Masanori Nasu

Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation

Abstract Peripheral nerve injury activates satellite cells to produce interleukin 1β (IL‐1β) which mediates inflammation and hyperalgesia. This study investigated the hypothesis that activation of satellite glial cells modulates the excitability of trigeminal ganglion (TRG) neurons via IL‐1β following inflammation. Inflammation was induced by injection of complete Freund’s adjuvant (CFA) into the whisker pad area. The threshold for escape from mechanical stimulation applied to the whisker pad in inflamed rats was significantly lower than that in control. Two days post‐CFA injection, the mean percentage of TRG neurons encircled by glial fibrillary acidic protein (GFAP)‐/IL‐1β‐immunoreactive cells was significantly increased compared to controls. GFAP and IL‐1β immunoreactivities were coexpressed in the same cells. Fluorogold (FG) labeling identified the site of inflammation. The number of FG‐labeled IL‐receptor type I (IL‐1RI) TRG neurons in inflamed rats was significantly greater than in controls. In FG‐labeled small TRG neurons, the size of IL‐1β (1 nM) induced‐depolarization in inflamed rats was larger than in controls. IL‐1β application significantly increased firing rates evoked by depolarizing pulses in the neurons of inflamed rats, compared to controls. The response to IL‐1β was abolished by treatment with the IL‐1RI antagonist. These results suggest that activation of satellite glial cells modulates the excitability of small‐diameter TRG neurons via IL‐1β following inflammation, and that the upregulation of IL‐1RI in the soma may contribute to the mechanism underlying inflammatory hyperalgesia. Therefore IL‐1β blockers are potential therapeutic agents for prevention of trigeminal hyperalgesia.

Activation of NK1 receptor of trigeminal root ganglion via substance P paracrine mechanism contributes to the mechanical allodynia in the temporomandibular joint inflammation in rats

&NA; The aim of this study was to investigate whether under in vivo conditions, temporomandibular joint (TMJ) inflammation alters the excitability of Aβ‐trigeminal root ganglion (TRG) neuronal activity innervating the facial skin by using extracellular electrophysiological recording with multibarrel‐electrodes. Complete Freunds adjuvant (CFA) was injected into the rat TMJ. Threshold for escape from mechanical stimulation applied to the whisker pad area in inflamed rats (2 days) was significantly lower than that in control rats. A total of 36 Aβ‐TRG neurons responding to electrical stimulation of the whisker pad was recorded in pentobarbital‐anesthetized rats. The number of Aβ‐TRG neurons with spontaneous firings and their firing rate in TMJ inflamed rats were significantly larger than those in control rats. The firing rates of their spontaneous activity in the Aβ‐TRG neurons were current‐dependently decreased by local iontophoretic application of an NK1 receptor antagonist (L‐703,606) in inflamed, but not non‐inflamed rats. Their spontaneous activities were current‐dependently increased by local iontophoretic application of substance P (SP) in control and inflamed rats. The mechanical response threshold of Aβ‐TRG neurons in inflamed rats was significantly lower than that in control rats. The mechanical response threshold in inflamed rats after iontophoretic application of L‐703,606 was not different from that in control rats. These results suggest that TMJ inflammation modulate the excitability of Aβ‐TRG neurons innervating the facial skin via paracrine mechanism due to SP released from TRG neuronal cell body. Such a SP release may play an important role in determining the trigeminal inflammatory allodynia concerning the temporomandibular disorder.

Prostaglandin E2-induced modification of tetrodotoxin-resistant Na+ currents involves activation of both EP2 and EP4 receptors in neonatal rat nodose ganglion neurones.

