Mouse Trigeminal Ganglia Research Articles

Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway.

The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization.

The temperature-sensitive receptors TRPV4 and TRPM8 have important roles in the pruritus of rosacea

BackgroundCertain sensations are the secondary phenotypes of rosacea and affect patients’ quality of life. Transient receptor potential (TRP) channels may be involved in its occurrence. However, there is a lack of research independently discussing itch in rosacea. ObjectivesOur study aimed to investigate risk factors for pruritus in rosacea patients and to discover the molecular mechanism of pruritus. MethodsA binary logistic regression model was used to identify significant variables affecting pruritus in 782 rosacea patients. The LL-37 was injected intradermally into the face of mice to establish the animal model. qRT–PCR, immunohistochemistry and immunofluorescence were used to analyse the expression differences in pruritus-related molecules in mouse skin and the corresponding trigeminal ganglion (TG) between pruritus and nonpruritus groups. ResultsThe incidence of pruritus in rosacea was 42.46%, and the incidence of other symptoms increased with pruritus. Temperature effects were prominently related to the itch sensation of rosacea. Intradermal injection of LL-37 not only caused rosacea-like facial lesions but also induced a behavioural pattern indicative of pruritus. Increased expression of the temperature-sensitive receptors TRPV4 and TRPM8 was found in pruritic mouse skin and TG and human skin samples. ConclusionsIn rosacea patients, pruritus occurs frequently along with burning, flushing and sensitivity, most likely due to changes in temperature. The temperature-sensitive receptors TRPV4 and TRPM8 are both involved in the mechanism of pruritus in rosacea.

Single-cell RNA sequencing reveals distinct transcriptional features of the purinergic signaling in mouse trigeminal ganglion.

Trigeminal ganglion (TG) is the first station of sensory pathways in the orofacial region. The TG neurons communicate with satellite glial cells (SGCs), macrophages and other cells forming a functional unit that is responsible for processing of orofacial sensory information. Purinergic signaling, one of the most widespread autocrine and paracrine pathways, plays a crucial role in intercellular communication. The multidirectional action of purinergic signaling in different cell types contributes to the neuromodulation and orofacial sensation. To fully understand the purinergic signaling in these processes, it is essential to determine the shared and unique expression patterns of genes associated with purinergic signaling in different cell types. Here, we performed single-cell RNA sequencing of 22,969 cells isolated from normal mouse TGs. We identified 18 distinct cell populations, including 6 neuron subpopulations, 3 glial subpopulations, 7 immune cell subpopulations, fibroblasts, and endothelial cells. We also revealed the transcriptional features of genes associated with purinergic signaling, including purinergic receptors, extracellular adenosine triphosphate (eATP) release channels, eATP metabolism-associated enzymes, and eATP transporters in each cell type. Our results have important implications for understanding and predicting the cell type-specific roles of the purinergic signaling in orofacial signal processing in the trigeminal primary sensory system.

Spontaneous activity in whisker-innervating region of neonatal mouse trigeminal ganglion

Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

Interactions Among Non-Coding RNAs and mRNAs in the Trigeminal Ganglion Associated with Neuropathic Pain.

BackgroundRecent studies have demonstrated the contribution of non-coding RNAs (ncRNAs) to neuropathic pain. However, the expression profile of ncRNAs in the trigeminal ganglion (TG) and their functional mechanism underlying trigeminal neuropathic pain are still unclear.MethodsIn the present study, the trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve (CCI-ION) was used to study the expression profile and potential regulatory mechanism of miRNAs, lncRNAs, circRNAs, and mRNAs in the TG by RNA-sequencing (RNA-seq) and bioinformatics analysis. CCI-ION mice suffered from mechanical allodynia from 3 days to 28 days after surgery.ResultsThe RNA-seq results discovered 67 miRNAs, 216 lncRNAs, 14 circRNAs, 595 mRNAs, and 421 genes were differentially expressed (DE) in the TG of CCI-ION mice 7 days after surgery. And 39 DEGs were known pain genes. Besides, 5 and 35 pain-related DE mRNAs could be targeted by 6 DE miRNAs and 107 DE lncRNAs, respectively. And 23 pain-related DEGs had protein–protein interactions (PPI) with each other. GO analysis indicated membrane-related cell components and binding-related molecular functions were significantly enriched. KEGG analysis showed that nociception-related signaling pathways were significantly enriched for DE ncRNAs and DEGs. Finally, the competing endogenous RNA (ceRNA) regulatory network of DE lncRNA/DE circRNA-DE miRNA-DE mRNA existed in the TG of mice with trigeminal neuropathic pain.ConclusionOur findings demonstrate ncRNAs are involved in the development of trigeminal neuropathic pain, possibly through the ceRNA mechanism, which brings a new bright into the study of trigeminal neuropathic pain and the development of novel treatments targeting ncRNAs.

Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties

PurposeExtracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems.MethodHuman bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for β3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites.ResultsThe NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (β3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 μm vs. 1860.5 μm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05).ConclusionThis study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

Mechanistic contribution of CaV3.2 calcium channels to trigeminal neuralgia pathophysiology not clarified

Trigeminal neuralgia (TN) is a rare, yet debilitating trigeminal pain disorder, with jolts of supramaximal-debilitating pain in one or more of the three trigeminal branches. Familial TN is now recognized, with a recent report describing several human genetic polymorphisms. One affected gene is the voltage-gated calcium channel, CaV3.2 (CACNA1H), with 19 polymorphisms first described. A recent study in PAIN by Gambeta-et-al (DOI:10.1097/j.pain.0000000000002651) is entitled "CaV3.2 calcium channels contribute to trigeminal neuralgia ". Here, I call into question their claim. My main arguments are 1)-3): 1) Gambeta-et-al studied 4/19 mutations reported in heterologous cellular expression, with two mutations showing gain-of-function of CaV3.2, two mutations not showing gain-of-function. Therefore the exemplary picks of familial TN-associated CaV3.2 mutations do not show a uniform change of channel function, such as gain-of-function. 2) In Gambeta-et-al, one gain-of-function mutation, CaV3.2(G563R) was directed to mouse trigeminal ganglion (TG) neurons, and their resulting hyperexcitability was demonstrated. A critical control of a non-gain-of-function channel was not included here, it was unclear whether neurons were separated by sex, and human sensory neurons were not used. Importantly, it is not clear that TG neurons are the critical cellular site of CaV3.2 mutations. 3) Gambeta-et-al used CaV3.2-/- pan-null knockout mice. Human TN-associated CaV3.2 mutations were not over-expressed. They used a infraorbital nerve constriction injury and measured facial heat hyperalgesia. CaV3.2-/- show a pain phenotype similar to control, yet are not affected by a CaV3-inhibitory compound, Z944. My argument is that when starting with TN-associated human mutations, use of a trigeminal neuropathic pain model is of limited value, and that human mutations have to be expressed against a mouse null background. Re thermal cue, Gambeta-et-al failed to study cold-evoked pain which is a TN clinical hallmark. Thus, Gambeta-et-al's 2022 PAIN-paper offers little new mechanistic evidence why CaV3.2 polymorphisms are associated with trigeminal neuralgia.

VEGF receptor heterodimers and homodimers are differentially expressed in neuronal and endothelial cell types.

PurposeWe have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells.MethodsPC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/μL) or VEGF-B (50 ng/μL) or “vehicle” (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells.ResultsTIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells.ConclusionsDifferences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Decoding gene expression signatures in mice trigeminal ganglion across trigeminal neuropathic pain stages via high-throughput sequencing

Trigeminal neuropathic pain (TNP) arises due to peripheral nerve injury, the mechanisms underlying which are little known. The altered gene expression profile in sensory ganglia is critical for neuropathic pain generation and maintenance. We, therefore, assessed the transcriptome of the trigeminal ganglion (TG) from mice at different periods of pain progression. Trigeminal neuropathic pain was established by partial infraorbital nerve transection (pIONT). High-throughput RNA sequencing was applied to detect the mRNA profiles of TG collected at 3 and 10 days after modeling. Injured TG displayed dramatically altered mRNA expression profiles compared to Sham. Different gene expression profiles were obtained at 3 and 10 days after pIONT. Moreover, 314 genes were significantly upregulated, and 81 were significantly downregulated at both 3 and 10 days post-pIONT. Meanwhile, enrichment analysis of these persistent differentially expressed genes (DEGs) showed that the MAPK pathway was the most significantly enriched pathway for upregulated DEGs, validated by immunostaining. In addition, TG cell populations defined by single-nuclei RNA sequencing displayed cellular localization of DEGs at a single-cell resolution. Protein-protein interaction (PPI) and sub-PPI network analyses constructed networks and identified the top 10 hub genes for DEGs at different time points. The present data provide novel information on the gene expression signatures of TG during the development and maintenance phases of TNP, and the identified hub genes and pathways may serve as potential targets for treatment.

Mechanistic contribution of CaV3.2 calcium channels to trigeminal neuralgia pathophysiology not clarified.

