The role of voltage-gated calcium channel α2δ-1 in the occurrence and development in myofascial orofacial pain.

Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess Whether Voltage-gated calcium channel α2δ-1(Cavα2δ-1) is related to myofascial orofacial pain. Rats were divided into the masseter tendon ligation group and the sham group. Compared with the sham group, the mechanical pain threshold of the masseter tendon ligation group was reduced on the 4th, 7th, 10th and 14th day after operation(P < 0.05). On the 14th day after operation, Cavα2δ-1 mRNA expression levels in trigeminal ganglion (TG) and the trigeminal spinal subnucleus caudalis and C1-C2 spinal cervical dorsal horn (Vc/C2) of the masseter tendon ligation group were increased (PTG=0.021, PVc/C2=0.012). Rats were divided into three groups. On the 4th day after ligating the superficial tendon of the left masseter muscle of the rats, 10 ul Cavα2δ-1 antisense oligonucleotide, 10 ul Cavα2δ-1 mismatched oligonucleotides and 10 ul normal saline was separately injected into the left masseter muscle of rats in Cavα2δ-1 antisense oligonucleotide group, Cavα2δ-1 mismatched oligonucleotides group and normal saline control group twice a day for 4 days. The mechanical pain threshold of the Cavα2δ-1 antisense oligonucleotides group was higher than Cavα2δ-1 mismatched oligonucleotides group on the 7th and 10th day after operation (P < 0.01). After PC12 cells were treated with lipopolysaccharide, Cavα2δ-1 mRNA expression level increased (P < 0.001). Cavα2δ-1 may be involved in the occurrence and development in myofascial orofacial pain.

Cannabidiol and it fluorinate analog PECS-101 reduces hyperalgesia and allodynia in trigeminal neuralgia via TRPV1 receptors.

Trigeminal neuralgia (TN) is an intense and debilitating orofacial pain. The gold standard treatment for TN is carbamazepine. This antiepileptic drug provides pain relief with limited efficacy and side effects. To study the antinociceptive potential of cannabidiol (CBD) and its fluorinated analog PECS-101 (former HUF-101), we induced unilateral chronic constriction injury of the infraorbital nerve (IoN-CCI) in male Wistar rats. Seven days of treatment with CBD (30 mg/kg), PECS-101 (3, 10, and 30 mg/kg), or carbamazepine (10 and 30 mg/kg) reduced allodynia and hyperalgesia responses. Unlike carbamazepine, CBD and PECS-101 did not impair motor activity. The relief of the hypersensitive reactions has been associated with transient receptor potential vanilloid type 1 (TRPV1) modulation in the trigeminal spinal nucleus. CBD (30 mg/kg) and PECS-101 (10 and 30 mg/kg) reversed the increased expression of TRPV1 induced by IoN-CCI in this nucleus. Using a pharmacological strategy, the combination of the selective TRPV1 antagonist (capsazepine-CPZ - 5 mg/kg) with sub-effective doses of CBD (3 and 10 mg/kg) is also able to reverse the IoN-CCI-induced allodynia and hyperalgesia responses. This effect was accompanied by reduced TRPV1 protein expression in the trigeminal spinal nucleus. Our results suggest that CBD and PECS-101 may benefit trigeminal neuralgia without motor coordination impairments. PECS-101 is more potent against the hypernociceptive and motor impairment induced by TN compared to CBD and carbamazepine. The antinociceptive effect of these cannabinoids is partially mediated by TRPV1 receptors in the caudal part of the trigeminal spinal nucleus, the first central station of orofacial pain processing.

The purinergic receptor P2X3 promotes facial pain by activating neurons and cytokines in the trigeminal ganglion.

