Robert M. Caudle

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.

Adenoviral gene transfer to spinal cord neurons: intrathecal vs. intraparenchymal administration

The spinal cord is the site of many chronic, debilitating, neurological disorders that may be amenable to gene therapy. The present study, using quantitative and anatomical methods, examines the ability of replication deficient adenovirus to transfer a transcription cassette composed of the cytomegalovirus promoter driving the expression of the LacZ reporter gene (AdCMVbetagal) to spinal-cord neurons. Rats were microinjected with AdCMVbetagal into the spinal-cord parenchyma or subarachnoid space and sacrificed between 1 and 60 days post-infusion. The spinal cord was assayed for beta-galactosidase (beta-gal) activity fluorometrically (MUG). Intraparenchymal injection resulted in significant beta-gal activity at day 1, which peaked at day 7, and decreased at day 14 (21-, 57- and 9.8-fold of control respectively). The spatial distribution of beta-gal activity on day 7 was confined to the 1-cm section containing the injection site but was detected 2 cm caudal to this section by day 14. Histochemical staining and immunocytochemistry revealed a prominent reaction product in neurons, particularly motor neurons, and glia within the ventral grey matter bilaterally. Intrathecal viral injections showed comparatively modest, yet significant increases in beta-gal activity throughout the spinal cord with the greatest activity (170% control) closest to the catheter tip. This study demonstrates that AdCMVbetagal injected into the ventral spinal cord results in extensive in vivo neuronal gene transfer with beta-gal activity reaching a peak by day 7 and remaining detectable at 60 days. Intrathecal viral injections result in greater spatial distribution but a comparatively lower level of expression.

Diverse immunocytochemical expression of opioid receptors in electrophysiologically defined cells of rat dorsal root ganglia

The development of opiate analgesics that do not produce adverse side effects is hampered by the difficulty in developing drugs that are tissue/sensory cell-specific. Previously, our laboratory has demonstrated that small- and medium-diameter dorsal root ganglia (DRG) cells can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents [Petruska, J.C., Napaporn, J., Johnson, R.D., Gu, J.G., Cooper, B.Y., 2000. Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents. J. Neurophysiol. 84 (5), 2365-2379; Petruska, J.C., Napaporn, J., Johnson, R.D., Cooper, B.Y., 2002. Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion. Neuroscience 115 (1), 15-30.] Based on their responses to algesic compounds and histochemical phenotype, eight of the nine subtypes are likely nociceptors. In the present study, we examined the immunoreactivity (IR) of delta-, kappa- and mu-opioid receptors (DOR, KOR and MOR, respectively), in 164 electrophysiologically subclassified DRG neurons. The expression of opioid receptors in the DRG cell types was diverse. Type 1 (25-30 microm cell diameter) and type 9 (35-45 microm) expressed MOR-IR, but were negative for DOR-IR and KOR-IR. Type 2 (25-30 microm) co-expressed DOR-IR and MOR-IR, but did not express KOR-IR. Type 3 (15-20 microm), the non-nociceptive cell type, was not immunoreactive. Type 4 (35-45 microm), type 6 (35-45 microm), and type 7 (15-20 microm) expressed all three opioid receptors. Type 5 (35-45 microm) and type 8 (35-45 microm), co-expressed KOR-IR and MOR-IR, but did not express DOR-IR. The co-expression of opioid receptors in some of the cell types suggests that these sensory afferents might contain heteromeric opioid receptors. Additionally, the diverse expression patterns of opioid receptors between cell types and the consistency of these patterns maintained within each cell type provides further evidence of distinct functional properties of DRG nociceptors.

Visceral and somatic hypersensitivity in a subset of rats following TNBS-induced colitis.

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 (200–250 g) were treated with either 20 mg/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 16 weeks after TNBS treatment following complete resolution of the colitis. Results. At 16 weeks, 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.

Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation

BackgroundSpinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury.ResultsLumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation.ConclusionThese data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

Placebo-induced analgesia in an operant pain model in rats.

Summary Rats show canonical features of human placebo‐induced analgesia in an assay designed to evaluate pain affect. ABSTRACT 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 1 mg/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 (5 mg/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.

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.

N-Methyl-D-aspartate receptor subunit expression and phosphorylation following excitotoxic spinal cord injury in rats

The role of NMDA receptor expression and post-translational modification in the pathological and behavioral consequences of injury were examined in rats receiving spinal injections of quisqualate. Spinal cords were removed 3 days following the development of excessive grooming behavior or, if the spontaneous pain-like behavior was not observed, 13 days following injections. Western blots from the spinal tissue demonstrated that non-grooming animals had elevated protein levels of the NR1 subunit of the NMDA receptor. These subunits did not demonstrate an enhanced level of phosphorylation. NR1 protein in grooming rats was not elevated, but there was a significant increase in NR1 serine phosphorylation. These findings suggest that excitotoxic lesions of the spinal cord induce both NR1 expression and NR1 serine phosphorylation. However, the injury-induced excessive grooming behavior is only associated with phosphorylation of the NR1 subunit.

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.