Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

Activation of cyclin-dependent kinase 5 broadens action potentials in human sensory neurons.

Chronic pain is one of the most devastating and unpleasant conditions, associated with many pathological states. Tissue or nerve injuries induce extensive neurobiological plasticity in nociceptive neurons, which leads to chronic pain. Recent studies suggest that cyclin-dependent kinase 5 (CDK5) in primary afferents is a key neuronal kinase that modulates nociception through phosphorylation under pathological conditions. However, the impact of the CDK5 on nociceptor activity especially in human sensory neurons is not known. To determine the CDK5-mediated regulation of human dorsal root ganglia (hDRG) neuronal properties, we have performed the whole-cell patch clamp recordings in neurons dissociated from hDRG. CDK5 activation induced by overexpression of p35 depolarized the resting membrane potential (RMP) and reduced the rheobase currents as compared to the control neurons. CDK5 activation changed the shape of the action potential (AP) by increasing AP -rise time, -fall time, and -half width. The application of a prostaglandin E2 (PG) and bradykinin (BK) cocktail in control hDRG neurons induced the depolarization of RMP and the reduction of rheobase currents along with increased AP rise time. However, PG and BK applications failed to induce any significant changes in the p35-overexpressing group. We conclude that, in dissociated hDRGs neurons, CDK5 activation through the overexpression of p35 broadens the AP and that CDK5 may play important roles in the modulation of AP properties in human primary afferents under the condition in which CDK5 is upregulated, contributing to chronic pain.

Piezo channels for skeletal development and homeostasis: Insights from mouse genetic models.

Piezo1 and Piezo2 are recently discovered mechanosensory ion channels. Piezo channels transduce mechanical stimulation into cellular signaling in a variety of tissues and organ systems. The functional roles of Piezo1 and Piezo2 have been revealed in both developmental and physiological scenarios by using mouse genetic models. Mechanotransduction by Piezo1 channels regulates osteoblast/osteocyte activity and, thus, strengthens the skeleton enabling it to adapt to a wide range of mechanical loadings. Deletion of the Piezo1 gene in the developing skeleton causes bone malformations that lead to spontaneous bone fractures, while inactivity of Piezo1 in adulthood results in osteoporosis. Furthermore, Piezo2 channels in sensory neurons might provide another route of skeletal regulation. Piezo channels also regulate the proliferation and differentiation of various types of stem cells. PIEZO1 and PIEZO2 mutations and channel malfunctions have been implicated in an increasing number of human diseases, and PIEZO channels are currently emerging as potential targets for disease treatment. This review summarizes the important findings of Piezo channels for skeletal development and homeostasis using the mouse genetic model system.

Nociceptive signaling through transient receptor potential vanilloid 1 is regulated by Cyclin Dependent Kinase 5-mediated phosphorylation of T407 in vivo.

Cyclin dependent kinase 5 (Cdk5) is a key neuronal kinase whose activity can modulate thermo-, mechano-, and chemo-nociception. Cdk5 can modulate nociceptor firing by phosphorylating pain transducing ion channels like the transient receptor potential vanilloid 1 (TRPV1), a thermoreceptor that is activated by noxious heat, acidity, and capsaicin. TRPV1 is phosphorylated by Cdk5 at threonine-407 (T407), which then inhibits Ca2+ dependent desensitization. To explore the in vivo implications of Cdk5-mediated TRPV1 phosphorylation on pain perception, we engineered a phospho-null mouse where we replaced T407 with alanine (T407A). The T407A point mutation did not affect the expression of TRPV1 in nociceptors of the dorsal root ganglia and trigeminal ganglia (TG). However, behavioral tests showed that the TRPV1T407A knock-in mice have reduced aversion to oral capsaicin along with a trend towards decreased facial displays of pain after a subcutaneous injection of capsaicin into the vibrissal pad. In addition, the TRPV1T407A mice display basal thermal hypoalgesia with increased paw withdrawal latency while tested on a hot plate. These results indicate that phosphorylation of TRPV1 by Cdk5 can have important consequences on pain perception, as loss of the Cdk5 phosphorylation site reduced capsaicin- and heat-evoked pain behaviors in mice.

Acute and Chronic Pain from Facial Skin and Oral Mucosa: Unique Neurobiology and Challenging Treatment.