The mechanism underlying allodynia/hyperalgesia caused by dental pulpitis has remained enigmatic. This investigation endeavored to characterize the influence of the purinergic receptor P2X3 on pain caused by experimental pulpitis and the mechanism involved. An experimental model of irreversible pulpitis was produced by the drilling and exposure of the dental pulp of the left upper first and second molars in rats, followed by measuring nociceptive responses in the oral and maxillofacial regions. Subsequently, neuronal activity and the expression of P2X3 and pertinent cytokines in the trigeminal ganglion (TG) were meticulously examined and analyzed. Histological evidence corroborated that significant pulpitis was produced in this model, which led to a distinct escalation in nociceptive responses in rats. The activation of neurons, coupled with the upregulated expression of c-fos, P2X3, p-p38, TNF-α and IL-1ß, was identified subsequent to the pulpitis surgery within the TG. The selective inhibition of P2X3 with A-317491 effectively restrained the abnormal allodynia/hyperalgesia following the pulpitis surgery and concurrently inhibited the upregulation of p-p38, TNF-α and IL-1ß within the TG. These findings suggest that the P2X3 signaling pathway plays a pivotal role in instigating and perpetuating pain subsequent to the induction of pulpitis in rats, implicating its association with the p38 MAPK signaling pathway and inflammatory factors.

Pain and salivary biomarkers of stress in temperomandibular disorders were affected by maxillary splints.

BACKGROUND: Longitudinal intervention studies on treatment options in temporomandibular dysfunction (TMD) including self reports and salivary biomarkers of stress are rare and the exact therapeutic function of occlusal splints widely unknown. METHODS: We examined the therapeutic effects of a Michigan splint with occlusal relevance in patients with TMD using a placebo-controlled, delayed-start design. Two intervention groups received a Michigan splint, while one of them had a placebo palatine splint for the first 3 weeks. We collected pain intensities (at rest and after five occlusal movements), salivary measures associated with stress (cortisol and alpha-amylase) and self-reported psychological distress (stress, anxiety, catastrophizing) at baseline and 3 and 7 weeks after onset of intervention. RESULTS: At baseline, we observed increased pain intensity and psychological distress in TMD patients compared to 11 matched healthy controls. Baseline anxiety was linked to movement pain intensity through stress. Over therapy reductions in pain intensity and morning cortisol were more pronounced in those patients starting immediately with the Michigan splint, while psychological distress decreased similarly in both groups. CONCLUSION: Our results suggest that perceived stress plays a role for the association between anxiety and TMD pain and underlines the need for an interdisciplinary perspective on the pathogenesis and therapy of TMD in a setting where psychotherapeutic knowledge is still scarce or rarely applied.

In vivo imaging of cathepsin B in activated glia in the brain after orofacial formalin test.

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.

Transplantation of olfactory ensheathing cells can alleviate neuroinflammatory responses in rats with trigeminal neuralgia.

Trigeminal neuralgia (TN) is a common form of facial pain, which primarily manifests as severe pain similar to facial acupuncture and electric shock. Olfactory ensheathing cells (OECs) are glial cells with high bioactivity; these cells are essential for the periodic regeneration of the olfactory nerve and have been utilized for the repair of nerve injuries. A member of the P2X receptor family, P2X7R, is an ion channel type receptor that has been confirmed to participate in various pain response processes. In this study, we transplanted OECs into trigeminal nerve-model rats with distal infraorbital nerve ligation to observe the therapeutic effect of transplanted OECs in rats. Additionally, we utilized the P2X7R-specific inhibitor brilliant blue G (BBG) to study the therapeutic mechanisms of cell transplantation. The facial mechanical pain threshold of these rats significantly increased following cell transplantation. The immunohistochemistry, immunoblotting, and RT-qPCR results demonstrated that the levels of P2X7R, (NOD)-like receptor protein-3 (NLRP3), nuclear factor-κB (NF-κB), interleukin (IL)-1ß, and IL-18 in the trigeminal ganglion of rats treated with OEC transplantation or BBG treatment were significantly lower than those in the injured group without treatment. Overall, our results demonstrate that OEC transplantation can alleviate TN in rats, and it can reduce the expression of P2X7R related inflammatory factors in TN rats, reducing neuroinflammatory response in TG.

Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury.

