Experimental Study on Involvement of the Central Nervous System in Inferior Alveolar Nerve Damage-Associated Hyperalgesia of the Mental Region.

PURPOSE: Involvement of the central nervous system in sensory disturbances of the mental region occurring after inferior alveolar nerve damage was investigated using a rat model of inferior alveolar nerve damage. PATIENTS AND METHODS: The rat inferior alveolar nerve was damaged by ligation with thread, and the course of behavioral changes after surgery was observed for 42 days. In addition, activation of microglia and astroglia in the trigeminal spinal subnucleus caudalis (Vc) was analyzed using immunohistochemistry. c-Fos-positive cells were quantitatively evaluated to analyze the state of neuron excitement. RESULTS: The withdrawal threshold was significantly decreased 5 days after surgery in the inferior alveolar nerve-ligated (IANL) group compared with that in the sham group and subsequently recovered over time. In addition, microglia and astroglia were activated in the Vc region 5 days after surgery in the model group, and c-fos-positive cells were also significantly more frequent in the IANL group. However, no significant difference in the withdrawal threshold was seen between the IANL and sham groups on day 42, nor were any significant differences seen in the amounts of microglia, astroglia, or c-fos-positive cells. CONCLUSIONS: Interactions among microglia, astroglia, and neurons in the central nervous system might be involved in the progression of inferior alveolar nerve damage-associated mental hyperalgesia to a chronic state.

Distinctive features of Phox2b-expressing neurons in the rat reticular formation dorsal to the trigeminal motor nucleus.

Phox2b encodes a paired-like homeodomain-containing transcription factor essential for development of the autonomic nervous system. Phox2b-expressing (Phox2b+) neurons are present in the reticular formation dorsal to the trigeminal motor nucleus (RdV) as well as the nucleus of the solitary tract and parafacial respiratory group. However, the nature of Phox2b+ RdV neurons is still unclear. We investigated the physiological and morphological properties of Phox2b+ RdV neurons using postnatal day 2-7 transgenic rats expressing yellow fluorescent protein under the control of Phox2b. Almost all of Phox2b+ RdV neurons were glutamatergic, whereas Phox2b-negative (Phox2b-) RdV neurons consisted of a few glutamatergic, many GABAergic, and many glycinergic neurons. The majority (48/56) of Phox2b+ neurons showed low-frequency firing (LF), while most of Phox2b- neurons (35/42) exhibited high-frequency firing (HF) in response to intracellularly injected currents. All, but one, Phox2b+ neurons (55/56) did not fire spontaneously, whereas three-fourths of the Phox2b- neurons (31/42) were spontaneously active. K+ channel and persistent Na+ current blockers affected the firing of LF and HF neurons. The majority of Phox2b+ (35/46) and half of the Phox2b- neurons (19/40) did not respond to stimulations of the mesencephalic trigeminal nucleus, the trigeminal tract, and the principal sensory trigeminal nucleus. Biocytin labeling revealed that about half of the Phox2b+ (5/12) and Phox2b- RdV neurons (5/10) send their axons to the trigeminal motor nucleus. These results suggest that Phox2b+ RdV neurons have distinct neurotransmitter phenotypes and firing properties from Phox2b- RdV neurons and might play important roles in feeding-related functions including suckling and possibly mastication.

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

BACKGROUND: Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. RESULTS: Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1ß over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. CONCLUSIONS: These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Fractalkine signaling in microglia contributes to ectopic orofacial pain following trapezius muscle inflammation.

