MiR-21-5p alleviates trigeminal neuralgia in rats through down-regulation of voltage-gated potassium channel Kv1.1. / MiR-21-5p通过下调电压门控钾离子通道Kv1.1è¡¨è¾¾å‡è½»å¤§é¼ ä¸‰å‰ç¥žç»ç—›.

OBJECTIVES: Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN. METHODS: A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. RESULTS: Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. CONCLUSIONS: Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.

Causal association between adiponectin and risk of trigeminal neuralgia: A Mendelian randomization study.

OBJECTIVE: To determine whether adiponectin levels and the risk of trigeminal neuralgia (TN) were causally related, a two-sample Mendelian Randomization (MR) study design was used. METHODS: We obtained data regarding adiponectin from the UK Biobank genome wide association studies (GWAS) (n = 39,883) as the exposure and TN, using GWAS summary statistics generated from FinnGen, (total n = 195 847 159; case = 800, control = 195 047) as the outcome. We conducted a two-sample Mendelian randomization analysis employing inverse variance-weighted (IVW), MR-Egger regression, weighted median, and weighted mode analyses. RESULTS: We selected 14 single nucleotide polymorphisms (SNPs) with genome-wide significance from the GWAS on adiponectin as instrumental variables. Based on the IVW method, a causal association between adiponectin levels and TN was evidenced (OR= 0.577, 95 %CI: 0.393-0.847). MR-Egger regression revealed that directional pleiotropy was unlikely to be biasing the result (intercept = -0.01; P = 0.663), but it showed no causal association between adiponectin and TN (OR=0.627, 95 %CI: 0.369-1.067). However, the weighted median (OR=0.569, 95 %CI: 0.353-0.917) and Weighted mode (OR= 0.586, 95 %CI: 0.376-0.916) approach yielded evidence of a causal association between adiponectin and TN. Cochran's Q-statistics and funnel plots indicated no evidence of heterogeneity or asymmetry, indicating no directional pleiotropy. CONCLUSION: The results of the MR analysis suggested that adiponectin may be causally associated with an increased TN risk.

Whole Blood Transcriptome Analysis in Patients with Trigeminal Neuralgia: a Prospective Clinical Study.

Trigeminal neuralgia (TN) brings a huge burden to patients, without long-term effective treatment. This study aimed to explore the differentially expressed genes (DEGs) and related enrichment pathways in patients with TN. This was a study of transcriptome sequencing and bioinformatics analysis of human samples. Whole blood samples were collected from the TN patients and pain-free controls. RNA was extracted to conduct the RNA-sequencing and the subsequent bioinformatics analysis. DEGs between the two groups were derived. Kyoto encyclopedia of genes and genomes (KEGG) and Gene ontology (GO) was used to find the enrichment pathways of DEGs. Protein protein interaction (PPI) network was used to depict the interaction between DEGs and find the most important gene, hub gene. Compared with the control group, there were 117 up-regulated DEGs and 103 down-regulated DEGs in the whole blood of patients in the TN group. Pathway enrichment analysis showed that DEGs were mainly enriched in the neuroimmune and metabolic pathways. The PPI network demonstrated that colony stimulating factor 2 (CSF2) was the most important hub gene in the whole blood of TN patients. This study shows the expression of the transcriptome in the whole blood samples of TN patients. The neuroimmune responses and key hub gene CSF2 in the whole blood cells play a vital role in the occurrence of TN. Our research provides a theoretical basis for the diagnosis and treatments of TN. This study was registered at clinicaltrials.gov in June 2021 (No. NCT04923399).

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

OBJECTIVE: To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. BACKGROUND: Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. METHODS: We examined trigeminal ganglion ex vivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. RESULTS: The trigeminal ganglion demonstrates strong circadian rhythms in both ex vivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. CONCLUSION: Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. PLAIN LANGUAGE SUMMARY: Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.

In situ tissue profile of rat trigeminal nerve in trigeminal neuralgia using spatial transcriptome sequencing.

