Osteoarthritis (OA) pain can arise from inflammatory cytokines sensitizing neurons that innervate the temporomandibular joint (TMJ) and knee. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenome editing enables targeted repression of inflammatory receptors and offers a promising strategy to modify disease mechanisms. This study tested whether CRISPR epigenome editing of interleukin-1 receptor type 1 (IL1R1) in trigeminal ganglia (TG; TMJ-innervating) and dorsal root ganglia (DRG; knee-innervating) neurons could reduce OA-associated sensitization. OA cartilage was collected from knee replacement patients and compared with healthy cadaveric cartilage. Rat TG and DRG neurons were cultured with IL-1β on tissue culture plastic or cartilage explants, loaded with calcium dye, and subjected to thermal stimulation. Neurons were transduced with lentiviral CRISPR-dCas9-KRAB vectors targeting IL1R1 or with nontargeting controls, and heat-evoked calcium transients were measured. Exposure to IL-1β and OA cartilage both increased the proportion of TG and DRG neurons exhibiting heat-induced calcium transients compared with controls. CRISPR epigenome editing of IL1R1 abolished sensitization in DRG neurons, restoring responses to healthy cartilage levels. In TG neurons, editing reduced maximum calcium responses to baseline but did not fully normalize the percentage of sensitized cells, suggesting additional OA factors contribute to TMJ pain. CRISPR epigenome editing of IL1R1 in joint-innervating neurons reduces OA cartilage-induced sensitization. These results highlight differential mechanisms underlying OA cartilage driven DRG and TG neuron sensitization and establish epigenome editing as a potential therapeutic strategy to target OA-associated sensitization in the knee and TMJ.

Hypertension has been associated with altered nociceptive thresholds in humans and animal models, but the relationship between blood pressure (BP) and pain sensitivity remains inconsistent. Here, we investigated trigeminal nociception in male spontaneously hypertensive rats (SHRs) compared with normotensive Wistar-Kyoto (WKY) rats using operant facial pain assays, trigeminal ganglion (TG) electrophysiology, and bulk RNA sequencing with cell-type deconvolution. SHRs exhibited reduced thermal and mechanical facial pain sensitivity relative to WKY controls; however, measured systolic BP did not robustly explain the strain difference in thermal pain behavior. Whole-cell recordings of TG neurons revealed increased hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents and voltage-gated potassium currents in SHRs, while voltage-gated sodium currents were unchanged despite reduced expression of Scn8a, Scn9a, and Scn10a. Transcriptomic analysis further demonstrated broad downregulation of ion channel and sensory transduction genes in SHRs, including Hcn1, Hcn4, Kcnq3, Kcnq4, Piezo2, and Trpm8. Cell-type deconvolution revealed strain-dependent shifts in TG composition, including alterations in sensory neurons and non-neuronal populations such as immune cells, satellite glia, endothelial cells, pericytes, and Schwann cells. Together, these findings show that reduced trigeminal nociception in SHRs is associated with changes in TG ion channel function, sensory gene programs, and cell-type composition, while not being robustly explained by measured systolic BP. PERSPECTIVE: This study shows that reduced trigeminal pain in SHRs is associated with changes in ion channel function, gene expression, and TG cell-type composition, while not being robustly explained by measured systolic BP.

Cannabidiol, a major non-intoxicating constituent of cannabis, has generated interest as a novel therapeutic for managing several pathological conditions including chronic pain and opioid use disorder. Here, we evaluated the effects of cannabidiol (3.2 or 10.0 mg/kg) on the antinociceptive and the reward-related effects of the opioid analgesic oxycodone (0.56 mg/kg) in rats (male and female Sprague-Dawley) using an operant facial pain assay, locomotor activity monitoring, and the conditioned place preference paradigm. Cannabidiol enhanced the antinociceptive effect of oxycodone without affecting oxycodone-induced rearing behavior, or the acquisition and expression of oxycodone conditioned place preference under the conditions tested. Together, these findings suggest that cannabidiol potentiates the analgesic effects of oxycodone without affecting its reward-related properties. These results support the potential of cannabidiol as an adjunctive, opioid-sparing agent in pain management. PERSPECTIVE: Opioids remain important for treating moderate to severe pain, but adverse effects and misuse liability limit their use. These preclinical findings suggest cannabidiol may enhance oxycodone antinociception under acute painful conditions, without increasing abuse-relevant effects under the conditions tested, supporting further study as an opioid-sparing adjunct.