The oral cavity is a portal into the digestive system, which exhibits unique sensory properties. Like facial skin, the oral mucosa needs to be exquisitely sensitive and selective, in order to detect harmful toxins versus edible food. Chemosensation and somatosensation by multiple receptors, including transient receptor potential channels, are well-developed to meet these needs. In contrast to facial skin, however, the oral mucosa rarely exhibits itch responses. Like the gut, the oral cavity performs mechanical and chemical digestion. Therefore, the oral mucosa needs to be insensitive, to some degree, in order to endure noxious irritation. Persistent pain from the oral mucosa is often due to ulcers, involving both tissue injury and infection. Trigeminal nerve injury and trigeminal neuralgia produce intractable pain in the orofacial skin and the oral mucosa, through mechanisms distinct from those seen in the spinal area, which is particularly difficult to predict or treat. The diagnosis and treatment of idiopathic chronic pain, such as atypical odontalgia (idiopathic painful trigeminal neuropathy or post-traumatic trigeminal neuropathy) and burning mouth syndrome, remain especially challenging. The central integration of gustatory inputs might modulate chronic oral and facial pain. A lack of pain in chronic inflammation inside the oral cavity, such as chronic periodontitis, involves the specialized functioning of oral bacteria. A more detailed understanding of the unique neurobiology of pain from the orofacial skin and the oral mucosa should help us develop novel methods for better treating persistent orofacial pain.

Injectable Capsaicin for the Management of Pain Due to Osteoarthritis.

Capsaicin is a potent agonist of the TRPV1 channel, a transduction channel that is highly expressed in nociceptive fibers (pain fibers) throughout the peripheral nervous system. Given the importance of TRPV1 as one of several transduction channels in nociceptive fibers, much research has been focused on the potential therapeutic benefits of using TRPV1 antagonists for the management of pain. However, an antagonist has two limitations. First, an antagonist in principle generally only affects one receptor. Secondly, most antagonists must have an ongoing presence on the receptor to have an effect. Capsaicin overcomes both liabilities by disrupting peripheral terminals of nociceptive fibers that express TRPV1, and thereby affects all of the potential means of activating that pain fiber (not just TRPV1 function). This disruptive effect is dependent on the dose and can occur within minutes. Thus, unlike a typical receptor antagonist, continued bioavailability at the level of the receptor is not necessary. By disrupting the entire terminal of the TRPV1-expressing nociceptive fiber, capsaicin blocks all the activation mechanisms within that fiber, and not just TRPV1 function. Topical capsaicin, an FDA approved treatment for neuropathic pain, addresses pain from abnormal nociceptor activity in the superficial layers of the skin. Effects after a single administration are evident over a period of weeks to months, but in time are fully reversible. This review focuses on the rationale for using capsaicin by injection for painful conditions such as osteoarthritis (OA) and provides an update on studies completed to date.

Phosphorylation of TRPV1 S801 Contributes to Modality-Specific Hyperalgesia in Mice.

Transient receptor potential vanilloid subtype 1 (TRPV1) is a nonselective cationic channel activated by painful stimuli such as capsaicin and noxious heat, and enriched in sensory neurons of the pain pathway. During inflammation, chemical mediators activate protein kinases (such as PKC) that phosphorylate TRPV1 and thereby enhance its function, with consequent increases in nociceptor sensitization. However, the causal relationships between TRPV1 phosphorylation and pathological pain remain unexplored. To directly investigate the roles of one specific TRPV1 phosphorylation event in vivo, we genetically altered a major PKC phosphorylation site, mouse TRPV1 S801, to alanine. The TRPV1 expression pattern in sensory neurons of S801A knock-in (KI) mice was comparable to that in WT controls. However, sensitization of capsaicin-mediated currents after the activation of PKC was substantially impaired in sensory neurons from KI mice. Thermal hyperalgesia induced by PMA or burn injury in KI was identical to WT. Inflammatory thermal hyperalgesia was only marginally attenuated in KI mice. In contrast, PMA-evoked nocifensive responses and sensitization of capsaicin responses were significantly attenuated in the hindpaws of KI mice. Ongoing pain from inflamed masseter muscle was also reduced in KI mice, and was further inhibited by the TRPV1 antagonist AMG9810. These results suggest that PKC-mediated phosphorylation of TRPV1 S801 contributes to inflammation-mediated sensitization of TRPV1 to ligand, but not heat, in vivo Further, this suggests that interference with TRPV1 S801 phosphorylation might represent one potential way to attenuate inflammatory pain, yet spare basal sensitivity and produce fewer side effects than more general TRPV1 inhibition.SIGNIFICANCE STATEMENT Transient receptor potential vanilloid subtype 1 (TRPV1) has been considered a potential target for pain intervention. Global inhibitors of TRPV1 function, however, produce side effects which could compromise their clinical utility. By precisely removing a unique PKC phosphorylation site (TRPV1 S801) in mice through CRISPR/Cas9 editing, we provide in vivo evidence for a highly specific inhibition that leaves basal TRPV1 function intact, yet alleviates some forms of hyperalgesia. These findings support inhibition of TRPV1 S801 phosphorylation as a potential intervention for pain management.

