Heather L. Rossi

Use of a novel thermal operant behavioral assay for characterization of orofacial pain sensitivity

&NA; Orofacial pain has been well‐characterized clinically, but evaluation of orofacial pain in animals has not kept pace. The objective of this study was to describe behavioral responses to facial thermal stimulation and inflammation with/without an analgesic using a novel operant paradigm. Animals were trained to voluntarily place their face against a stimulus thermode (37.7–57.2 °C) providing access to positive reinforcement. These contingencies present a conflict between positive reward and tolerance for nociceptive stimulation. Inflammation was induced and morphine was provided as an analgesic in a subset of animals. Six outcome measures were determined: reward intake, reward licking contacts, stimulus facial contacts, facial contact duration, ratio of reward/stimulus contacts, and ratio of facial contact duration/event. Animals displayed aversive behaviors to the higher temperatures, denoted by a significant decrease in reward intake, total facial contact duration, and reward licking events. The number of facial contacts increased with increasing temperature, replacing long drinking bouts with more frequent short drinks, as reflected by a low ratio of facial contact duration/event. The number of reward licking/facial contact events was significantly decreased as the thermal stimulus intensity increased, providing another pain index derived from this operant method. These outcomes were significantly affected in the direction of increased nociception following inflammation, and these indices of hyperalgesia were reversed with morphine administration. These data reflect an orofacial pain behavior profile that was based on an animals responses in an operant escape paradigm. This technique allows evaluation of nociceptive processing and modulation throughout the neuraxis.

Sex Differences in Thermal Pain Sensitivity and Sympathetic Reactivity for Two Strains of Rat

UNLABELLED Human females are more sensitive than males to brief nociceptive stimuli such as heat and cold. However, a more pronounced peripheral vasoconstriction by females than by males during prolonged nociceptive stimulation predicts that females would be more sensitive to prolonged cold but not heat stimulation. We tested this possibility with reflex (lick/guard) and operant escape and preference tests of sensitivity to prolonged stimulation of Long-Evans and Sprague-Dawley rats. Escape responses to cold stimulation revealed a greater sensitivity of females. In contrast, males were more sensitive to nociceptive heat stimulation. An operant preference test of relative sensitivity to cold or heat stimulation confirmed these results. Cold was more aversive than heat for females, but heat was more aversive than cold for males. Recordings of skin temperature during nociceptive heat stimulation were consistent with the results of operant testing. A reduction in skin temperature (peripheral vasoconstriction) during nociceptive stimulation should increase cold sensitivity as observed for females relative to males. Lick/guard testing did not confirm the results of operant testing. Lick/guard (L/G) responding to nociceptive heat stimulation was greater for females than for males. Female escape responses to heat were more variable than males, but L/G responding of males to the same stimulus was more variable than for females. PERSPECTIVE A variety of chronic pain conditions are more prevalent for females, and psychological stress (with attendant sympathetic activation) is implicated in development and maintenance of these conditions. Therefore, understanding relationships between gender differences in pain sensitivity and sympathetic activation could shed light on mechanisms for some varieties of chronic pain.

Differentiation between capsaicin-induced allodynia and hyperalgesia using a thermal operant assay

Investigations of new analgesic treatments ideally are coupled with the use of compassionate methods for pain testing in animals. Recently, we described a novel operant thermal testing device that can be used to quantify orofacial pain. The objective of the current study was to differentiate thermal allodynia from hyperalgesia using this operant thermal assay. Rats were trained to complete a task whereby they had a conflict between a positive reward and tolerance for thermal nociceptive stimulation. They were subjected to cool to hot temperatures (24-45 degrees C) and evaluated under naïve (untreated), capsaicin cream (0.075%), capsaicin/morphine, or morphine test conditions. The following outcome measures were evaluated: reward intake; licking contacts; facial contacts; time to complete 25, 50, and 75% of the events (licks and face contacts); facial contact duration; ratio of reward/stimulus contacts; and ratio of facial contact duration/event. Capsaicin produced an increase in mechanical sensitivity and a significant thermal allodynic effect at 42 degrees C and hyperalgesic effect at 45 degrees C. These effects were blocked with morphine pre-treatment. The temporal profile for completing the task was also significantly altered following capsaicin treatment. These data demonstrate that using the operant orofacial assay in conjunction with capsaicin cream can provide a reproducible, sensitive, minimally invasive, and powerful approach for quantifying and studying enhanced thermal pain within the trigeminal system. This technique provides an alternative to reflex tests of orofacial sensitivity, and it presents a pivotal link for translating basic pain research into clinic trial strategies.

Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior

BackgroundRodent 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).ResultsAs 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.ConclusionTaken 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.

Characterization of Cold Sensitivity and Thermal Preference Using an Operant Orofacial Assay

BackgroundA 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°C) and in a thermal preference task where rats chose between -4 and 48°C stimulation. We also evaluated the effects of menthol, a TRPM8 agonist, on operant responses to cold stimulation (24, 10, and -4°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.ResultsWe 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°C. In the preference task, males preferred 48°C to -4°C, despite the fact that 48°C and -4°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°C, but not at 24 or -4°C.ConclusionOur 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.

Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors

BackgroundManagement 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.MethodsAs 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.ResultsWe 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 × 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.ConclusionThis 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.

Effects of Environmental Enrichment on Thermal Sensitivity in an Operant Orofacial Pain Assay

Environmental enrichment reduces reactivity to stressor and could also modulate pain perception. In this study we sought to compare the effects of enriched and standard housing on temperature perception. In an operant assay, rats housed in an enriched environment exhibited significantly lower sensitivities to thermal stimuli and displayed less exploratory behavior in a rearing chamber. These findings indicate that environmental enrichment can significantly affect temperature perception, likely through stress-related mechanisms.

Characterization of bilateral trigeminal constriction injury using an operant facial pain assay.

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.

Obesity increases nociceptive activation of the trigeminal system

Obesity is a risk factor associated with several pain syndromes. However, the mechanisms underlying the association between obesity and pain are not known. The aim of this study was to test the hypothesis that obesity enhances neuronal responses to nociceptive stimulation within the trigeminal nucleus caudalis (TNC).

Photophobia in primary headaches.

Photophobia is a debilitating feature of many headache disorders.