Fractalkine (FKN) signaling is involved in mechanical allodynia in the facial skin following trapezius muscle inflammation. Complete Freund's adjuvant (CFA) injection into the trapezius muscle produced mechanical allodynia in the ipsilateral facial skin that was not associated with facial skin inflammation and resulted in FKN but not FKN receptor (CX3CR1) expression, and microglial activation was enhanced in trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2). Intra-cisterna magna anti-CX3CR1 or anti-interleukin (IL)-1ß neutralizing antibody administration decreased the enhanced excitability of Vc and C1-C2 neurons in CFA-injected rats, whereas intra-cisterna magna FKN administration induced microglial activation and mechanical allodynia in the facial skin. IL-1ß expression and p38 mitogen-activated protein kinase phosphorylation were enhanced in activated microglia after CFA injection. The excitability of neurons whose receptive fields was located in the facial skin was significantly enhanced in CFA-injected rats, and the number of cells expressing phosphorylated extracellular signal-regulated kinase (pERK) following noxious mechanical stimulation of the facial skin was significantly increased in Vc and C1-C2. We also observed mechanical allodynia of the trapezius muscle as well as microglial activation and increased pERK expression in C2-C6 after noxious stimulation of the trapezius muscle in facial skin-inflamed rats. These findings suggest that FKN expression was enhanced in Vc and C1-C2 or C2-C6 following trapezius muscle or facial skin inflammation, microglia are activated via FKN signaling, IL-1ß is released from the activated microglia, and the excitability of neurons in Vc and C1-C2 or C2-C6 is enhanced, resulting in the ectopic mechanical allodynia.

Involvement of ERK phosphorylation of trigeminal spinal subnucleus caudalis neurons in thermal hypersensitivity in rats with infraorbital nerve injury.

To evaluate the involvement of the mitogen-activated protein kinase (MAPK) cascade in orofacial neuropathic pain mechanisms, this study assessed nocifensive behavior evoked by mechanical or thermal stimulation of the whisker pad skin, phosphorylation of extracellular signal-regulated kinase (ERK) in trigeminal spinal subnucleus caudalis (Vc) neurons, and Vc neuronal responses to mechanical or thermal stimulation of the whisker pad skin in rats with the chronic constriction nerve injury of the infraorbital nerve (ION-CCI). The mechanical and thermal nocifensive behavior was significantly enhanced on the side ipsilateral to the ION-CCI compared to the contralateral whisker pad or sham rats. ION-CCI rats had an increased number of phosphorylated ERK immunoreactive (pERK-IR) cells which also manifested NeuN-IR but not GFAP-IR and Iba1-IR, and were significantly more in ION-CCI rats compared with sham rats following noxious but not non-noxious mechanical stimulation. After intrathecal administration of the MEK1 inhibitor PD98059 in ION-CCI rats, the number of pERK-IR cells after noxious stimulation and the enhanced thermal nocifensive behavior but not the mechanical nocifensive behavior were significantly reduced in ION-CCI rats. The enhanced background activities, afterdischarges and responses of wide dynamic range neurons to noxious mechanical and thermal stimulation in ION-CCI rats were significantly depressed following i.t. administration of PD98059, whereas responses to non-noxious mechanical and thermal stimulation were not altered. The present findings suggest that pERK-IR neurons in the Vc play a pivotal role in the development of thermal hypersensitivity in the face following trigeminal nerve injury.

Metabotropic glutamate receptor 5 contributes to inflammatory tongue pain via extracellular signal-regulated kinase signaling in the trigeminal spinal subnucleus caudalis and upper cervical spinal cord.

BACKGROUND: In the orofacial region, limited information is available concerning pathological tongue pain, such as inflammatory pain or neuropathic pain occurring in the tongue. Here, we tried for the first time to establish a novel animal model of inflammatory tongue pain in rats and to investigate the roles of metabotropic glutamate receptor 5 (mGluR5)-extracellular signal-regulated kinase (ERK) signaling in this process. METHODS: Complete Freund's adjuvant (CFA) was submucosally injected into the tongue to induce the inflammatory pain phenotype that was confirmed by behavioral testing. Expression of phosphorylated ERK (pERK) and mGluR5 in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were detected with immunohistochemical staining and Western blotting. pERK inhibitor, a selective mGluR5 antagonist or agonist was continuously administered for 7 days via an intrathecal (i.t.) route. Local inflammatory responses were verified by tongue histology. RESULTS: Submucosal injection of CFA into the tongue produced a long-lasting mechanical allodynia and heat hyperalgesia at the inflamed site, concomitant with an increase in the pERK immunoreactivity in the Vc and C1-C2. The distribution of pERK-IR cells was laminar specific, ipsilaterally dominant, somatotopically relevant, and rostrocaudally restricted. Western blot analysis also showed an enhanced activation of ERK in the Vc and C1-C2 following CFA injection. Continuous i.t. administration of the pERK inhibitor and a selective mGluR5 antagonist significantly depressed the mechanical allodynia and heat hyperalgesia in the CFA-injected tongue. In addition, the number of pERK-IR cells in ipsilateral Vc and C1-C2 was also decreased by both drugs. Moreover, continuous i.t. administration of a selective mGluR5 agonist induced mechanical allodynia in naive rats. CONCLUSIONS: The present study constructed a new animal model of inflammatory tongue pain in rodents, and demonstrated pivotal roles of the mGluR5-pERK signaling in the development of mechanical and heat hypersensitivity that evolved in the inflamed tongue. This tongue-inflamed model might be useful for future studies to further elucidate molecular and cellular mechanisms of pathological tongue pain such as burning mouth syndrome.