BACKGROUND: Trigeminal neuralgia (TN) is the most common neuropathic disorder in the maxillofacial region. The etiology and pathogenesis of TN have not been clearly determined to date, although there are many hypotheses. OBJECTIVE: The goal of this study was to investigate the interactions between different types of cells in TN, particularly the impact and intrinsic mechanism of demyelination on the trigeminal ganglion, and to identify new important target genes and regulatory pathways in TN. METHODS: TN rat models were prepared by trigeminal root compression, and trigeminal nerve tissues were isolated for spatial transcriptome sequencing. The gene expression matrix was reduced dimensionally by PCA and presented by UMAP. Gene function annotation was analyzed by Metascape. The progression of certain clusters and the developmental pseudotime were analyzed using the Monocle package. Modules of the gene coexpression network between different groups were analyzed based on weighted gene coexpression network analysis and assigned AddModuleScore values. The intercellular communication of genes in these networks via ligand-receptor interactions was analyzed using CellPhoneDB analysis. RESULTS: The results suggested that the trigeminal ganglion could affect Schwann cell demyelination and remyelination responses through many ligand-receptor interactions, while the effect of Schwann cells on the trigeminal ganglion was much weaker. Additionally, ferroptosis may be involved in the demyelination of Schwann cells. CONCLUSIONS: This study provides spatial transcriptomics sequencing data on TN, reveals new markers, and redefines the relationship between the ganglion and myelin sheath, providing a theoretical basis and supporting data for future mechanistic research and drug development.

The pathophysiology of trigeminal neuralgia: a molecular review.

OBJECTIVE: The goal of this study was to provide a comprehensive overview of the current understanding of molecular and genetic mechanisms underlying the pathophysiology of trigeminal neuralgia (TN). METHODS: The authors searched PubMed systematically for primary research literature investigating specific molecular mechanisms from samples derived from patients with TN. The genes/molecules of interest from the selected literature were then cross-referenced with corresponding studies in animal models of TN. RESULTS: From approximately 345 articles, a total of 12 articles were selected and included in the review, focusing on ionotropic channel expressivity and mutations, reactive oxygen species expressivity, inflammatory marker expressivity, and microRNA expressivity. Of the 12 included articles, only 4 had studies completed in other animal models regarding the corresponding TN mechanism found in humans. CONCLUSIONS: The current literature does not suggest a conclusive disease mechanism for TN in humans. In addition to neurovascular conflict/compression of the trigeminal nerve, recent studies have indicated that TN may be linked to inflammatory and reactive oxygen species signaling as well. Recent genetic studies in patients with TN have yet to be investigated further in animal models.

Electrophysiological characterization of a CaV3.1 calcium channel mutation linked to trigeminal neuralgia.

Trigeminal neuralgia is a rare and debilitating disorder that affects one or more branches of the trigeminal nerve, leading to severe pain attacks and a poor quality of life. It has been reported that the CaV3.1 T-type calcium channel may play an important role in trigeminal pain and a recent study identified a new missense mutation in the CACNA1G gene that encodes the pore forming α1 subunit of the CaV3.1 calcium channel. The mutation leads to a substitution of an Arginine (R) by a Glutamine (Q) at position 706 in the I-II linker region of the channel. Here, we used whole-cell voltage-clamp recordings to evaluate the biophysical properties of CaV3.1 wild-type and R706Q mutant channels expressed in tsA-201 cells. Our data indicate an increase in current density in the R706Q mutant, leading to a gain-of-function effect, without changes in the voltage for half activation. Moreover, voltage clamp using an action potential waveform protocol revealed an increase in the tail current at the repolarization phase in the R706Q mutant. No changes were observed in the voltage-dependence of inactivation. However, the R706Q mutant displayed a faster recovery from inactivation. Hence, the gain-of-function effects in the R706Q CaV3.1 mutant have the propensity to impact pain transmission in the trigeminal system, consistent with a contribution to trigeminal neuralgia pathophysiology.

[Analysis of potential pathogenic factors of trigeminal neuralgia in rats].