Orthodontic force induces nerve injury-like transcriptomic changes driven by TRPV1-expressing afferents in mouse trigeminal ganglia.

Orthodontic force produces mechanical irritation and localized inflammation in the periodontium, which causes pain in most patients. Nocifensive behaviors resulting from orthodontic force in mice can be substantially attenuated by intraganglionic injection of resiniferatoxin (RTX), a neurotoxin that specifically ablates a subset of neurons expressing transient receptor potential vanilloid 1 (TRPV1). In the current study, we determined changes in the transcriptomic profiles in the trigeminal ganglia (TG) following the application of orthodontic force, and assessed the roles of TRPV1-expressing afferents in these transcriptomic changes. RTX or vehicle was injected into the TG of mice a week before the placement of an orthodontic spring exerting 10 g of force. After 2 days, the TG were collected for RNA sequencing. The application of orthodontic force resulted in 1279 differentially expressed genes (DEGs) in the TG. Gene ontology analysis showed downregulation of gliogenesis and ion channel activities, especially of voltage-gated potassium channels. DEGs produced by orthodontic force correlated more strongly with DEGs resulting from nerve injury than from inflammation. Orthodontic force resulted in the differential expression of multiple genes involved in pain regulation, including upregulation of Atf3, Adcyap1, Bdnf, and Csf1, and downregulation of Scn10a, Kcna2, Kcnj10, and P2ry1. Orthodontic force-induced DEGs correlated with DEGs specific to multiple neuronal and non-neuronal subtypes following nerve injury. These transcriptomic changes were abolished in the mice that received the RTX injection. These results suggest that orthodontic force produces transcriptomic changes resembling nerve injury in the TG and that nociceptive inputs through TRPV1-expressing afferents leads to subsequent changes in gene expression not only in TRPV1-positive neurons, but also in TRPV1-negative neurons and non-neuronal cells throughout the ganglia. Orthodontic force-induced transcriptomic changes might be an active regenerative program of trigeminal ganglia in response to axonal injury following orthodontic force.

Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1.

Capsaicin is an ingredient in spicy peppers that produces burning pain by activating transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel in nociceptors. Capsaicin has also been used as an analgesic, and its topical administration is approved for the treatment of certain pain conditions. The mechanisms underlying capsaicin-induced analgesia likely involve reversible ablation of nociceptor terminals. However, the mechanisms underlying these effects are not well understood. To visualize TRPV1-lineage axons, a genetically engineered mouse model was used in which a fluorophore is expressed under the TRPV1 promoter. Using a combination of these TRPV1-lineage reporter mice and primary afferent cultures, we monitored capsaicin-induced effects on afferent terminals in real time. We found that Ca2+ influx through TRPV1 is necessary for capsaicin-induced ablation of nociceptive terminals. Although capsaicin-induced mitochondrial Ca2+ uptake was TRPV1-dependent, dissipation of the mitochondrial membrane potential, inhibition of the mitochondrial transition permeability pore, and scavengers of reactive oxygen species did not attenuate capsaicin-induced ablation. In contrast, MDL28170, an inhibitor of the Ca2+-dependent protease calpain, diminished ablation. Furthermore, overexpression of calpastatin, an endogenous inhibitor of calpain, or knockdown of calpain 2 also decreased ablation. Quantitative assessment of TRPV1-lineage afferents in the epidermis of the hind paws of the reporter mice showed that EGTA and MDL28170 diminished capsaicin-induced ablation. Moreover, MDL28170 prevented capsaicin-induced thermal hypoalgesia. These results suggest that TRPV1/Ca2+/calpain-dependent signaling plays a dominant role in capsaicin-induced ablation of nociceptive terminals and further our understanding of the molecular mechanisms underlying the effects of capsaicin on nociceptors.

Role of the outer pore domain in transient receptor potential vanilloid 1 dynamic permeability to large cations.

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.