Background: Trigeminal nerve injury causes orofacial pain that can interfere with activities of daily life. However, the underlying mechanism remains unknown, and the appropriate treatment has not been established yet. This study aimed to examine the involvement of interferon gamma (IFN-γ) signaling in the spinal trigeminal caudal subnucleus (Vc) in orofacial neuropathic pain. Methods: Infraorbital nerve (ION) injury (IONI) was performed in rats by partial ION ligation. The head-withdrawal reflex threshold (HWT) to mechanical stimulation of the whisker pad skin was measured in IONI or sham rats, as well as following a continuous intracisterna magna administration of IFN-γ and a mixture of IFN-γ and fluorocitrate (inhibitor of astrocytes activation) in naïve rats, or an IFN-γ antagonist in IONI rats. The IFN-γ receptor immunohistochemistry and IFN-γ Western blotting were analyzed in the Vc after IONI or sham treatment. The glial fibrillary acid protein (GFAP) immunohistochemistry and Western blotting were also analyzed after administration of IFN-γ and the mixture of IFN-γ and fluorocitrate. Moreover, the change in single neuronal activity in the Vc was examined in the IONI, sham, and IONI group administered IFN-γ antagonist. Results: The HWT decreased after IONI. The IFN-γ and IFN-γ receptor were upregulated after IONI, and the IFN-γ receptor was expressed in Vc astrocytes. IFN-γ administration decreased the HWT, whereas the mixture of IFN-γ and fluorocitrate recovered the decrement of HWT. IFN-γ administration upregulated GFAP expression, while the mixture of IFN-γ and fluorocitrate recovered the upregulation of GFAP expression. IONI significantly enhanced the neuronal activity of the mechanical-evoked responses, and administration of an IFN-γ antagonist significantly inhibited these enhancements. Conclusions: IFN-γ signaling through the receptor in astrocytes is a key mechanism underlying orofacial neuropathic pain associated with trigeminal nerve injury. These findings will aid in the development of therapeutics for orofacial neuropathic pain.

Photic sensitization is mediated by cortico-accumbens pathway in rats with trigeminal neuropathic pain.

Exposure to light stimuli may trigger or exacerbate perception of pain, also known as a common yet debilitating symptom of photophobia in patient with chronic orofacial pain. Mechanism underlying this phenomenon of photic sensitization in neuropathic condition remains elusive. Here, we found that rats developed hypersensitivity to normal light illumination after establishment of chronic constriction injury of infraorbital nerve (ION-CCI) model, which can be attenuated by blocking the exposure of photic stimulation. Additionally, this behavioral phenotype of light-sensitivity impairment was associated with overexpression of anterior cingulate cortex (ACC) c-fos positive neurons, enhancement of neural excitability in the ACC neurons and its excitatory synaptic transmission between nucleus accumbens (NAc). Optogenetic and chemogenic silencing of ACC-NAc pathway improved trigeminal sensitization in responses to light stimuli by decreasing spontaneous pain-like episodes in ION-CCI animals. In contrast, selective activation of ACC-to-NAc circuits enhanced photic hypersensitivity in dark environment. Thus, our data provided novel role of ACC and its projection to NAc in bidirectional modulation of photic sensation, which may contribute to the understanding of photic allodynia in trigeminal neuropathic pain status.

Emerging role of microglia and astrocyte in the affective-motivational response induced by a rat model of persistent orofacial pain.

Few studies are approaching the neural basis underlying the aggregation of emotional disorders in orofacial pain despite the stress, depression, and anxiety are some of the most commonly reported risk factors. Using a persistent orofacial pain rat model induced by complete Freund's adjuvant (CFA) injection into the temporomandibular joint, we have investigated the plasticity astrocytes and microglia key brain regions for the affective-emotional component of pain. We measured the expression and morphologic pattern of reactivation of glial fibrillary acidic protein (GFAP, astrocyte marker) and Iba-1 (microglial marker) by western blotting and immunohistochemistry analysis. The results showed no alterations on motor activity during inflammatory pain, indicating an exclusive effect of nociceptive behavior on the plasticity of limbic regions. CFA-induced temporomandibular inflammation changed GFAP and Iba-1 expression in distinct regions related to emotional behavior in a time-dependent manner. A significant increase in GFAP and Iba-1 expression was observed in the central nucleus of the amygdala, hippocampus and periaqueductal grey matter from day 3 to day 10 post-CFA injection. Moreover, a positive correlation between GFAP and Iba-1 upregulation and an increased mechanical hypersensitivity was observed. Conversely, no change on GFAP and Iba-1 expression was observed in the hypothalamus and colliculus during orofacial inflammatory pain. Our data suggest an important role for glial cells in the affective-motivational dimension of orofacial pain beyond their well-explored role in the traditional nociceptive transmission circuits.