1 The aim of the present study was to investigate which EP receptor subtypes (EP1–EP4) act predominantly on the modification of the tetrodotoxin‐resistant Na+ current (INaR) in acutely isolated neonatal rat nodose ganglion (NG) neurones. 2 Of the four EP receptor agonists ranging from 0.01 to 10 μM, the EP2 receptor agonist (ONO‐AE1‐259, 0.1–10 μM) and the EP4 receptor agonist (ONO‐AE1‐329, 1 μM) significantly increased peak INaR. The responses were associated with a hyperpolarizing shift in the activation curve. 3 Neither the EP1 receptor agonist ONO‐DI‐004 nor the EP3 receptor agonist ONO‐AE‐248 significantly modified the properties of INaR. 4 In PGE2 applications ranging from 0.01 to 10 μM, 1 μM PGE2 produced a maximal increase in the peak INaR amplitude. The PGE2 (1 μM)‐induced increase in the GV1/2 baseline (% change in G at baseline V1/2) was significantly attenuated by either intracellular application of the PKA inhibitor PKI or extracellular application of the protein kinase C inhibitor staurosporine (1 μM). However, the slope factor k was not significantly altered by PGE2 applications at 0.01–10 μM. In addition, the hyperpolarizing shift of V1/2 by PGE2 was not significantly altered by either PKI or staurosporine. 5 In other series of experiments, reverse transcription–polymerase chain reaction (RT–PCR) of mRNA from nodose ganglia indicated that all four EP receptors were present. 6 The NG contained many neuronal cell bodies (diameter <30 μm) with intense or moderate EP2, EP3, and EP4 receptor‐immunoreactivities. 7 These results suggest that the PGE2‐induced modification of INaR is mainly mediated by activation of both EP2 and EP4 receptors.

Temporomandibular joint inflammation decreases the voltage-gated K+ channel subtype 1.4-immunoreactivity of trigeminal ganglion neurons in rats.

Voltage‐gated K+ (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freunds Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4‐/neurofilament (NF) 200(myelinated fiber marker) positive‐ and negative‐immunoreactivities FG‐labeled small‐/medium‐diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small‐/medium sized (Aδ‐/C‐) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.

Peripheral inflammation suppresses inward rectifying potassium currents of satellite glial cells in the trigeminal ganglia

Summary Inflammation suppresses satellite glial inward rectifying K+ (Kir) currents in the trigeminal ganglia. Kir channel may be a potential therapeutic target for treatment of pain. ABSTRACT Previous studies indicate that silencing Kir4.1, a specific inward rectifying K+ (Kir) channel subunit, in sensory ganglionic satellite glial cells (SGCs) induces behavioral hyperalgesia. However, the function of Kir4.1 channels in SGCs in vivo under pathophysiological conditions remains to be determined. The aim of the present study was to examine whether peripheral inflammation in anesthetized rats alters the SGC Kir4.1 current using in vivo patch clamp and immunohistochemical techniques. Inflammation was induced by injection of complete Freund’s adjuvant into the whisker pad. The threshold of escape from mechanical stimulation applied to the orofacial area in inflamed rats was significantly lower than in naïve rats. The mean percentage of small/medium diameter trigeminal ganglion (TRG) neurons encircled by Kir4.1‐immunoreactive SGCs in inflamed rats was also significantly lower than in naïve rats. In vivo whole‐cell recordings were made using SGCs in the trigeminal ganglia (TRGs). Increasing extracellular K+ concentrations resulted in significantly smaller potentiation of the mean peak amplitude of the Kir current in inflamed compared with naïve rats. In addition, the density of the Ba2+‐sensitive Kir current associated with small‐diameter TRG neurons was significantly lower in inflamed rats compared with naïve rats. Mean membrane potential in inflamed rats was more depolarized than in naïve rats. These results suggest that inflammation could suppress Kir4.1 currents of SGCs in the TRGs and that this impairment of glial potassium homeostasis in the TRGs contributes to trigeminal pain. Therefore, the Kir4.1 channel in SGCs may be a new molecular target for the treatment of trigeminal inflammatory pain.

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.

Immunohistochemical co-expression of carbonic anhydrase II with Kv1.4 and TRPV1 in rat small-diameter trigeminal ganglion neurons

The co-expression of carbonic anhydrase II (CAII) with the voltage-gated potassium channel subtype 1.4 (Kv1.4) or the vanilloid receptor (TRPV1) was examined in adult rat trigeminal ganglion (TG) neurons by using the immunofluorescence method. The small-diameter Kv.1.4-positive TG neurons co-expressed CAII immunoreactivity (47%). Most TRPV1-positive TG neurons (79%) had the CAII immunoreactivity, but showed a lack of immunoreactivity for a neurofilament protein (NF200), a maker of large TG neurons with myelinated axons. The fact that CAII-immunoreactive TG neurons revealed a common expression of both Kv1.4 and TRPV1 leads us to suggest that CAII may be one of the nociceptive neuronal markers.