Transcriptome analysis of trigeminal ganglia following masseter muscle inflammation in rats.

BACKGROUND: Chronic pain in masticatory muscles is a major medical problem. Although mechanisms underlying persistent pain in masticatory muscles are not fully understood, sensitization of nociceptive primary afferents following muscle inflammation or injury contributes to muscle hyperalgesia. It is well known that craniofacial muscle injury or inflammation induces regulation of multiple genes in trigeminal ganglia, which is associated with muscle hyperalgesia. However, overall transcriptional profiles within trigeminal ganglia following masseter inflammation have not yet been determined. In the present study, we performed RNA sequencing assay in rat trigeminal ganglia to identify transcriptome profiles of genes relevant to hyperalgesia following inflammation of the rat masseter muscle. RESULTS: Masseter inflammation differentially regulated >3500 genes in trigeminal ganglia. Predominant biological pathways were predicted to be related with activation of resident non-neuronal cells within trigeminal ganglia or recruitment of immune cells. To focus our analysis on the genes more relevant to nociceptors, we selected genes implicated in pain mechanisms, genes enriched in small- to medium-sized sensory neurons, and genes enriched in TRPV1-lineage nociceptors. Among the 2320 candidate genes, 622 genes showed differential expression following masseter inflammation. When the analysis was limited to these candidate genes, pathways related with G protein-coupled signaling and synaptic plasticity were predicted to be enriched. Inspection of individual gene expression changes confirmed the transcriptional changes of multiple nociceptor genes associated with masseter hyperalgesia (e.g., Trpv1, Trpa1, P2rx3, Tac1, and Bdnf) and also suggested a number of novel probable contributors (e.g., Piezo2, Tmem100, and Hdac9). CONCLUSION: These findings should further advance our understanding of peripheral mechanisms involved in persistent craniofacial muscle pain conditions and provide a rational basis for identifying novel genes or sets of genes that can be potentially targeted for treating such conditions.

Carboxyl-terminal domain of transient receptor potential vanilloid 1 contains distinct segments differentially involved in capsaicin- and heat-induced desensitization.

Multiple Ca(2+)-dependent processes are involved in capsaicin-induced desensitization of transient receptor potential vanilloid 1 (TRPV1), but desensitization of TRPV1 by heat occurs even in the absence of extracellular Ca(2+), although the mechanisms are unknown. In this study, we tested the hypothesis that capsaicin and heat desensitize TRPV1 through distinct mechanisms involving distinct structural segments of TRPV1. In HEK293 cells that heterologously express TRPV1, we found that heat-induced desensitization was not affected by the inclusion of intracellular ATP or alanine mutation of Lys(155), both of which attenuate capsaicin-induced desensitization, suggesting that heat-induced desensitization occurs through mechanisms distinct from capsaicin-induced desensitization. To determine protein domains involved in heat-induced desensitization, we generated chimeric proteins between TRPV1 and TRPV3, a heat-gated channel lacking heat-induced desensitization. We found that TRPV1 with the carboxyl-terminal domain (CTD) of TRPV3 retained heat activation but was impaired in heat-induced desensitization. Further experiments using chimeric or deletion mutants within TRPV1 CTD indicated that the distal half of CTD regulates the activation and desensitization of TRPV1 in modality-specific manners. Within the distal CTD, we identified two segments that distinctly regulated capsaicin- and heat-induced desensitization. The results suggest that the activation and desensitization of TRPV1 by capsaicin and heat can be modulated differentially and disproportionally through different regions of TRPV1 CTD. Identifying the domains involved in thermal regulation of TRPV1 may facilitate the development of novel anti-hyperalgesic approaches aimed at attenuating activation and enhancing desensitization of TRPV1 by thermal stimuli.

Nociceptor mechanisms underlying pain and bone remodeling via orthodontic forces: toward no pain, big gain.