Oral biosciences: The annual review 2022.

BACKGROUND: The Journal of Oral Biosciences is devoted to advancing and disseminating fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review features review articles in the fields of "Bone Cell Biology," "Tooth Development & Regeneration," "Tooth Bleaching," "Adipokines," "Milk Thistle," "Epithelial-Mesenchymal Transition," "Periodontitis," "Diagnosis," "Salivary Glands," "Tooth Root," "Exosome," "New Perspectives of Tooth Identification," "Dental Pulp," and "Saliva" in addition to the review articles by the winner of the "Lion Dental Research Award" ("Plastic changes in nociceptive pathways contributing to persistent orofacial pain") presented by the Japanese Association for Oral Biology. CONCLUSION: The review articles in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge about various aspects of oral biosciences. The current editorial review introduces these exciting review articles.

Age-related Changes in Trigeminal Ganglion Macrophages Enhance Orofacial Ectopic Pain After Inferior Alveolar Nerve Injury.

BACKGROUND/AIM: The ectopic pain associated with inferior alveolar nerve (IAN) injury has been reported to involve macrophage expression in the trigeminal ganglion (TG). However, the effect of age-related changes on this abnormal pain conditions are still unknown. This study sought to clarify the involvement of age-related changes in macrophage expression and phenotypic conversion in the TG and how these changes enhance ectopic mechanical allodynia after IAN transection (IANX). MATERIALS AND METHODS: We used senescence-accelerated mouse (SAM)-prone 8 (SAMP8) and SAM-resistance 1 (SAMR1) mice, which are commonly used to study ageing-related changes. Mechanical stimulation was applied to the whisker pad skin under light anaesthesia; the mechanical head withdrawal threshold (MHWT) was measured for 21 d post-IANX. We subsequently counted the numbers of Iba1 (macrophage marker)-immunoreactive (IR) cells, Iba1/CD11c (M1-like inflammatory macrophage marker)-co-IR cells, and Iba1/CD206 (M2-like anti-inflammatory macrophage marker)-co-IR cells in the TG innervating the whisker pad skin. After continuous intra-TG administration of liposomal clodronate Clophosome®-A (LCCA) to IANX-treated SAMP8-mice, the MHWT values of the whisker pad skin were examined. RESULTS: Five days post-IANX, the MHWT had significantly decreased in SAMP8 mice compared to SAMR1-mice. Iba1-IR and Iba1/CD11c-co-IR cell counts were significantly increased in SAMP8 mice compared to SAMR1 mice 5 d post-IANX. LCCA administration significantly restored MHWT compared to control-LCCA administration. CONCLUSION: Ectopic mechanical allodynia of whisker pad skin after IANX is exacerbated by ageing, which involves increases in M1-like inflammatory macrophages in the TG.

Astrocytic and microglial interleukin-1ß mediates complement C1q-triggered orofacial mechanical allodynia.

Glial cells, such as microglia and astrocytes, in the trigeminal spinal subnucleus caudalis (Vc) are activated after trigeminal nerve injury and interact with Vc neurons to contribute to orofacial neuropathic pain. Complement C1q released from microglia has been reported to activate astrocytes and causes orofacial mechanical allodynia. However, how C1q-induced phenotypic alterations in Vc astrocytes are involved in orofacial pain remains to be elucidated. Intracisternal administration of C1q caused mechanical allodynia in the whisker pad skin and concurrent significant upregulation of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 in the Vc. Immunohistochemical analyses clarified that C1q induces a significant increase in the cytokine interleukin (IL)-1ß, predominantly in Vc astrocytes and partially in Vc microglia. The number of c-Fos-positive neurons in the Vc increased significantly in response to C1q. IL-1 receptor antagonist (IL-1Ra) was used to analyze the involvement of IL-1ß in C1q-induced mechanical allodynia. Intracisternal administration of IL-1Ra ameliorated C1q-induced orofacial mechanical allodynia. The present findings suggest that IL-1ß released from activated astrocytes and microglia in the Vc mediates C1q-induced orofacial pain.