Trigeminal neuralgia (TN) is a rare, yet debilitating trigeminal pain disorder, with jolts of supramaximal-debilitating pain in one or more of the three trigeminal branches. Familial TN is now recognized, with a recent report describing several human genetic polymorphisms. One affected gene is the voltage-gated calcium channel, CaV3.2 ( CACNA1H), with 19 polymorphisms first described. A recent study in PAIN by Gambeta-et-al (DOI:10.1097/j.pain.0000000000002651) is entitled " CaV3.2 calcium channels contribute to trigeminal neuralgia ". Here, I call into question their claim. My main arguments are 1)-3): 1) Gambeta-et-al studied 4/19 mutations reported in heterologous cellular expression, with two mutations showing gain-of-function of CaV3.2, two mutations not showing gain-of-function. Therefore the exemplary picks of familial TN-associated CaV3.2 mutations do not show a uniform change of channel function, such as gain-of-function. 2) In Gambeta-et-al, one gain-of-function mutation, CaV3.2(G563R) was directed to mouse trigeminal ganglion (TG) neurons, and their resulting hyperexcitability was demonstrated. A critical control of a non-gain-of-function channel was not included here, it was unclear whether neurons were separated by sex, and human sensory neurons were not used. Importantly, it is not clear that TG neurons are the critical cellular site of CaV3.2 mutations. 3) Gambeta-et-al used CaV3.2-/- pan-null knockout mice. Human TN-associated CaV3.2 mutations were not over-expressed. They used a infraorbital nerve constriction injury and measured facial heat hyperalgesia. CaV3.2-/- show a pain phenotype similar to control, yet are not affected by a CaV3-inhibitory compound, Z944. My argument is that when starting with TN-associated human mutations, use of a trigeminal neuropathic pain model is of limited value, and that human mutations have to be expressed against a mouse null background. Re thermal cue, Gambeta-et-al failed to study cold-evoked pain which is a TN clinical hallmark. Thus, Gambeta-et-al's 2022 PAIN-paper offers little new mechanistic evidence why CaV3.2 polymorphisms are associated with trigeminal neuralgia.

Transcriptional Alterations of Mouse Trigeminal Ganglion Neurons Following Orofacial Inflammation Revealed by Single-Cell Analysis.

Orofacial inflammation leads to transcriptional alterations in trigeminal ganglion (TG) neurons. However, diverse alterations and regulatory mechanisms following orofacial inflammatory pain in different types of TG neurons remain unclear. Here, orofacial inflammation was induced by injection of complete Freund’s adjuvant (CFA) in mice. After 7 days, we performed single-cell RNA-sequencing on TG cells of mice from control and treatment groups. We identified primary sensory neurons, Schwann cells, satellite glial cells, oligodendrocyte-like cells, immune cells, fibroblasts, and endothelial cells in TG tissue. After principal component analysis and hierarchical clustering, we identified six TG neuronal subpopulations: peptidergic nociceptors (PEP1 and PEP2), non-peptidergic nociceptors (NP1 and NP2), C-fiber low-threshold mechanoreceptors (cLTMR) and myelinated neurons (Nefh-positive neurons, NF) based on annotated marker gene expression. We also performed differential gene expression analysis among TG neuronal subtypes, identifying several differential genes involved in the inflammatory response, neuronal excitability, neuroprotection, and metabolic processes. Notably, we identified several potential novel targets associated with pain modulation, including Arl6ip1, Gsk3b, Scn7a, and Zbtb20 in PEP1, Rgs7bp in PEP2, and Bhlha9 in cLTMR. The established protein–protein interaction network identified some hub genes, implying their critical involvement in regulating orofacial inflammatory pain. Our study revealed the heterogeneity of TG neurons and their diverse neuronal transcriptomic responses to orofacial inflammation, providing a basis for the development of therapeutic strategies for orofacial inflammatory pain.

Seq-ing the mechanisms of migraine

The trigeminal ganglia (TG) play a crucial role in migraine pathophysiology. In this issue of Neuron, Yang etal. developed a single-cell atlas profiling the transcriptome and epigenome of mouse and human TG, thus providing a roadmap for therapeutic targeting.

CGRP and the Calcitonin Receptor are Co-Expressed in Mouse, Rat and Human Trigeminal Ganglia Neurons.