Orthodontic forces are strongly associated with pain, the primary complaint among patients wearing orthodontic braces. Compared to other side effects of orthodontic treatment, orthodontic pain is often overlooked, with limited clinical management. Orthodontic forces lead to inflammatory responses in the periodontium, which triggers bone remodeling and eventually induces tooth movement. Mechanical forces and subsequent inflammation in the periodontium activate and sensitize periodontal nociceptors and produce orthodontic pain. Nociceptive afferents expressing transient receptor potential vanilloid subtype 1 (TRPV1) play central roles in transducing nociceptive signals, leading to transcriptional changes in the trigeminal ganglia. Nociceptive molecules, such as TRPV1, transient receptor potential ankyrin subtype 1, acid-sensing ion channel 3, and the P2X3 receptor, are believed to mediate orthodontic pain. Neuropeptides such as calcitonin gene-related peptides and substance P can also regulate orthodontic pain. While periodontal nociceptors transmit nociceptive signals to the brain, they are also known to modulate alveolar bone remodeling in periodontitis. Therefore, periodontal nociceptors and nociceptive molecules may contribute to the modulation of orthodontic tooth movement, which currently remains undetermined. Future studies are needed to better understand the fundamental mechanisms underlying neuroskeletal interactions in orthodontics to improve orthodontic treatment by developing novel methods to reduce pain and accelerate orthodontic tooth movement-thereby achieving "big gains with no pain" in clinical orthodontics.

Neurobiological mechanisms of botulinum neurotoxin-induced analgesia for neuropathic pain.

Botulinum neurotoxins (BoNTs) are a family of neurotoxins produced by Clostridia and other bacteria that induce botulism. BoNTs are internalized into nerve terminals at the site of injection and cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to inhibit the vesicular release of neurotransmitters. BoNTs have been approved for multiple therapeutic applications, including the treatment of migraines. They have also shown efficacies for treating neuropathic pain, such as diabetic neuropathy, and postherpetic and trigeminal neuralgia. However, the mechanisms underlying BoNT-induced analgesia are not well understood. Peripherally administered BoNT is taken up by the nerve terminals and reduces the release of glutamate, calcitonin gene-related peptide, and substance P, which decreases neurogenic inflammation in the periphery. BoNT is retrogradely transported to sensory ganglia and central terminals in a microtubule-dependent manner. BoNTs decrease the expression of pronociceptive genes (ion channels or cytokines) from sensory ganglia and the release of neurotransmitters and neuropeptides from primary afferent central terminals, which likely leads to decreased central sensitization in the dorsal horn of the spinal cord or trigeminal nucleus. BoNT-induced analgesia is abolished after capsaicin-induced denervation of transient receptor potential vanilloid 1 (TRPV1)-expressing afferents or the knockout of substance P or the neurokinin-1 receptor. Although peripheral administration of BoNT leads to changes in the central nervous system (e.g., decreased phosphorylation of glutamate receptors in second-order neurons, reduced activation of microglia, contralateral localization, and cortical reorganization), whether such changes are secondary to changes in primary afferents or directly mediated by trans-synaptic, transcytotic, or the hematogenous transport of BoNT is controversial. To enhance their therapeutic potential, BoNTs engineered for specific targeting of nociceptive pathways have been developed to treat chronic pain. Further mechanistic studies on BoNT-induced analgesia can enhance the application of native or engineered BoNTs for neuropathic pain treatment with improved safety and efficacy.

Survival analysis of teeth following clinical crown lengthening and crown insertion procedures up to 14 years: A retrospective cohort study.

BACKGROUND: The purpose of this study was to conduct survival analysis of teeth following clinical crown lengthening procedures (CLPs) and crown insertions via a retrospective cohort study. METHODS: Patient- and tooth-related data were collected from 268 participants who received CLPs from 2009 to 2015. The Kaplan-Meier curve and the log-rank tests were used to estimate the probability of survival and compare the survival probabilities among different variables. A Cox multivariate proportional hazard regression model was used to investigate the collective effects of root canal treatment (RCT) and the types of opposing dentition. RESULTS: The rate of tooth loss was 21.6% during the observation period from 1 to 14 years, with 58 teeth extracted. The most attributable reason for tooth extraction was coronal tooth fracture, followed by endodontic failure such as root fracture. The survival probability was 0.87 at 5 years and 0.7 at 10 years. No significant differences in the survival probabilities were found among different providers and locations, the presence of a post, and the types of crowns. The hazard ratio for tooth loss was 6.3, 95% confidence interval (CI) [2.6 to 20.9] in the teeth with RCT (p < 0.001) and 2.4, 95% CI [1.1 to 4.8] in the teeth occluding implant-retained prostheses (p = 0.016). CONCLUSIONS: Tooth loss following CLPs and crown insertions appeared least among the teeth without RCT when occluding natural teeth, while tooth loss was most among the teeth with RCT when occluding implants.

Elucidation of neuronal activity in mouse models of TMJ injury by in vivo GCaMP Ca 2+ imaging of intact trigeminal ganglion neurons.

