painful and analgesic responses to mechanical stimuli. These findings allow comparison of both mechanical and thermal stimuli using the same outcome measures., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Sweet solutions are commonly used in animal research to deliver drugs to test for addictive capacity and efficacy. In this study we compared the effects of a range of sucrose and saccharin concentrations on the performance of an operant assay. Our findings demonstrate that across a range of sucrose solutions some produce a success ratio which could mistakenly be labeled allodynic demonstrating the importance of choosing the correct reward solution., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Unlabelled: Individuals with chronic craniofacial pain experience symptoms that are consistent with central sensitization. In fact, central sensitization may constitute the major disease process in these conditions, particularly if the original injury has healed or the condition is idiopathic. To understand central sensitization we have developed a conjugate of substance P and cholera toxin (SP-CTA). SP-CTA is selectively taken up by cells that express neurokinin receptors. Twenty-four hours following intracisternal administration of SP-CTA, wild-type rats and mice demonstrated signs of persistent background nociception, but when tested for facial cold sensitivity, they did not differ from controls. However, treating the SP-CTA-injected animals with naloxone exposed cold hypersensitivity in the face. Mu-opioid receptor knockout mice treated with SP-CTA demonstrated hypersensitivity without naloxone treatment. These findings suggest that central sensitization leads to activation of an endogenous opioid system. The data also demonstrate that the intracisternal administration of SP-CTA in rodents is a useful model for studying central sensitization as a disease process without having to induce a peripheral injury., Perspective: Central sensitization is a concern in many craniofacial pain conditions. In this project, we utilize a conjugate of substance P and the catalytic subunit of cholera toxin to induce central sensitization in the nucleus caudalis of rodents. The data indicate that the injected animals become hypersensitive in the face., (Copyright 2010 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Background: Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing., Results: CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs., Conclusion: Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation.

Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of hypersensitivity to visceral and somatic stimuli following inflammation or tissue injury. Our objective was to investigate the role of NMDA NR1 receptors in the spinal cord (T10-L1; L4-S1) of a subset of rats that remain hypersensitive following the histological resolution of TNBS-induced colitis compared to saline treated rats and rats that had recovered both behaviorally and histologically. We hypothesized that NMDA NR1 subunit expression mediates hypersensitivity following transient TNBS colitis., Methods: Male Sprague-Dawley rats (150 g-250 g) received 20 mg/rat intracolonic trinitrobenzene sulfonic acid (TNBS) in 50% ethanol or saline. Animals underwent nociceptive visceral/somatic pain testing 16 weeks after resolution of TNBS colitis. Animals were sacrificed and their spinal cords (T10-L1; L4-S1) were retrieved and 2-dimensional polyacrylamide gel electrophoresis and immunohistocytochemistry techniques were used to investigate spinal-NMDA receptor expression., Results: NR1(001) was the only NMDA NR1 receptor subunit that was expressed in recovered and control rats, whereas hypersensitive animals expressed NR1(011) and NR1(111) as well as NR1(001) subunits. Immunohistochemistry analysis demonstrated increased expression of NMDA NR1-N1, C1, and C2-plus expression in laminae I and II of the spinal cord (T10-L1; L4-S1) in hypersensitive rats but not in recovered/control rats., Conclusions: Selective increases in the expression of the NMDA NR1 splice variants occur in hypersensitive rats following resolution of TNBS colitis. This suggests that the NMDA NR1 receptor plays an important role in the development of neuronal plasticity and central sensitization. The recombination of NR1 splice variants may serve as a key functional protein that maintains hypersensitivity following resolution of TNBS colitis.

Unlabelled: Icilin induces wet dog shakes (WDS) in rodents when injected systemically and activates the cold receptor TRPM8 and putative cold receptor TRPA1. It is assumed that WDS reflect an enhanced cold sensitivity; however, none have examined the relationship between WDS and cutaneous cold sensitivity following systemic icilin. In this study, we sought to characterize the effect of systemic and central icilin administration on WDS and thermal preference with either hindpaw or facial stimulation. It was found that a low dose of icilin (.025 mg), which transiently elevated WDS, decreased preference for cold with hindpaw stimulation (15 and 45 degrees C) when administered ip or it. Intracisternal administration of this dose produced similar results for facial stimulation (10 and 48 degrees C), but had no effect when administered ip. In contrast a high dose of icilin (.25 mg), which persistently elevated WDS, strongly increased preference for cold with hindpaw stimulation and had no effect on thermal preference with facial stimulation. These findings indicate that at the low concentration, systemic and central icilin enhances cold sensitivity, likely via TRPM8 and TRPA1 activation. In contrast, systemic icilin at the high concentration produces peripheral and/or central effects that diminish cold sensitivity, while WDS is maintained at a persistent rate., Perspective: Icilin is a unique compound that produces dissociable effects on an innate behavior (WDS) and on operant behaviors related to thermal perception. This compound could help clarify the relationship between peripheral cold transduction and the central induction of thermogenesis and nocifensive behaviors, as well as alterations that produce pathological pain.

