Peroxynitrite Contributes to Behavioral Responses, Increased Trigeminal Excitability, and Changes in Mitochondrial Function in a Preclinical Model of Migraine.

Administration of a nitric oxide (NO) donor triggers migraine attacks, but the mechanisms by which this occurs are unknown. Reactive nitroxidative species, including NO and peroxynitrite (PN), have been implicated in nociceptive sensitization, and neutralizing PN is antinociceptive. We determined whether PN contributes to nociceptive responses in two distinct models of migraine headache. Female and male mice were subjected to 3 consecutive days of restraint stress or to dural stimulation with the proinflammatory cytokine interleukin-6. Following resolution of the initial poststimulus behavioral responses, animals were tested for hyperalgesic priming using a normally non-noxious dose of the NO donor sodium nitroprusside (SNP) or dural pH 7.0, respectively. We measured periorbital von Frey and grimace responses in both models and measured stress-induced changes in 3-nitrotyrosine (3-NT) expression (a marker for PN activity) and trigeminal ganglia (TGs) mitochondrial function. Additionally, we recorded the neuronal activity of TGs in response to the PN generator SIN-1 [5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride]. We then tested the effects of the PN decomposition catalysts Fe(III)5,10,15,20-tetrakis(N-methylpyridinium-4-yl) porphyrin (FeTMPyP) and FeTPPS [Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride], or the PN scavenger MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin] against these behavioral, molecular, and neuronal changes. Neutralizing PN attenuated stress-induced periorbital hypersensitivity and priming to SNP, with no effect on priming to dural pH 7.0. These compounds also prevented stress-induced increases in 3-NT expression in both the TGs and dura mater, and attenuated TG neuronal hyperexcitability caused by SIN-1. Surprisingly, FeTMPyP attenuated changes in TG mitochondrial function caused by SNP in stressed males only. Together, these data strongly implicate PN in migraine mechanisms and highlight the therapeutic potential of targeting PN.SIGNIFICANCE STATEMENT Among the most reliable experimental triggers of migraine are nitric oxide donors. The mechanisms by which nitric oxide triggers attacks are unclear but may be because of reactive nitroxidative species such as peroxynitrite. Using mouse models of migraine headache, we show that peroxynitrite-modulating compounds attenuate behavioral, neuronal, and molecular changes caused by repeated stress and nitric oxide donors (two of the most common triggers of migraine in humans). Additionally, our results show a sex-specific regulation of mitochondrial function by peroxynitrite following stress, providing novel insight into the ways in which peroxynitrite may contribute to migraine-related mechanisms. Critically, our data underscore the potential in targeting peroxynitrite formation as a novel therapeutic for the treatment of migraine headache.

HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia.

IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

SARS-CoV-2 Rapidly Infects Peripheral Sensory and Autonomic Neurons, Contributing to Central Nervous System Neuroinvasion before Viremia.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells.

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal-glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10-P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-ßIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and ßIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and ßIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal-glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.

Elucidating the structure and functions of Resolvin D6 isomers on nerve regeneration with a distinctive trigeminal transcriptome.

Innervation sustains cornea integrity. Pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15- or 5-LOX inhibitors, suggesting that the 4- and 17-hydroxy of DHA have an RR- or SR-configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1-4 from tears were resolved. The RR-RvD6 standard eluted as a single peak with RvD61 while pure SR-RvD6 eluted with RvD63 . The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that RR-RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. RR-RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

Stem cells from human exfoliated deciduous teeth relieve pain via downregulation of c-Jun in a rat model of trigeminal neuralgia.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) have excellent immunomodulatory and neuroprotective abilities. It is possible that systemic SHED transplantation could ameliorate trigeminal neuralgia. The phosphorylation of c-Jun contributes to the development of hyperalgesia and allodynia. OBJECTIVE: The present study aimed to evaluate whether systemic SHED transplantation could lead to analgesic effects by regulating peripheral c-Jun in the trigeminal ganglia (TG) in a rat model of trigeminal neuralgia. METHODS: Chronic constriction injury of the infraorbital nerve (CCI-ION) was performed to establish a rat pain model. SHED were obtained from discarded exfoliated deciduous teeth from children and transplanted by a single infusion through the tail vein. SHED were labelled with the PKH26 red fluorescent cell linker mini kit for tract distribution. The mechanical threshold was determined using von Frey filaments. The mRNA levels of c-Jun in the ipsilateral TG were quantified. The phosphorylation of c-Jun in the ipsilateral TG was assessed by immunohistochemistry and Western blotting. RESULTS: PKH26-labelled SHED were distributed to both sides of TG, lung, liver and spleen. Systemic SHED transplantation significantly elevated the mechanical thresholds in CCI-ION rats and blocked the upregulation of c-Jun mRNA levels in the TG caused by nerve ligation. The activation of c-Jun in the TG was blocked by SHED transplantation. CONCLUSIONS: These findings demonstrate that systemic SHED administration reverts trigeminal neuralgia via downregulation of c-Jun in the TG.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

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.