Patients with temporomandibular disorders (TMD) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca 2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo , specifically in TMJ animal models. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of forced mouth open (FMO) mice. An inhibitor of the CGRP receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of CGRP antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca 2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain animal models of TMD, bringing us closer understanding the pathophysiological processes underlying TMD. Our study also illustrates the utility of in vivo GCaMP3 Ca 2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMD.

Inflammation, brain connectivity, and neuromodulation in post-traumatic headache.

Post-traumatic headache (PTH) is a debilitating condition that affects individuals with different levels of traumatic brain injury (TBI) severity. The difficulties in developing an effective treatment are related to a lack of understanding the complicated mechanisms and neurobiological changes in brain function after a brain injury. Preclinical studies have indicated that peripheral and central sensitization of the trigeminal nociceptive pathways contributes to PTH. While recent brain imaging studies have uncovered widespread changes in brain functional connectivity following trauma, understanding exactly how these networks contribute to PTH after injury remains unknown. Stimulation of peripheral (trigeminal or vagus) nerves show promising efficacies in PTH experimental animals, likely mediated by influencing TBI-induced pathological plasticity by decreasing neuroinflammation and neuronal apoptosis. Non-invasive brain stimulations, such as transcranial magnetic or direct current stimulations, show analgesia for multiple chronic pain conditions, including PTH. Better mechanistic understanding of analgesia achieved by neuromodulations can define peripheral and central mechanisms involved in the development, the resolution, and the management of PTH.

Therapeutic effectiveness of keratinized mucosa augmentation for functioning dental implants: A systematic review and meta-analysis.

PURPOSE: This study aimed to provide the latest updates on the therapeutic effectiveness of keratinized mucosa (KM) augmentation using autogenous soft tissue grafts for dental implants retaining prostheses. STUDY SELECTION: A systematic search of electronic databases was conducted on autogenous soft tissue grafts to create and/or augment KM for functioning dental implants. Two investigators independently extracted data from the selected 11 clinical studies, including 290 participants, from the initially retrieved 573 publications. RESULTS: A lack of KM surrounding dental implants was associated with greater mucosal inflammation. A free gingival graft (FGG) was used to increase the KM width, and a connective tissue graft (CTG) was used to manage peri-implant mucosal recession (MR). The weighted mean gain in KM was 2.6 mm from the selected FGG studies, with a significant reduction in mucosal inflammation and no changes in crestal bone levels for up to 4 years. The weighted mean reduction in MR was 2 mm in selected CTG studies. CONCLUSIONS: A lack of KM negatively affects soft tissue health around dental implants. FGG was effective in increasing KM and reducing mucosal inflammation, whereas CTG was effective in decreasing MR.

Genetic labeling of the nucleus of tractus solitarius neurons associated with electrical stimulation of the cervical or auricular vagus nerve in mice.

INTRODUCTION: Vagus nerve stimulation (VNS) is clinically useful for treating epilepsy, depression, and chronic pain. Currently, cervical VNS (cVNS) treatment is well-established, while auricular VNS (aVNS) is under development. Vagal stimulation regulates functions in diverse brain regions; therefore, it is critical to better understand how electrically-evoked vagal inputs following cVNS and aVNS engage with different brain regions. OBJECTIVE: As vagus inputs are predominantly transmitted to the nucleus of tractus solitarius (NTS), we directly compared the activation of NTS neurons by cVNS or aVNS and the brain regions directly projected by the activated NTS neurons in mice. METHODS: We adopted the targeted recombination in active populations method, which allows for the activity-dependent, tamoxifen-inducible expression of mCherry-a reporter protein-in neurons specifically associated with cVNS or aVNS. RESULTS: cVNS and aVNS induced comparable bilateral mCherry expressions in neurons within the NTS, especially in its caudal section (cNTS). However, the numbers of mCherry-expressing neurons within different subdivisions of cNTS was distinctive. In both cVNS and aVNS, anterogradely labeled mCherry-expressing axonal terminals were similarly observed across different areas of the forebrain, midbrain, and hindbrain. These terminals were enriched in the rostral ventromedial medulla, parabrachial nucleus, periaqueductal gray, thalamic nuclei, central amygdala, and the hypothalamus. Sex difference of cVNS- and aVNS-induced labeling of NTS neurons was modest. CONCLUSION: The central projections of mCherry-expressing cNTS terminals are comparable between aVNS and cVNS, suggesting that cVNS and aVNS activate distinct but largely overlapping projections into the brain through the cNTS.