Prescription opioid misuse is a significant public health concern among individuals with chronic pain. Treating severe pain often requires high doses of opioids, increasing the risk of developing an opioid use disorder. Cannabidiol (CBD) is a non-intoxicating component of cannabis that has shown therapeutic potential without abuse liability. This study investigated the effects of CBD on oxycodone self-administration and hyperalgesia in an animal model of chronic neuropathic pain. Adult male rats were trained to self-administer intravenous oxycodone (0.06 mg/kg/infusion). Subsequently, they underwent chronic constriction injury (CCI) of the sciatic nerve or received sham surgery. Paw withdrawal latency was measured using the Hargreaves test as an indicator of thermal pain sensitivity. CBD (0, 1, 3, and 10 mg/kg, IP) was administered before the self-administration sessions, and pain testing was conducted afterward. The rats acquired oxycodone self-administration, as indicated by more active than inactive lever presses. CCI surgery decreased the paw withdrawal latency, confirming the induction of neuropathic pain. CCI alone did not affect oxycodone self-administration, suggesting that neuropathic pain does not substantially influence opioid intake at the dose tested. Treatment with CBD reduced oxycodone self-administration in both the sham and CCI rats. Oxycodone self-administration in the CCI rats reversed the CCI-induced decrease in paw withdrawal latency. However, CBD did not affect the antinociceptive effect of oxycodone in CCI rats. Taken together, these findings demonstrate that CBD reduces oxycodone self-administration without affecting the antinociceptive effects of oxycodone in neuropathic pain. This study supports the potential of CBD to reduce opioid use and misuse, regardless of pain status.

Tissue clearing techniques, combined with immunolabeling and three-dimensional (3-D) high-resolution imaging, have emerged as powerful tools for mapping the architecture of nerves that supply a specific tissue. However, despite significant advances in these techniques, visualizing nerve fibers within joint structures remains a technically challenging task. Moreover, most current protocols are optimized for use in mice, limiting their application in other animal species with greater tissue size, such as rats, which offer advantages for anatomical studies combined with behavioral models of sensory innervation and pain. Therefore, continued refinement of tissue clearing and imaging methods in rat tissue is essential for improving the resolution and translational relevance of joint innervation studies. In this work, we assessed and compared two tissue clearing protocols, a modified polyethylene glycol-associated solvent system (PEGASOS) and a newly developed hybrid method that combines CUBIC/3DISCO for tissue clearing (c-Clear), to visualize neurofilament-positive (NF+ ) nerve fibers in rat knees using 3-D fluorescence imaging. In summary, c-Clear resulted in a better option to detect NF+ fibers in the rat knee, whereas PEGASOS cleared tissues revealed greater autofluorescence within the muscle and bone marrow, compromising neurofilament visualization. Additionally, we highlight the importance of multi-angle imaging approaches when using cutting-edge light-sheet microscopy to capture the spatial context of neural innervation patterns within the complex knee microenvironment.

Temporomandibular disorders (TMDs) are complex conditions characterized by orofacial pain and dysfunction, affecting a significant portion of the population. TMDs may involve joint and/or muscle pain, dysfunction (e.g., noise, limited or altered jaw movements), or both, leading to a marked decrease in quality of life. Patients often experience functional limitations that hinder eating, speaking, and daily activities. Additionally, TMDs are frequently associated with psychological distress, including anxiety and depression, which further impacts overall well-being. Despite the profound individual and societal impact of TMDs, effective therapies remain elusive, partly due to deficiencies in translational research. A primary limitation in the TMD field is the scarcity of animal models that accurately replicate disease features in humans. This may ultimately be due to species differences, but likely also reflects the etiological and symptomatic heterogeneities of TMDs, as there are over 30 different conditions in this umbrella term. Both factors pose a significant challenge in developing and using animal models for TMD research. This review highlights preclinical TMD research to enhance clinical care, focusing on anatomy/physiology, pain and behavior models, functional and tissue modeling, biopsychosocial factors, and technological considerations. The "TMD Research Community" collaborated to produce this review, with the Discussion offering a proposal for a path forward

Previous studies have driven the notion that the cannabis constituent cannabidiol could be an effective adjunct to opioid administration for managing pain. Most of these studies have used experimental rodents with routes of administration, such as subcutaneous and intraperitoneal, that do not correspond with the routes used in clinical practice. In response to this, we tested the ability of cannabidiol co-administration to augment opioid analgesia via the more clinically-relevant oral route of administration. To this end, male and female rats were orally gavaged with cannabidiol (25 mg/kg), oxycodone (1.4 mg/kg), or a combination of both, after which they were tested in an operant thermal orofacial pain assay in which they voluntarily exposed their faces to cutaneous thermal pain to receive a palatable reward. All three drug conditions produced analgesic effects of varying degrees, being most profound in the combination group where a statistically significant enhancement over oxycodone-induced analgesia alone was evident. Additionally, oxycodone administration decreased lick frequencies - a measure of motor coordination of rhythmic movements - which too was magnified by co-administration of cannabidiol. Together these studies provide further support of an ability of cannabidiol to augment opioid effects, particularly analgesia, when administered by a route relevant to human pain management. As such, they encourage the notion that cannabidiol could find utility as an opioid-sparing approach to treating pain.