PAC1, VPAC1, and VPAC2 Receptor Expression in Rat and Human Trigeminal Ganglia: Characterization of PACAP-Responsive Receptor Antibodies.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the trigeminal ganglia (TG). The TG conducts nociceptive signals in the head and may play roles in migraine. PACAP infusion provokes headaches in healthy individuals and migraine-like attacks in patients; however, it is not clear whether targeting this system could be therapeutically efficacious. To effectively target the PACAP system, an understanding of PACAP receptor distribution is required. Therefore, this study aimed to characterize commercially available antibodies and use these to detect PACAP-responsive receptors in the TG. Antibodies were initially validated in receptor transfected cell models and then used to explore receptor expression in rat and human TG. Antibodies were identified that could detect PACAP-responsive receptors, including the first antibody to differentiate between the PAC1n and PAC1s receptor splice variants. PAC1, VPAC1, and VPAC2 receptor-like immunoreactivity were observed in subpopulations of both neuronal and glial-like cells in the TG. In this study, PAC1, VPAC1, and VPAC2 receptors were detected in the TG, suggesting they are all potential targets to treat migraine. These antibodies may be useful tools to help elucidate PACAP-responsive receptor expression in tissues. However, most antibodies exhibited limitations, requiring the use of multiple methodologies and the careful inclusion of controls.

The zinc-finger transcription factor GLI3 is a regulator of precerebellar neuronal migration.

Hindbrain precerebellar neurons arise from progenitor pools at the dorsal edge of the embryonic hindbrain: the caudal rhombic lip. These neurons follow distinct migratory routes to establish nuclei that provide climbing or mossy fiber inputs to the cerebellum. Gli3, a zinc-finger transcription factor in the Sonic hedgehog signaling pathway, is an important regulator of dorsal brain development. We demonstrate that in Gli3-null mutant mice, disrupted neuronal migratory streams lead to a disorganization of precerebellar nuclei. Precerebellar progenitors are properly established in Gli3-null embryos and, using conditional gene inactivation, we provide evidence that Gli3 does not play a cell-autonomous role in migrating precerebellar neurons. Thus, GLI3 likely regulates the development of other hindbrain structures, such as non-precerebellar nuclei or cranial ganglia and their respective projections, which may in turn influence precerebellar migration. Although the organization of non-precerebellar hindbrain nuclei appears to be largely unaffected in absence of Gli3, trigeminal ganglia and their central descending tracts are disrupted. We show that rostrally migrating precerebellar neurons are normally in close contact with these tracts, but are detached in Gli3-null embryos.

N/OFQ modulates orofacial pain induced by tooth movement through CGRP-dependent pathways.

BACKGROUND: Nociceptin/orphanin FQ (N/OFQ) has been revealed to play bidirectional roles in orofacial pain modulation. Calcitonin gene-related peptide (CGRP) is a well-known pro-nociceptive molecule that participates in the modulation of orofacial pain. We aimed to determine the effects of N/OFQ on the modulation of orofacial pain and on the release of CGRP. METHODS: Orofacial pain model was established by ligating springs between incisors and molars in rats for the simulation of tooth movement. The expression level of N/OFQ was determined and pain level was scored in response to orofacial pain. Both agonist and antagonist of N/OFQ receptor were administered to examine their effects on pain and the expression of CGRP in trigeminal ganglia (TG). Moreover, gene therapy based on the overexpression of N/OFQ was delivered to validate the modulatory role of N/OFQ on pain and CGRP expression. RESULTS: Tooth movement elicited orofacial pain and an elevation in N/OFQ expression. N/OFQ exacerbated orofacial pain and upregulated CGRP expression in TG, while UFP-101 alleviated pain and downregulated CGRP expression. N/OFQ-based gene therapy was successful in overexpressing N/OFQ in TG, which resulted in pain exacerbation and elevation of CGRP expression in TG. CONCLUSIONS: N/OFQ exacerbated orofacial pain possibly through upregulating CGRP.

Glia and Orofacial Pain: Progress and Future Directions.