PURPOSES: Transcriptomics-based analysis of key transcriptional molecules in the pathogenesis of trigeminal neuropathic pain was conducted to screen key molecules in the pathogenesis of trigeminal neuralgia. METHODS: Rat trigeminal nerve pathological pain model, namely chronic constriction injury of distal infraorbital nerve (IoN-CCI), was constructed and animal behaviors postsurgery were observed and analyzed. Trigeminal ganglia were collected for RNA-seq transcriptomics analysis. StringTie was used to annotate and quantify genome expression. DESeq2 was applied to compare between groups with P value less than 0.05 and fold change greater than 2 times and less than 0.5 times to screen differential genes, and display them with volcano graphs and cluster graphs. ClusterProfiler software was used to perform GO function enrichment analysis of differential genes. RESULTS: On the fifth postoperative day (POD5), the rat's face-grooming behavior increased to a peak; on the seventh postoperative day (POD7), the von-frey value dropped to the lowest value, indicating that the mechanical pain threshold of rats was significantly decreased. RNA-seq analysis of IoN-CCI rat ganglia found that the significantly up-regulated signaling pathways included B cell receptor signaling pathway, cell adhesion, complement and coagulation cascade pathways; significantly down-regulated pathways were related to systemic lupus erythematosus. Multiple genes among Cacna1s, Cox8b, My1, Ckm, Mylpf, Myoz1, Tnnc2 were involved in mediating the occurrence of trigeminal neuralgia. CONCLUSIONS: B cell receptor signaling pathway, cell adhesion, complement and coagulation cascade pathways, neuroimmune pathways are closely related to the occurrence of trigeminal neuralgia. The interaction of multiple genes among Cacna1s, Cox8b, My11, Ckm, Mylpf, Myoz1, Tnnc2 leads to the occurrence of trigeminal neuralgia.

MARS1 mutations linked to familial trigeminal neuralgia via the integrated stress response.

BACKGROUND: While new genetic analysis methods are widely used in the clinic, few researchers have focused on trigeminal neuralgia (TN) with familial clustering (≥ 2 TN patients in one kindred family). Previous literature suggests that familial trigeminal neuralgia (FTN) may be associated with inherited genetic factors. To date, few next-generation sequencing studies have been reported for FTN. This study investigated the pathogenic mechanism of FTN by using whole-exome sequencing (WES) technology, which may enhance our understanding of human TN pathophysiology.  METHOD: We performed WES for 7 probands from families of FTN. Sanger sequencing was performed for two control groups (FTN family members group and nonfamilial TN subject group) to potentially identify new FTN-related gene mutations. In families where FTN probands carried potentially pathogenic gene mutations, the ribonucleic acid (RNA) of FTN probands and related family members, as well as nonfamilial TN patients were analysed by RNA sequencing (RNA-seq) to confirm differential gene expression. RESULTS: Seven probands were derived from 3 Chinese families. WES and Sanger sequencing identified MARS1 mutation c.2398C > A p.(Pro800Thr) in Family 1. MARS1 mutation was confirmed in 14/26 [53.8%] members of Family 1 in FTN family member group, while none of nonfamilial TN subjects had this MARS1 mutation. RNA-seq showed that 3 probands in Family 1 had higher expression of Fosl1 (Fos-like antigen 1) and NFE2 (Nuclear factor, erythroid 2) than 3 subjects in the nonfamilial TN subject group. Fosl1 and NFE2 are genes related to integrated stress response (ISR). CONCLUSION: MARS1 mutations may cause chronic activation of ISR, contribute to ISR pathophysiological changes in FTN, and cause/accelerate peripheral nerve degeneration. The findings of this study can enrich our knowledge of the role of molecular genetics in TN in humans.

Awareness of HLA-B* 15:02 screening in trigeminal neuralgia and the gene screening policy among dentists in Southern Thailand.

OBJECTIVES: To determine the factors associated with public hospital dentists' awareness of HLA-B*15:02 screening in trigeminal neuralgia (TN) and the national gene screening policy in Thailand. METHODS: Cross-sectional study. A validated questionnaire was distributed to public hospital dentists with at least 1 year of practice in Southern Thailand (n = 760) to assess their knowledge of TN, carbamazepine (CBZ) use, awareness of HLA-B*15:02 screening, and the gene screening policy. RESULTS: A total of 385 dentists participated (50.7% response rate); 81.3% of respondents were aware of HLA-B*15:02 screening. However, 18.7% of dentists were not aware of the importance of gene testing. Furthermore, dentists who were aware of gene screening had significantly better knowledge of TN diagnosis and CBZ use than "unaware" dentists. Awareness of HLA-B*15:02 screening was also significantly associated with dental specialty. Moreover, 80.5% of respondents were not aware of the gene screening policy. The primary problems related to the policy were its inefficient publication, poor implementation, and lack of clinical practice guidelines (CPGs) to encourage dentists to follow the policy and prescribe gene tests. CONCLUSION: While most hospital dentists were aware of the necessity of HLA-B*15:02 screening prior to prescribing CBZ in TN, the majority were unaware of the national gene screening policy. Dental specialty and knowledge were associated with awareness of HLA-B*15:02 screening.

Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia.

Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.