We present simple method to assess dental pain in the awake rat. Using a sensitive strain gauge we examined changes in bite strength and bite pattern in rats following dental injury. Rats with dental injury displayed a significant reduction in mean peak bite strength and an altered bite cluster pattern. Both changes in the dental injury rats were reversed by an analgesic dose of morphine, and this could be reversed with naloxone. These changes were not observed in naive control animals. This simple method significantly improves our ability to evaluate dental pain syndromes.

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 degrees 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 degrees C). All three strains displayed nocifensive behaviors at 55 degrees 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 degrees 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.

The purpose of this study was to evaluate the levels of sRAGE in primary Sjögren's syndrome (SS), and to assess whether there is an association between sRAGE levels and disease characteristics. Thirteen patients were randomly selected from three subgroups: primary SS, (n = 6), secondary Sjögren's, (n = 4), and ANA(+) but lacking criteria for further disease classification (n = 3). Levels of serum sRAGE were measured in triplicate using an enzyme-linked immunosorbent assay kit. Mean sRAGE levels were significantly lower in the primary Sjögren's group. Logistic regression analysis indicated that plasma sRAGE level was a significant predictor of diagnostic status. Analyses using routine serological tests for diagnosing autoimmune disorders failed to reach statistical significance. This preliminary study supports the hypothesis that the RAGE system might participate in the disease pathway of primary SS, and that sRAGE may be a potential biomarker to aid in the diagnosis of primary SS.

The classification, morphology and function of enteric neurons have been extensively studied in the small and large intestine. However, little is known about enteric neurons that directly project to the CNS. Previous studies have identified these unique neurons in the rectum, rectospinal neurons, but little was done to characterize them. Therefore, the aim of this study was to identify and characterize enteric neurons in the rat colon that directly project to the CNS by using retrograde neuronal tracing and immunohistochemistry. By applying the retrograde tracers 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and Fluorogold (FG) to the L6/S1 segments of the spinal cord, we identified these neurons in both the myenteric and submucosal plexuses of the colon. These neurons were immunoreactive for neurofilament (NF) a marker for Adelta-fibers and isolectin-B4 (IB(4)) a marker for C-fibers. These neurons expressed the enzyme neuronal nitric oxide synthase (nNOS) as well as peptides associated with sensory neurons such as substance P (SP) and vasoactive intestinal polypeptide (VIP) but did not express calcitonin gene-related peptide (CGRP). The N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2D and proteinase-activated receptor-2 (PAR2) were also found in these neurons. However they did not express the transient receptor potential receptor V1 (TRPV1) or neurokinin 1 receptor (NK1). The expression of the peptides and receptors suggests that there are at least two separate populations of neurons projecting from the colon to the CNS. The data suggest that these colospinal afferent neurons (CANs) might be involved in nociception. Whether sensory information from CANs is perceived by the animal or is part of the parasympathetic reflex is currently not known.

Inflammation of visceral structures in rats has been shown to produce visceral/somatic hyperalgesia. Our objectives were to determine if trinitrobenzene sulfonic acid (TNBS) induced colitis in rats leads to visceral/somatic hypersensitivity. Male Sprague-Dawley rats (200-250 g) were treated with 20 mg of TNBS in 50% ethanol (n = 40) or an equivalent volume of ethanol (n = 40) or saline (n = 25) via the colon. Colonic distension, Von Frey, Hargreaves, and tail reflex tests were used to evaluate for visceral, mechanical, and thermal sensitivity. The rats demonstrated visceral hypersensitivity at 2-28 days following TNBS administration (P < 0.0001). The ethanol-treated rats also demonstrated visceral hypersensitivity that resolved after day 14. TNBS-treated rats demonstrated somatic hypersensitivity at days 14-28 (P < 0.0001) in response to somatic stimuli of the hind paw. TNBS colitis is associated with visceral and somatic hypersensitivity in areas of somatotopic overlap. This model of colitis should allow further investigation into the mechanisms of visceral and somatic hypersensitivity.