P2X3 receptor mediates ectopic mechanical allodynia with inflamed lower lip in mice.

Ectopic pain in other orofacial regions develops with local inflammation in separated orofacial structures. However, the basis for the spreading of pain to adjacent orofacial areas after local inflammation is still unknown. In the present study, we determined if the P2X(3) receptor (P2X(3)R) was associated with altered mechanical sensitivity of the whisker pad skin following complete Freund's adjuvant (CFA) injection into the lower lip. Mice with local inflammation induced by CFA injection into the lower lip demonstrated significant mechanical allodynia of whisker pad skin. The mechanical allodynia was reversed by P2X(3)R antagonist, A-317491 administration into whisker pad skin. The number of P2X(3)R and calcitonin gene-related peptide (CGRP) positive trigeminal ganglion (TG) neurons that innervates the whisker pad skin and lower lip was increased after CFA injection into the lower lip. CGRP protein expression in TG ipsilateral to CFA injection was also significantly greater than that of the saline-injected mice. The present findings suggest that induced CGRP by local inflammation in the lower lip increases P2X(3)R in TG neurons, the increased P2X(3)Rs are involved in the sensitization of primary afferent neurons in the whisker pad skin. This P2X(3)R overexpression may underlie ectopic mechanical allodynia in the whisker pad skin after CFA injection into the lower lip.

Organization of pERK-immunoreactive cells in trigeminal spinal nucleus caudalis, upper cervical cord, NTS and Pa5 following capsaicin injection into masticatory and swallowing-related muscles in rats.

Many phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) cells are expressed in the trigeminal spinal subnucleus caudalis (Vc), upper cervical spinal cord (C1-C2), nucleus tractus solitarii (NTS) and paratrigeminal nucleus (Pa5) after capsaicin injection into the whisker pad (WP), masseter muscle (MM), digastric muscle (DM) or sternohyoideus muscle (SM). The pERK-IR cells also showed NeuN immunoreactivity, indicating that ERK phosphorylation occurs in neurons. The pERK-IR cells were significantly reduced after intrathecal injection of MEK 1/2 inhibitor PD98059. The pERK-IR cells expressed bilaterally in the Vc and C1-C2 after capsaicin injection into the unilateral DM or SM, whereas unilaterally in the Vc and C1-C2 after unilateral WP or MM injection. After capsaicin injection into the WP or MM, the pERK-IR cell expression in the Vc was restricted rostrocaudally within a narrow area. However, the distribution of pERK-IR cells was more wide spread without a clear peak in the Vc and C1-C2 after capsaicin injection into the DM or SM. In the NTS, the unimodal pERK-IR cell expression peaked at 0-720µm rostral from the obex following capsaicin injection into WP, MM, DM or SM. In the ipsilateral Pa5, many pERK-IR cells were observed following capsaicin injection into the SM. The number of swallows elicited by distilled water administration was significantly smaller after capsaicin injection into the WP, MM or DM but not SM compared to that of vehicle-injected rats. Various noxious inputs due to the masticatory or swallowing-related muscle inflammation may be differentially involved in muscle pain and swallowing reflex activity.