A TRPM7 mutation linked to familial trigeminal neuralgia: Omega current and hyperexcitability of trigeminal ganglion neurons.

Trigeminal neuralgia (TN) is a unique pain disorder characterized by intense paroxysmal facial pain within areas innervated by the trigeminal nerve. Although most cases of TN are sporadic, familial clusters of TN suggest that genetic factors may contribute to this disorder. Whole-exome sequencing in patients with TN reporting positive family history demonstrated a spectrum of variants of ion channels including TRP channels. Here, we used patch-clamp analysis and Ca2+ and Na+ imaging to assess a rare variant in the TRPM7 channel, p.Ala931Thr, within transmembrane domain 3, identified in a man suffering from unilateral TN. We showed that A931T produced an abnormal inward current carried by Na+ and insensitive to the pore blocker Gd3+. Hypothesizing that replacement of the hydrophobic alanine at position 931 with the more polar threonine destabilizes a hydrophobic ring, near the voltage sensor domain, we performed alanine substitutions of F971 and W972 and obtained results suggesting a role of A931-W972 hydrophobic interaction in S3-S4 hydrophobic cleft stability. Finally, we transfected trigeminal ganglion neurons with A931T channels and observed that expression of this TRPM7 variant lowers current threshold and resting membrane potential, and increases evoked firing activity in TG neurons. Our results support the notion that the TRPM7-A931T mutation located in the S3 segment at the interface with the transmembrane region S4, generates an omega current that carries Na+ influx in physiological conditions. A931T produces hyperexcitability and a sustained Na+ influx in trigeminal ganglion neurons that may underlie pain in this kindred with trigeminal neuralgia.

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.

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.

Characterization of Acetylation of Histone H3 at Lysine 9 in the Trigeminal Ganglion of a Rat Trigeminal Neuralgia Model.

Trigeminal neuralgia (TN) is a chronic neuropathic pain disorder characterized by spontaneous and elicited paroxysms of electric-shock-like or stabbing pain in a region of the face. The epigenetic regulation of TN is still obscure. In current study, a rat TN model subject to carbamazepine (CBZ) treatment was established, and transcriptome- and genome-scale profiling of H3K9ac and HDAC3 was performed by RNA-seq and ChIP-seq. We observed that H3K9ac levels in the trigeminal ganglion were lower in the TN rats compared with those in the control, and CBZ treatment led to recovery of H3K9ac levels. Further, we found that HDAC3 was overactivated, which interfered with H3K9 acetylation due to higher phosphorylation in TN compared with that in the control. Finally, the phosphokinase leucine-rich repeat kinase 2 (LRRK2) was demonstrated to contribute to HDAC3 activity via the MAPK signaling pathway. Taken together, we identified a regulatory mechanism in which the phosphate groups transferred from activated ERK and LRRK2 to HDAC3 caused genome-scale deacetylation at H3K9 and resulted in the silencing of a large number of genes in TN. The kinases or important enzymes within this regulatory axis may represent important targets for TN therapy and prevention.

MiR-223-3p alleviates trigeminal neuropathic pain in the male mouse by targeting MKNK2 and MAPK/ERK signaling.

BACKGROUND: Trigeminal neuralgia (TN) is a neuropathic pain that occurs in branches of the trigeminal nerve. MicroRNAs (miRNAs) have been considered key mediators of neuropathic pain. This study was aimed to elucidate the pathophysiological function and mechanisms of miR-223-3p in mouse models of TN. METHODS: Infraorbital nerve chronic constriction injury (CCI-ION) was applied in male C57BL/6J mice to establish mouse models of TN. Pain responses were assessed utilizing Von Frey method. The expression of miR-223-3p, MKNK2, and MAPK/ERK pathway protein in trigeminal ganglions (TGs) of CCI-ION mice was measured using RT-qPCR and Western blotting. The concentrations of inflammatory cytokines were evaluated using Western blotting. The relationship between miR-223-3p and MKNK2 was tested by a luciferase reporter assay. RESULTS: We found that miR-223-3p was downregulated, while MKNK2 was upregulated in TGs of CCI-ION mice. MiR-223-3p overexpression by an intracerebroventricular injection of Lv-miR-223-3p attenuated trigeminal neuropathic pain in CCI-ION mice, as well as reduced the protein levels of pro-inflammatory cytokines in TGs of CCI-ION mice. MKNK2 was verified to be targeted by miR-223-3p. Additionally, miR-223-3p overexpression decreased the phosphorylation levels of ERK1/2, JNK, and p38 protein in TGs of CCI-ION mice to inhibit MAPK/ERK signaling. CONCLUSIONS: Overall, miR-223-3p attenuates the development of TN by targeting MKNK2 to suppress MAPK/ERK signaling.