Background: Chronic abdominal pain is one of the most common gastrointestinal symptoms experienced by patients. Visceral hypersensitivity has been shown to be a biological marker in many patients with chronic visceral pain. We have previously shown that IBS patients with visceral hypersensitivity also have evidence of thermal hyperalgesia of the hand/foot., Objective: The objective of the current study was to develop an animal model of chronic visceral and somatic hypersensitivity in rats treated with intracolonic trinitrobenzene sulfonic acid., Design: Male Sprague-Dawley rats (200-250g) were treated with either 20mg/rat trinitrobenzene sulfonic acid (TNBS, Sigma Chemical Co.) in 50% ethanol (n=75), an equivalent volume of 50% ethanol (n=20) or an equivalent volume of saline (n=20). The agents were delivered with a 24-gauge catheter inserted into the lumen of the colon. Mechanical and thermal behavioral tests were performed using an automated von Frey and Hargreaves device to evaluate somatic hyperalgesia. Colonic distension was performed using an automated distension device to evaluate visceral pain thresholds. All animals were tested 16weeks after TNBS treatment following complete resolution of the colitis., Results: At 16weeks, 24% of the treated rats (18/75 rats) still exhibited evidence of visceral as well as somatic hypersensitivity compared to saline- and ethanol-treated rats., Conclusion: Transient colonic inflammation leads to chronic visceral and somatic hypersensitivity in a subset of rats. These findings are similar to the subset of patients who develop chronic gastrointestinal symptoms following enteric infection.

Background: Management of pain involves a balance between inhibition of pain and minimization of side effects; therefore, in developing new analgesic compounds, one must consider the effects of treatment on both pain processing and behavior. The purpose of this study was to evaluate the effects of the mu and kappa-2 opioid receptor agonists on general and pain behavioral outcomes., Methods: As a general behavioral assessment, we modified the cylinder rearing assay and recorded the number and duration of rearing events. Thermal sensitivity was evaluated using either a reflexive measure of hindpaw withdrawal latency to a radiant heat source or using an orofacial operant thermal assay. Acetic acid-induced visceral pain and capsaicin-induced neurogenic inflammatory pain were used as painful stimuli. The mu-opioid receptor agonist, morphine or the kappa-2 receptor agonist GR89696 was administered 30 min prior to testing. A general linear model repeated measures analysis was completed for baseline session comparisons and an analysis of variance was used to evaluate the effects of treatment on each outcome measure (SPSS Inc). When significant differences were found, post-hoc comparisons were made using the Tukey honestly significant difference test. *P < 0.05 was considered significant in all instances., Results: We found that morphine and GR89,696 dose-dependently decreased the number of reaching events and rearing duration. Rearing behavior was not affected at 0.5 mg/kg for morphine, 1.25 x 10-4 mg/kg for GR89,696. Hindpaw thermal sensitivity was significantly increased only at the highest doses for each drug. At the highest dose that did not significantly influence rearing behavior, we found that visceral and neurogenic inflammatory pain was not affected following GR89,696 administration and morphine was only partially effective for blocking visceral pain., Conclusion: This study demonstrated that high levels of the opioids produced significant untoward effects and made distinguishing an analgesic versus a more general effect more difficult. Quantification of rearing behavior in conjunction with standard analgesic assays can help in gaining a better appreciation of true analgesic efficacy of experimental drugs.

N-methyl-D-aspartate (NMDA) receptors and the expression of their different splice variants and subunits were previously characterized in the brain and spinal cord. However, knowledge on the NMDA receptor expression and function in the enteric nervous system is limited. Previous work suggested that NMDA receptors were involved in a rat model of visceral hypersensitivity. The aim of this study was to characterize the expression of the NMDA receptor NR1 splice variants and the NR2 subunit subtypes in the rat colon. We visualized the expression of NR1 protein in the rat submucosal and myenteric plexuses. The NR1 splice variants found in the colon of rats lacked the N1 and C1 cassettes and contained the C2 and C2' cassettes (NR1(000) and NR1(001)). The NR2B and NR2D subunits were also found in the rat colon. Moreover, NMDA receptors in the rat colon were heteromeric, since NR1 was co-localized with NR2B and NR2D subunits using fluorescent immunohistochemistry. The identification of the NMDA receptors in the enteric nervous system could lead to the development of drugs that selectively modulate bowel function.

Background: Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA). This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing., Results: The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 microg) injected intrathecally produce thermal hyperalgesia. At higher doses (10 microg) peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons., Conclusion: The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury.