Trigeminal neuralgia (TN) is a severe orofacial pain condition with sex-specific differences in pain responses. Standard treatments offer limited efficacy and significant side effects. We hypothesized that cannabidiol (CBD) alleviates TN-induced allodynia more effectively than carbamazepine in a sex- and dose-dependent manner through neuroimmune mechanisms, including modulation of glia, Fos protein expression, and oxidative stress in the ventrolateral periaqueductal gray (vlPAG) and spinal trigeminal nucleus caudalis (Sp5c). In an infraorbital nerve constriction model, mechanical allodynia was evaluated in male and female Wistar-Hannover rats. Our study demonstrates the potent antinociceptive effects of CBD in reducing mechanical allodynia in both male and female models of trigeminal neuralgia, without affecting locomotor activity, unlike carbamazepine. Although CBD's analgesic effects were consistent across sexes, carbamazepine showed sex-dependent efficacy. Cannabidiol's effects on Fos-B were region- and sex-dependent: it inhibited Fos-B in the Sp5c in both sexes but only in males in the vlPAG, suggesting sexually dimorphic activation of descendent pain circuits. Cannabidiol prevented superoxide oxidation in the vlPAG in both sexes, with effects on microglia and astrocytes at similar doses, suggesting that glial cells produce the oxidative stress inhibited by CBD. In the Sp5c, CBD modulated Fos-B, superoxide oxidation, microglia, and astrocytes in both sexes, indicating a possible lack of sexual dimorphism in this region. These results highlight CBD's efficacy in managing TN by modulating ascending and descending nociceptive pathways. Beyond its neuronal effects, CBD's analgesic actions in TN may also involve significant modulation of glial cell activity, underscoring the complexity of its therapeutic mechanisms.

This assay is designed as an efficient method to evaluate nociceptive behaviors and orofacial pain sensitivity in rodents through voluntary facial contact by positive reinforcement using mechanical or thermal stimuli. The protocol includes step-by-step procedures for animal habituation, training, and testing, including device setup, sensor calibration, and optimal task performance of licking/contact detection in controlled mechanical or noxious thermal conditions. The aim is to conduct daily training sessions to ensure consistent licking and contact behavior to achieve accurate data during testing sessions, providing quantitative measures of pain-related responses with minimal stress and allowing reproducibility. This assessment is reliable for orofacial pain models in preclinical studies. Stoelting: OroFacial Pain Assessment Device (OPAD) Supplier

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.

Background: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.

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.

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.

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 (Nav), and menthol evoked TRPM8 currents. Voltage gated potassium channel (Kv) 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, Nav, 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.

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.

Introduction: Management of postoperative pain remains paramount for patients and surgeons. Therapeutic options that are novel and efficacious which provide alternatives to opioid analgesics; offer an opportunity to attenuate or block nocioceptive pathways directly associated with the surgical procedure. Modeling of surgical pain in animal models is critical to evaluate therapeuctics. We describe our preliminary experience with the Orofacial Pain Assessment Device (OPAD) to assess pain through a reward/conflict paradigm providing a humane way of testing a novel therapeutic implant systems. Methods: The Orofacial Pain Assessment Device (OPAD) uses a reward/conflict assay which allows a rodent to choose between receiving a reinforcing reward or escaping an aversive stimulus thus controlling the amount of pain it feels during a session Rodents are first trained to press their faces into temperature controlled thermodes in order to gain access to a food bottle containing a liquid reward. After training, the stimulus temperature can be heated or cooled and differences in responding can indicate the level of nociception or analgesia the animal perceives. Results: the OPAD protocol which incorporate various oral-facial surgical incisions reveals the applicability of and the potential to assess analgesic effect of surgical incisional implants. Conclusion: Use of the Orofacial Pain Assessment Device (OPAD) facilitates surgical modeling of oral-facial surgical pain and the utility in assessment of novel surgical incisional implants in analgesia for post operative care and management.

EDITORIAL article Front. Pharmacol., 08 October 2021 | https://doi.org/10.3389/fphar.2021.778880

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 ED50 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.

An was used in 13 patients as a result of the finding of retrograde flow, including administering flushes more distally in the colon (4/9 responded), changing to oral laxatives (2/2 responded), changing patient positioning during flushes (1/1 responded), and slowing flush administration (1/1 responded).One patient thought to have a cecostomy was found to have a tube placed in the ileum and symptoms resolved with conversion to appendicostomy.Retrograde flow was associated with a younger age (p<0.01)but not with sex or underlying diagnosis.Conclusion: Identifying retrograde flow into the small bowel during fluoroscopic studies with contrast administered via appendicostomy or cecostomy can be useful for children with a poor response to ACE or symptoms associated with ACE administration.In some patients, an intervention to deliver flushes more distally in the colon can improve response and decrease associated symptoms.