Ca V 3.2 calcium channels contribute to trigeminal neuralgia.

ABSTRACT: Trigeminal neuralgia (TN) is a rare but debilitating disorder characterized by excruciating facial pain, with a higher incidence in women. Recent studies demonstrated that TN patients present mutations in the gene encoding the Ca V 3.2 T-type calcium channel, an important player in peripheral pain pathways. We characterize the role of Ca V 3.2 channels in TN at 2 levels. First, we examined the biophysical properties of CACNA1H variants found in TN patients. Second, we investigated the role of Ca V 3.2 in an animal model of trigeminal neuropathic pain. Whole-cell patch-clamp recordings from 4 different mutants expressed in tsA-201 cells (E286K in the pore loop of domain I, H526Y, G563R, and P566T in the domain I-II linker) identified a loss of function in activation in the E286K mutation and gain of function in the G563R and P566T mutations. Moreover, a loss of function in inactivation was observed with the E286K and H526Y mutations. Cell surface biotinylation revealed no difference in channel trafficking among the variants. The G563R mutant also caused a gain of function in the firing properties of transfected trigeminal ganglion neurons. In female and male mice, constriction of the infraorbital nerve induced facial thermal heat hyperalgesia. Block of T-type channels with Z944 resulted in antihyperalgesia. The effect of Z944 was absent in Ca V 3.2 -/- mice, indicating that Ca V 3.2 is the molecular target of the antihyperalgesic Z944 effect. Finally, enzyme-linked immunosorbent assay analysis revealed increased Ca V 3.2 channel expression in the spinal trigeminal subnucleus caudalis. Altogether, the present study demonstrates an important role of Ca V 3.2 channels in trigeminal pain.

Emerging roles of lncRNAs in the pathogenesis, diagnosis, and treatment of trigeminal neuralgia.

Trigeminal neuralgia (TN) is one of the most common neuropathic pain disorders and is often combined with other comorbidities if managed inadequately. However, the present understanding of its pathogenesis at the molecular level remains lacking. Long noncoding RNAs (lncRNAs) play crucial roles in neuropathic pain, and many studies have reported that specific lncRNAs are related to TN. This review summarizes the current understanding of lncRNAs in the pathogenesis, diagnosis, and treatment of TN. Recent studies have shown that the lncRNAs uc.48+, Gm14461, MRAK009713 and NONRATT021972 are potential candidate loci for the diagnosis and treatment of TN. The current diagnostic system could be enhanced and improved by a workflow for selecting transcriptomic biomarkers and the development of lncRNA-based molecular diagnostic systems for TN. The discovery of lncRNAs potentially impacts drug selection for TN; however, the current supporting evidence is limited to preclinical studies. Additional studies are needed to further test the diagnostic and therapeutic value of lncRNAs in TN.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

Familial occurrence of classical and idiopathic trigeminal neuralgia.

Trigeminal neuralgia (TN) is a severe facial pain disease with unknown pathogenesis. It has been thought that the familial form of TN is rare with a prevalence of about 1-2% among affected individuals, but emerging evidence suggests a role of genetic factors. This study examined the occurrence of familial TN among patients with classical or idiopathic TN. Patients with TN recruited from a hospital registry received an informed consent form with a questionnaire, and individuals reporting other family members with TN underwent a structured phone-interview. For affected family members, type of TN, available clinical, imaging, management results and available hospital patient records were studied. Pedigrees for all affected families were established. This study included 268 patients with either classical or idiopathic TN. The familial form of TN was present in 41/268 (15.3%) patients, that is, 37/244 (15.2%) patients with classical TN and in 4/24 (16.7%) with idiopathic TN. Total 38 families were identified, with two affected members in 32/38 families (84.2%), three affected family members in 5/38 (13.2%) and four family members in 1/38 (2.6%) families. Comparing the 41 familial TN cases with the 227 sporadic TN patients showed significantly earlier onset of TN and a significantly higher occurrence of right-sided pain in familial cases, while there was no difference in gender distribution, occurrence of arterial hypertension or trigeminal branch involved. Among patients with classical or idiopathic TN, the occurrence of the familial form of the disease is more frequent than traditionally assumed.