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed in the trigeminal ganglia, a key site in craniofacial pain and migraine. CGRP potently activates two receptors: the CGRP receptor and the AMY1 receptor. These receptors are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with either the calcitonin receptor-like receptor (CLR) to form the CGRP receptor or the calcitonin receptor (CTR) to form the AMY1 receptor. The expression of the CGRP receptor in trigeminal ganglia has been described in several studies; however, there is comparatively limited data available describing AMY1 receptor expression and in which cellular subtypes it is found. This research aimed to determine the relative distributions of the AMY1 receptor subunit, CTR, and CGRP in neurons or glia in rat, mouse and human trigeminal ganglia. Antibodies against CTR, CGRP and neuronal/glial cell markers were applied to trigeminal ganglia sections to investigate their distribution. CTR-like and CGRP-like immunoreactivity were observed in both discrete and overlapping populations of neurons. In rats and mice, 30–40% of trigeminal ganglia neurons displayed CTR-like immunoreactivity in their cell bodies, with approximately 78–80% of these also containing CGRP-like immunoreactivity. Although human cases were more variable, a similar overall pattern of CTR-like immunoreactivity to rodents was observed in the human trigeminal ganglia. CTR and CGRP appeared to be primarily colocalized in small to medium sized neurons, suggesting that colocalization of CTR and CGRP may occur in C-fiber neurons. CGRP-like or CTR-like immunoreactivity were not typically observed in glial cells. Western blotting confirmed that CTR was expressed in the trigeminal ganglia of all three species. These results confirm that CTR is expressed in trigeminal ganglia neurons. The identification of populations of neurons that express both CGRP and CTR suggests that CGRP could act in an autocrine manner through a CTR-based receptor, such as the AMY1 receptor. Overall, this suggests that a trigeminal ganglia CTR-based receptor may be activated during migraine and could therefore represent a potential target to develop treatments for craniofacial pain and migraine.

Activation of the N-methyl-D-aspartate receptor contributes to orofacial neuropathic and inflammatory allodynia by facilitating calcium-calmodulin-dependent protein kinase II phosphorylation in mice

Neuropathic and inflammatory pain are major clinical challenges due to their ambiguous mechanisms and limited treatment approaches. N-methyl-D-aspartate receptor (NMDAR) and calcium-calmodulin-dependent protein kinase II (CaMKII) are responsible for nerve system sensation and are required for the induction and maintenance of pain. However, the roles of NMDAR and CaMKII in regulating orofacial pain are still less well known. Here, we established a neuropathic pain model by transecting a mouse inferior alveolar nerve (IAN) and an inflammatory pain model by injecting complete Freund’s adjuvant (CFA) into its whisker pad. The Cre/loxp site-specific recombination system was used to conditionally knock out (KO) NR2B in the trigeminal ganglion (TG). Von Frey filament behavioral tests showed that IANX and CFA-induced mechanical allodynia were altered in NR2B-deficient mice. CFA upregulated CaMKIIα and CaMKIIβ in the mouse TG and spinal trigeminal caudate nucleus (SpVc). CaMKIIα first decreased and then increased in the TG after IANX, and CaMKIIβ decreased in the TG and SpVc. CFA and IANX both greatly enhanced the expression of phospho (p)-NR2B, p-CaMKII, cyclic adenosine monophosphate (cAMP), p-ERK, and p-cAMP response element binding protein (CREB) in the TG and SpVc. These neurochemical signal pathway alterations were reversed by the conditional KO of NR2B and inhibition of CaMKII. Similarly, IANX- and CFA-related behavioral alterations were reversed by intra-ganglionic (i.g.) -application of inhibitors of CaMKII, cAMP, and ERK. These findings revealed novel molecular signaling pathways (NR2B-CaMKII-cAMP-ERK-CREB) in the TG- and SpVc-derived latent subsequent peripheral and spinal central sensitization under nerve injury and inflammation, which might be beneficial for the treatment of orofacial allodynia.

Sensory neuron-expressed TRPC3 mediates acute and chronic itch.

Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represents a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in most presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch-like and pain-like behaviors in naive mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), the Trpc3 mRNA expression level and function were upregulated in the TG after CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch through a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Transcriptomic Analysis of Trigeminal Ganglion and Spinal Trigeminal Nucleus Caudalis in Mice with Inflammatory Temporomandibular Joint Pain

BackgroundPersistent facial pain heavily impacts the quality of life in patients with temporomandibular joint (TMJ) disorders. Previous studies have demonstrated that long non-coding ribonucleic acid (lncRNA) is an important regulator of pain. In this study, we aimed to analyze lncRNA expression in the whole transcriptome of trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) in a chronic inflammatory TMJ pain mouse model.MethodsChronic inflammatory TMJ pain was induced by intra-TMJ injection of complete Freund’s adjuvant (CFA). Mouse TG and Sp5C tissues were harvested on day 4 after CFA injection. The lncRNA expression patterns in the whole transcriptome of TG and Sp5C were profiled with RNA sequencing.ResultsWe observed that 38 lncRNAs and 849 mRNAs were differentially expressed after CFA treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis further revealed relationships among those differentially expressed lncRNAs and mRNAs and their potential functions. Specific categories of biological process, cellular processes, and molecular function of the differentially expressed transcripts were ascertained.ConclusionOur results suggest that lncRNA expression in the whole transcriptome of trigeminal nociceptive system could contribute to the molecular mechanisms that underlie chronic inflammatory TMJ pain.