Background: The association between smoking and periodontal disease is well established; however, the mechanism by which smoking augments the destruction of periodontal tissue is not clear. We hypothesize that smoking is related to an increased expression of receptor of advanced glycation end products (RAGE) in gingival tissues of smokers., Methods: Gingival biopsies from five smokers and five age- and gender-matched non-smokers were examined. In addition, gingival epithelial cells (GECs) were reacted with 1 muM nornicotine for 4, 16, 24, and 48 hours for mRNA for RAGE and an additional 72 hours for protein expression. RAGE mRNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and expression of RAGE at the protein level in GECs was studied with Western blots., Results: In the gingival biopsies from all 10 subjects, RT-PCR with RAGE-specific primers produced a band of the predicted size. For all pairs, the smoker biopsies expressed a greater level of RAGE compared to the matched non-smokers. When viewed as groups, analysis of the band intensity indicated that RAGE mRNA in smokers was approximately 1.4-fold of the expression in non-smokers (Wilcoxon test; P = 0.031). In GECs treated with nornicotine, there was a time-dependent increase in RAGE expression up to two-fold at 48 hours. RAGE protein levels initially were reduced but increased to 1.4-fold after 48 hours., Conclusion: The ability of nornicotine to elevate RAGE expression in GECs, along with increased RAGE expression in inflamed gingival tissue from smokers, indicates that RAGE may be associated with periodontal disease linked to smoking.

Background: A hallmark of many orofacial pain disorders is cold sensitivity, but relative to heat-related pain, mechanisms of cold perception and the development of cold allodynia are not clearly understood. Molecular mediators of cold sensation such as TRPM8 have been recently identified and characterized using in vitro studies. In this study we characterized operant behavior with respect to individually presented cold stimuli (24, 10, 2, and -4 degrees C) and in a thermal preference task where rats chose between -4 and 48 degrees C stimulation. We also evaluated the effects of menthol, a TRPM8 agonist, on operant responses to cold stimulation (24, 10, and -4 degrees C). Male and female rats were trained to drink sweetened milk while pressing their shaved faces against a thermode. This presents a conflict paradigm between milk reward and thermal stimulation., Results: We demonstrated that the cold stimulus response function was modest compared to heat. There was a significant effect of temperature on facial (stimulus) contacts, the ratio of licking contacts to stimulus contacts, and the stimulus duration/contact ratio. Males and females differed only in their facial contacts at 10 degrees C. In the preference task, males preferred 48 degrees C to -4 degrees C, despite the fact that 48 degrees C and -4 degrees C were equally painful as based on their reward/stimulus and duration/contact ratios. We were able to induce hypersensitivity to cold using menthol at 10 degrees C, but not at 24 or -4 degrees C., Conclusion: Our results indicate a strong role for an affective component in processing of cold stimuli, more so than for heat, which is in concordance with human psychophysical findings. The induction of allodynia with menthol provides a model for cold allodynia. This study provides the basis for future studies involving orofacial pain and analgesics, and is translatable to the human experience.

N-Methyl-d-aspartic acid (NMDA) receptors are known to function in the mediation of pain and have a significant role in the development of hyperalgesia following inflammation. Serine phosphorylation regulation of NMDA receptor function occurs in a variety of conditions. No studies have demonstrated a change in phosphorylation of enteric NMDA receptors following colonic inflammation. We examined the levels of NMDA NR1 phosphorylation in trinitrobenzene sulfonic acid (TNBS) induced colitis in rats and compared it to protein translation and the development of visceral hypersensitivity. We have previously, demonstrated an increase in the C1 cassette of NR1 mRNA expression at 14, 21, and 28 days following TNBS administration. In this study, we examined the NR1 serine phosphorylation at 14 days following TNBS injection. Male Sprague-Dawley rats (200-250 g) were treated with TNBS (20mg per rat) diluted in 50% ethanol (n=3) and vehicle controls of 50% ethanol (n=3). TNBS and vehicle controls were administered with a 24 gauge catheter inserted into the lumen of the rat colon. The animals were sacrificed at 14 days after induction of the colitis and their distal colon was retrieved for two-dimensional (2D) western blot analysis. Serine phosphorylation of the NR1 subunit with C1 cassette appears at 14 days after TNBS injection. In contrast, there was no NR1-C1 expression in the vehicle controls and untreated normal controls. These results suggest a role for colonic-NMDA receptor phosphorylation in the development of neuronal plasticity following colonic inflammation. Phosphorylation of NR1 may partially explain visceral hypersensitivity present during colonic inflammation.