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

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

Phosphorylation of the AMPARs regulated by protein kinase C (PKC) and protein interacting with C-kinase 1 (PICK1) contribute to orofacial neuropathic pain.

The α-amino-3-hydroxy-5-methylisoxazole-4-isoxazolepropionic acid receptor (AMPAR) has been recognized to play a vital role in the development of neuropathic pain. Recent studies have indicated that protein kinase C (PKC) and protein interacting with C-kinase 1 (PICK1) are involved in the phosphorylation of AMPARs. However, whether PKC and PICK1 were involved in the AMPAR phosphorylation in the trigeminal ganglion (TG) to participate in orofacial neuropathic pain remains enigmatic. A behavioral test was utilized to evaluate the head withdrawal threshold (HWT) after chronic constriction injury of the infraorbital nerve (CCI-ION). The distribution and expression of GluA1, GluA2, PKC, and PICK1 were examined in the trigeminal ganglion (TG) by immunofluorescence, real-time reverse transcription-quantitative polymerase chain reaction, immunoblotting, and co-immunoprecipitation. Intra-ganglionic injections of drugs were performed to investigate the regulation mechanism. The present study demonstrated that CCI-ION-induced mechanical allodynia was maintained over at least 21 days. GluA1 and GluA2 were mainly expressed in the neurons. Trigeminal nerve injury potentiated the phosphorylation of GluA1, GluA2, and PKC in the TG, which was prevented by inhibiting PKC with chelerythrine chloride. Additionally, PICK1 colocalized and interacted with GluA2 in the TG. Following blocking PICK1 with FSC-231, the phosphorylation of GluA2 decreased. Finally, inhibition of PKC and PICK1 both alleviated mechanical allodynia in the whisker pad of CCI-ION mice. In conclusion, activation of PKC and PICK1 contribute to orofacial allodynia by regulating AMPAR phosphorylation in the TG of male mice, which provides potential therapeutic targets for alleviating orofacial neuropathic pain.

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 CaMKIIß in the mouse TG and spinal trigeminal caudate nucleus (SpVc). CaMKIIα first decreased and then increased in the TG after IANX, and CaMKIIß 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.

NMDARs mediate peripheral and central sensitization contributing to chronic orofacial pain.

Peripheral and central sensitizations of the trigeminal nervous system are the main mechanisms to promote the development and maintenance of chronic orofacial pain characterized by allodynia, hyperalgesia, and ectopic pain after trigeminal nerve injury or inflammation. Although the pathomechanisms of chronic orofacial pain are complex and not well known, sufficient clinical and preclinical evidence supports the contribution of the N-methyl-D-aspartate receptors (NMDARs, a subclass of ionotropic glutamate receptors) to the trigeminal nociceptive signal processing pathway under various pathological conditions. NMDARs not only have been implicated as a potential mediator of pain-related neuroplasticity in the peripheral nervous system (PNS) but also mediate excitatory synaptic transmission and synaptic plasticity in the central nervous system (CNS). In this review, we focus on the pivotal roles and mechanisms of NMDARs in the trigeminal nervous system under orofacial neuropathic and inflammatory pain. In particular, we summarize the types, components, and distribution of NMDARs in the trigeminal nervous system. Besides, we discuss the regulatory roles of neuron-nonneuronal cell/neuron-neuron communication mediated by NMDARs in the peripheral mechanisms of chronic orofacial pain following neuropathic injury and inflammation. Furthermore, we review the functional roles and mechanisms of NMDARs in the ascending and descending circuits under orofacial neuropathic and inflammatory pain conditions, which contribute to the central sensitization. These findings are not only relevant to understanding the underlying mechanisms, but also shed new light on the targeted therapy of chronic orofacial pain.

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

Background: Recent 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. Methods: In 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. Results: The 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. Conclusion: Our 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.