Human and mouse trigeminal ganglia cell atlas implicates multiple cell types in migraine

Sensitization of trigeminal ganglion neurons contributes to primary headache disorders such as migraine, but the specific neuronal and non-neuronal trigeminal subtypes that are involved remain unclear. We thus developed a cell atlas in which human and mouse trigeminal ganglia are transcriptionally and epigenomically profiled at single-cell resolution. These data describe evolutionarily conserved and human-specific gene expression patterns within each trigeminal ganglion cell type, as well as the transcription factors and gene regulatory elements that contribute to cell-type-specific gene expression. We then leveraged these data to identify trigeminal ganglion cell types that are implicated both by human genetic variation associated with migraine and two mouse models of headache. This trigeminal ganglion cell atlas improves our understanding of the cell types, genes, and epigenomic features involved in headache pathophysiology and establishes a rich resource of cell-type-specific molecular features to guide the development of more selective treatments for headache and facial pain.

Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5.

The mechanisms underlying facial pain are still incompletely understood, posing major therapeutic challenges. Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase involved in pain signaling. However, the regulatory roles of Cdk5 in facial pain signaling and the possibility of therapeutic intervention at the level of mouse trigeminal ganglion primary neurons remain elusive. In this study, we use optimized intravital imaging to directly compare trigeminal neuronal activities after mechanical, thermal, and chemical stimulation. We then test whether facial inflammatory pain in mice could be alleviated by the Cdk5 inhibitor peptide TFP5. We demonstrate regulation of total Ca2+ intensity by Cdk5 activity using transgenic and knockout mouse models. In mice with vibrissal pad inflammation, application of TFP5 specifically decreases total Ca2+ intensity in response to noxious stimuli. It also alleviates inflammation-induced allodynia by inhibiting activation of trigeminal peripheral sensory neurons. Cdk5 inhibitors may provide promising non-opioid candidates for pain treatment.

Neuronal Fc\u03b5RI\u03b1 directly mediates ocular itch via IgE-immune complex in a mouse model of allergic conjunctivitis

BackgroundClassical understanding of allergic conjunctivitis (ACJ) suggests that ocular itch results from a mast cell-dependent inflammatory process. However, treatments that target inflammatory mediators or immune cells are often unsatisfying in relieving the stubborn itch symptom. This suggests that additional mechanisms are responsible for ocular itch in ACJ. In this study, we aim to determine the role of neuronal FcεRIa in allergic ocular itch.MethodsCalcium imaging was applied to observe the effect of IgE-immune complex in trigeminal neurons. Genomic FcεRIa knockout mice and adeno-associated virus (AAV) mediated sensory neuron FcεRIa knockdown mice were used in conjunction with behavioral tests to determine ocular itch. In addition, immunohistochemistry, Western blot and quantitative RT-PCR were used for in vitro experiments.ResultsWe found that FcεRIα was expressed in a subpopulation of conjunctiva sensory neurons. IgE-IC directly activated trigeminal neurons and evoked acute ocular itch without detectible conjunctival inflammation. These effects were attenuated in both a global FcεRIa-knockout mice and after sensory neuronal-specific FcεRIa-knockdown in the mouse trigeminal ganglion. In an ovalbumin (OVA) induced murine ACJ model, FcεRIα was found upregulated in conjunctiva-innervating CGRP+ sensory neurons. Sensory neuronal-specific knockdown of FcεRIa significantly alleviated ocular itch in the ACJ mice without affecting the immune cell infiltration and mast cell activation in conjunctiva. Although FcεRIα mRNA expression was not increased by IgE in dissociated trigeminal ganglion neurons, FcεRIα protein level was enhanced by IgE in a cycloheximide-resistance manner, with concordant enhancement of neuronal responses to IgE-IC. In addition, incremental sensitization gradually enhanced the expression of FcεRIα in small-sized trigeminal neurons and aggravated OVA induced ocular itch.ConclusionsOur study demonstrates that FcεRIα in pruriceptive neurons directly mediates IgE-IC evoked itch and plays an important role in ocular itch in a mouse model of ACJ. These findings reveal another axis of neuroimmune interaction in allergic itch condition independent to the classical IgE-mast cell pathway, and might suggest novel therapeutic strategies for the treatment of pruritus in ACJ and other immune-related disorders.

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response.

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.