Trigeminal ganglion itself can be a viable target to manage trigeminal neuralgia.

Excruciating trigeminal neuralgia (TN) management is very difficult and severely affects the patient's quality of life. Earlier studies have shown that the trigeminal ganglion (TG) comprises several receptors and signal molecules that are involved in the process of peripheral sensitization, which influences the development and persistence of neuropathic pain. Targeting TG can modulate this sensitization pathway and mediate the pain-relieving effect. So far,there are few studies in which modulation approaches to TG itself have been suggested so far. "Trigeminal ganglion modulation" and "trigeminal neuralgia" were used as search phrases in the Scopus Index and PubMed databases to discover articles that were pertinent to the topic. In this review, we address the role of the trigeminal ganglion in TN and underlying molecules and neuropeptides implicated in trigeminal pain pathways in processing pathological orofacial pain. We also reviewed different modulation approaches in TG for TN management. Furthermore, we discuss the prospect of targeting trigeminal ganglion to manage such intractable pain.

Parkia platycephala Lectin (PPL) Inhibits Orofacial Nociception Responses via TRPV1 Modulation.

Lectins are a heterogeneous group of proteins that reversibly bind to simple sugars or complex carbohydrates. The plant lectin purified from the seed of Parkia platycephala (PPL) was studied. This study aimed to investigate the possible orofacial antinociceptive of PPL lectin in adult zebrafish and rodents. Acute nociception was induced by cinnamaldehyde (0.66 µg/mL), 0.1% acidified saline, glutamate (12.5 µM) or hypertonic saline (5 M NaCl) applied into the upper lip (5.0 µL) of adult wild zebrafish. Zebrafish were pretreated by intraperitoneal injection (20 µL) with vehicle (Control) or PPL (0.025; 0.05 or 0.1 mg/mL) 30 min before induction. The effect of PPL on zebrafish locomotor behaviour was evaluated in the open field test. Naive groups were included in all tests. In one experiment, animals were pre-treated with capsazepine to investigate the mechanism of antinociception. The involvement of central afferent C-fibres was also investigated. In another experiment, rats pre-treated with PPL or saline were submitted to the temporomandibular joint formalin test. Other groups of rats were submitted to infraorbital nerve transection to induce chronic pain, followed by induction of mechanical sensitivity using von Frey. PPL reduced nociceptive behaviour in adult zebrafish, and this is related to the activation of the TRPV1 channels since antinociception was effectively inhibited by capsazepine and by capsaicin-induced desensitization. PPL reduced nociceptive behaviour associated with temporomandibular joint and neuropathic pain. The results confirm the potential pharmacological relevance of PPL as an inhibitor of orofacial nociception in acute and chronic pain.

NGF-Induced Upregulation of CGRP in Orofacial Pain Induced by Tooth Movement Is Dependent on Atp6v0a1 and Vesicle Release.

The nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) play a crucial role in the regulation of orofacial pain. It has been demonstrated that CGRP increases orofacial pain induced by NGF. V-type proton ATPase subunit an isoform 1 (Atp6v0a1) is involved in the exocytosis pathway, especially in vesicular transport in neurons. The objective was to examine the role of Atp6v0a1 in NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars in Sprague-Dawley rats. Gene and protein expression levels were determined through real-time polymerase chain reaction, immunostaining, and fluorescence in situ hybridization. Lentivirus vectors carrying Atp6v0a1 shRNA were used to knockdown the expression of Atp6v0a1 in TG and SH-SY5Y neurons. The release of vesicles in SH-SY5Y neurons was observed by using fluorescence dye FM1-43, and the release of CGRP was detected by Enzyme-Linked Immunosorbent Assy. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that intraganglionic administration of NGF and Atp6v0a1 shRNA upregulated and downregulated CGRP in trigeminal ganglia (TG) and trigeminal subnucleus caudalis (Vc), respectively, and the orofacial pain was also exacerbated and alleviated, respectively, following administration of NGF and Atp6v0a1 shRNA. Besides, intraganglionic administration of NGF simultaneously caused the downregulation of Atp6v0a1 in TG. Moreover, the release of vesicles and CGRP in SH-SY5Y neurons was interfered by NGF and Atp6v0a1 shRNA. In conclusion, in the orofacial pain induced by experimental tooth movement, NGF induced the upregulation of CGRP in TG and Vc, and this process is dependent on Atp6v0a1 and vesicle release, suggesting that they are involved in the transmission of nociceptive information in orofacial pain.