Orofacial pain is a universal predicament, afflicting millions of individuals worldwide. Research on the molecular mechanisms of orofacial pain has predominately focused on the role of neurons underlying nociception. However, aside from neural mechanisms, non-neuronal cells, such as Schwann cells and satellite ganglion cells in the peripheral nervous system, and microglia and astrocytes in the central nervous system, are important players in both peripheral and central processing of pain in the orofacial region. This review highlights recent molecular and cellular findings of the glia involvement and glia-neuron interactions in four common orofacial pain conditions such as headache, dental pulp injury, temporomandibular joint dysfunction/inflammation, and head and neck cancer. We will discuss the remaining questions and future directions on glial involvement in these four orofacial pain conditions.

TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization.

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1ß gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1ß mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14-22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.

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.

Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

A variety of mouse strains and sexes are used in studies of corneal wound healing and nerve regeneration. However, there is a gap of knowledge about corneal nerve density and its function in different mouse strains and sexes. In this study, we report a strain divergence of total and substance P (SP) sensory corneal nerves in uninjured mice. The BALB/c mouse showed the highest nerve density, corneal sensitivity, and tear volume followed by CFW and then C57BL/6. No differences were found in total nerves and SP-positive nerves between sexes. After injury damaged the corneal nerves, an important role for mouse strains, biologic sex, and their association to corneal nerve regeneration was identified. All female mice have a faster nerve regeneration rate than males. The molecular mechanism of this sexual divergence involves higher secretion neurotrophic factors in tears, which in turn modulate gene expression in trigeminal ganglion neurons. An important upstream signaling regulator was ß-estradiol, and topical treatment with ß-estradiol confirmed its function in corneal nerve regeneration. In conclusion, our study shows that the strain and sex of laboratory mice significantly affect the different indicators of corneal innervation and nerve regeneration. Researchers investigating corneal diseases should carefully consider these factors.-Pham, T. L., Kakazu, A., He, J., Bazan, H. E. P. Mouse strains and sexual divergence in corneal innervation and nerve regeneration.

Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

BACKGROUND: Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS: We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58 days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS: Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS: These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.

CGRP Plasma Levels Decrease in Classical Trigeminal Neuralgia Patients Treated with Botulinum Toxin Type A: A Pilot Study.

OBJECTIVE: The aim of this study was to investigate the changes of calcitonin gene-related peptide (CGRP) plasma levels in patients with classical trigeminal neuralgia (TN) and if plasma CGRP concentrations could be used to predict the response to botulinum toxin type A (BTX-A). METHODS: Forty-seven patients with classical TN were recruited and treated with BTX-A. A patient was considered a responder when the visual analog scale (VAS) score and number of episodes were reduced by at least 50% compared with baseline data. Matched healthy subjects with no headache history served as controls. CGRP levels were measured by the enzyme-linked immunosorbent assay. RESULTS: A total of 45 patients and 30 healthy controls completed the study. Plasma CGRP concentrations after treatment with BTX-A (median [interquartile range {IQR}] = 28.86 [14.75-61.23] pg/mL) were significantly lower than before treatment (median [IQR] = 55.38 [22.59-71.67] pg/mL, P < 0.001). Plasma CGRP concentrations in responders after treatment with BTX-A (median [IQR] = 28.02 [12.78-57.28] pg/mL) were significantly lower than before treatment (median [IQR] = 50.57 [24.30-70.09] pg/mL, P < 0.001). In nonresponders, there were no significant differences between the levels before and after treatment (P = 0.938). Age, gender, VAS score, taking/not taking carbamazepine, and the number of trigeminal nerve branches involved had no significant influence on the median difference between plasma CGRP concentrations. The concentration of CGRP before treatment was not predictive of the treatment result. CONCLUSIONS: CGRP levels decrease significantly in patients with classical TN after treatment with BTX-A. Plasma levels of CGRP cannot be used to predict the response to BTX-A. This study indicates that CGRP is likely to be involved in the pathophysiology of classical TN. Moreover, the analgesic mechanism of BTX-A may be related to the inhibition of CGRP release.

Molecular basis of tactile specialization in the duck bill.

Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

An M2 Rather than a TH2 Response Contributes to Better Protection against Latency Reactivation following Ocular Infection of Naive Mice with a Recombinant Herpes Simplex Virus 1 Expressing Murine Interleukin-4.

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (TH) responses, with interleukin-4 (IL-4) inducing type 2 TH (TH2) as well as M2 responses and IFN-γ inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC+ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN-γ (an inducer of TH1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.