Effects of chitosan oligosaccharides on the femur trabecular structure in ovariectomized rats

ObjectivesDrugs used to treat osteoporosis are taken long-term and ideally have no adverse effects. Recent interest has focused on chitosan oligosaccharides as a bone-inducing substance for use as bone graft material. The present study was designed to investigate the effects of chitosan oligosaccharides on the femur trabecular structure in ovariectomized rats by three-dimensional imaging analysis using micro-computed tomography (CT).MethodsFemale 12-week-old Wistar rats were divided into three groups, which underwent ovariectomy (two groups) or sham surgery (one group). After 12 weeks, rats of one of the ovariectomized groups began treatment with chitosan oligosaccharides (500 mg/kg bodyweight) three times per week for 8 weeks. Then, the femurs of all rats were resected for bone analysis by micro-CT, and three-dimensional images of the femur were reconstructed from the axial CT images to evaluate the following parameters: bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.sp), trabecular bone pattern factor (TBPf), structure model index (SMI), number of nodes per unit tissue volume, number of termini per unit tissue volume, and the total strut length per unit tissue volume.ResultsThe BV/TV, Tb.Th, and Tb.N were significantly higher in the ovariectomized and treated (OVX + C) group versus the ovariectomized, untreated (OVX) group. Tb.Sp, TBPf, and SMI were significantly lower in the OVX + C group versus the OVX group.ConclusionsOur findings demonstrate that chitosan oligosaccharides prevent decreases in bone volume, trabecular number, trabecular thickness, and trabecular connectivity in the femurs of ovariectomized rats.

The effects of oral xylitol administration on bone density in rat femur.

To examine the effects of oral xylitol administration on rat femur bone density, 36 four-week-old male Wistar rats divided into three groups were fed CE-2 diet (control, n = 12) alone or supplemented with 10% (n = 12) or 20% (n = 12) dietary xylitol for 40 days. Biochemical, morphological, and histological analyses were performed. The 10% and 20% xylitol groups showed higher levels of both serum Ca and alkaline phosphatase activity and lower levels of serum tartrate-resistant acid phosphatase than the control group. Although no significant differences in the three-dimensional bone structure or trabecular bone structure of the femur were observed, both xylitol groups showed significantly higher bone density than the control group. Compared to the control group, the 10% and 20% xylitol groups showed an increase in trabeculae. Thus, oral administration of xylitol appears to affect bone metabolism, leading to increased bone density in rat femur.

Experimental study on wound healing of alveolar bone sockets in the rat maxilla after X-ray irradiation

Abstract The purpose of this study was to examine the effects of radiation on the healing process of tooth extraction wounds. X-ray doses of 10 Gy were delivered once to the maxillofacial area of Wistar strain rats, and the maxillary first molar was extracted 7 days later. Animals were killed 3, 7, and 14 days after odontectomy, and thin sections of the maxilla were prepared. The specimens were stained with hematoxylin and eosin, azan, alkaline phosphatase and tartrate-resistant acid phosphatase (TRAP) stains for microscopic observation. The ratio of the area of the bone tissue to the area of the alveolar socket (BS/TS) and the numbers of TRAP-positive cells were evaluated. Seven days after odontectomy, BS/TS was significantly lower in the irradiated group than in the nonirradiated group. Alkaline phosphatase staining was observed along the rim of the bone 7 and 14 days after odontectomy in both groups, but it was milder in the irradiated group than in the nonirradiated group. TRAP-positive cells were present on the floor of the postextraction socket and the margins of the septal alveolar bone, and, 3 days after odontectomy, the number of TRAP-positive cells was significantly greater in the irradiation group than in the nonirradiation group.