Development of a Nanoformulation for Oral Protein Administration: Characterization and Preclinical Orofacial Antinociceptive Effect.

Nanoencapsulation is a valid alternative for the oral administration of peptide drugs and proteins, as nanoparticles protect them from proteolytic degradation in the gastrointestinal tract and promote the absorption of these macromolecules. The orofacial antinociceptive effect of frutalin (FTL), through the intraperitoneal route, has already been proven. This study aimed to develop, characterize, and evaluate the orofacial antinociceptive activity of an oral formulation containing FTL in acute and neuropathic preclinical tests. Nanoencapsulated FTL was administered by oral route. The acute nociceptive behavior was induced by administering capsaicin to the upper lip and NaCl to the right cornea. The nociceptive behavior was also induced by formalin injected into the temporomandibular joint. The neuropathic pain model involved infraorbital nerve transection (IONX), which induced mechanical hypersensitivity and was assessed by von Frey stimulation. Trpv1 gene expression was analyzed in the trigeminal ganglion. The analyzed sample did not show any cytotoxicity; 52.2% of the FTL was encapsulated, and the size of the nanocapsule was less than 200 nm, the polydispersion was 0.361, and the zeta potential was - 5.87 and - 12.8 mV, with and without FTL, respectively. Nanoencapsulated FTL administered by oral route had an orofacial antinociceptive effect in acute and neuropathic rodent models. The antinociceptive effect of FTL was prevented by ruthenium red, but not by camphor. FTL reduced Trpv1 gene expression. FTL promotes orofacial antinociception, probably due to the antagonism of TRPV1 channels, and the nanoformulation represents an effective method for the oral administration of this protein. HIGHLIGHTS: • Nanoformulation for oral protein administration. • Nanocapsule containing FTL prevents orofacial nociceptive acute and neuropathic pain. • Frutalin promotes orofacial antinociception behavior antagonism of TRPV1 channels.

Differential roles of NMDAR subunits 2A and 2B in mediating peripheral and central sensitization contributing to orofacial neuropathic pain.

The spinal N-methyl-d-aspartate receptor (NMDAR), particularly their subtypes NR2A and NR2B, plays pivotal roles in neuropathic and inflammatory pain. However, the roles of NR2A and NR2B in orofacial pain and the exact molecular and cellular mechanisms mediating nervous system sensitization are still poorly understood. Here, we exhaustively assessed the regulatory effect of NMDAR in mediating peripheral and central sensitization in orofacial neuropathic pain. Von-Frey filament tests showed that the inferior alveolar nerve transection (IANX) induced ectopic allodynia behavior in the whisker pad of mice. Interestingly, mechanical allodynia was reversed in mice lacking NR2A and NR2B. IANX also promoted the production of peripheral sensitization-related molecules, such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, brain-derived neurotrophic factor (BDNF), and chemokine upregulation (CC motif) ligand 2 (CCL2), and decreased the inward potassium channel (Kir) 4.1 on glial cells in the trigeminal ganglion, but NR2A conditional knockout (CKO) mice prevented these alterations. In contrast, NR2B CKO only blocked the changes of Kir4.1, IL-1ß, and TNF-α and further promoted the production of CCL2. Central sensitization-related c-fos, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) were promoted and Kir4.1 was reduced in the spinal trigeminal caudate nucleus by IANX. Differential actions of NR2A and NR2B in mediating central sensitization were also observed. Silencing of NR2B was effective in reducing c-fos, GFAP, and Iba-1 but did not affect Kir4.1. In contrast, NR2A CKO only altered Iba-1 and Kir4.1 and further increased c-fos and GFAP. Gain-of-function and loss-of-function approaches provided insight into the differential roles of NR2A and NR2B in mediating peripheral and central nociceptive sensitization induced by IANX, which may be a fundamental basis for advancing knowledge of the neural mechanisms' reaction to nerve injury.

P2Y14 receptor in trigeminal ganglion contributes to neuropathic pain in mice.

Trigeminal nerve injury is a common complication of various dental and oral procedures, which could induce trigeminal neuropathic pain but lack effective treatments. P2 purinergic receptors have emerged as novel therapeutic targets for such pain. Recent reports implied that the P2Y14 receptor (P2Y14R) was activated and promoted orofacial inflammatory pain and migraine. However, the role and mechanism of P2Y14R in trigeminal neuropathic pain remain unknown. We induced an orofacial neuropathic pain model by chronic constriction injury of the infraorbital nerve (CCI-ION). Von-Frey tests showed that CCI-ION induced orofacial mechanical hypersensitivity. The increased activating transcription factor 3 (ATF3) expression in the trigeminal ganglion (TG) measured by immunofluorescence confirmed trigeminal nerve injury. Immunofluorescence showed that P2Y14R was expressed in trigeminal ganglion neurons (TGNs) and satellite glial cells (SGCs). RT-qPCR and Western blot identified increased expression of P2Y14R in TG after CCI-ION. CCI-ION also upregulated interleukin-1ß (IL-1ß), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-α (TNF-α) in TG. Notably, CCI-ION-induced mechanical hypersensitivity and pro-inflammatory cytokines production were decreased by a P2Y14R antagonist (PPTN). Trigeminal administration of P2Y14R agonist (UDP-glucose) evoked orofacial mechanical hypersensitivity and increased pro-inflammatory cytokines above in TG. Furthermore, CCI-ION induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 in TG, which also were reduced by PPTN. The inhibitors of ERK1/2 (U0126) and p38 (SB203580) decreased these upregulated pro-inflammatory cytokines after CCI-ION. Collectively, this study revealed that P2Y14R in TG contributed to trigeminal neuropathic pain via ERK- and p38-dependent neuroinflammation. Thus, P2Y14R may be a potential drug target against trigeminal neuropathic pain.

Loss of Elp1 disrupts trigeminal ganglion neurodevelopment in a model of familial dysautonomia.

Familial dysautonomia (FD) is a sensory and autonomic neuropathy caused by mutations in elongator complex protein 1 (ELP1). FD patients have small trigeminal nerves and impaired facial pain and temperature perception. These signals are relayed by nociceptive neurons in the trigeminal ganglion, a structure that is composed of both neural crest- and placode-derived cells. Mice lacking Elp1 in neural crest derivatives ('Elp1 CKO') are born with small trigeminal ganglia, suggesting Elp1 is important for trigeminal ganglion development, yet the function of Elp1 in this context is unknown. We demonstrate that Elp1, expressed in both neural crest- and placode-derived neurons, is not required for initial trigeminal ganglion formation. However, Elp1 CKO trigeminal neurons exhibit abnormal axon outgrowth and deficient target innervation. Developing nociceptors expressing the receptor TrkA undergo early apoptosis in Elp1 CKO, while TrkB- and TrkC-expressing neurons are spared, indicating Elp1 supports the target innervation and survival of trigeminal nociceptors. Furthermore, we demonstrate that specific TrkA deficits in the Elp1 CKO trigeminal ganglion reflect the neural crest lineage of most TrkA neurons versus the placodal lineage of most TrkB and TrkC neurons. Altogether, these findings explain defects in cranial gangliogenesis that may lead to loss of facial pain and temperature sensation in FD.

Chronic facial inflammatory pain-induced anxiety is associated with bilateral deactivation of the rostral anterior cingulate cortex.

Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10-11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.