Expression of Ascorbate Peroxidase Derived from Cyanidioschyzon merolae in Mammalian Cells

Background: Ascorbate peroxidase (APX) derived from Cyanidioschyzon merolae, a primitive red alga living in high temperature and acidic environments, has a greater anti-oxidative capacity than similar peroxidases occurring in other plants. In the present study, we examined the ability of Cyanidioschyzon merolae-derived APX (cAPX) to increase anti-oxidative capacity when expressed in mammalian cells. Materials and Methods: The cAPX gene was introduced into the mouse fibroblast-like cell line C3H10T1/2. Production of reactive oxygen species (ROS) and/or cell viability was assessed after heat, H2O2 and acid stimulation. Results: Heat and H2O2 stimulation caused ROS production. cAPX-expressing cells were more tolerant to oxidative stress induced by heat, H2O2 and acid stimulations than control cells lacking cAPX. Conclusion: Introduction of cAPX increases anti-oxidative capacity in mammalian cells

Mechanisms involved in extraterritorial facial pain following cervical spinal nerve injury in rats

<p>Abstract</p> <p>Background</p> <p>The aim of this study is to clarify the neural mechanisms underlying orofacial pain abnormalities after cervical spinal nerve injury. Nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) expression and astroglial cell activation in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal dorsal horn (C1-C2) neurons were analyzed in rats with upper cervical spinal nerve transection (CNX).</p> <p>Results</p> <p>The head withdrawal threshold to mechanical stimulation of the lateral facial skin and head withdrawal latency to heating of the lateral facial skin were significantly lower and shorter respectively in CNX rats compared to Sham rats. These nocifensive effects were apparent within 1 day after CNX and lasted for more than 21 days. The numbers of pERK-like immunoreactive (LI) cells in superficial laminae of Vc and C1-C2 were significantly larger in CNX rats compared to Sham rats following noxious and non-noxious mechanical or thermal stimulation of the lateral facial skin at day 7 after CNX. Two peaks of pERK-LI cells were observed in Vc and C1-C2 following mechanical and heat stimulation of the lateral face. The number of pERK-LI cells in C1-C2 was intensity-dependent and increased when the mechanical and heat stimulations of the face were increased. The decrements of head withdrawal latency to heat and head withdrawal threshold to mechanical stimulation were reversed during intrathecal (i.t.) administration of MAPK/ERK kinase 1/2 inhibitor PD98059. The area of activated astroglial cells was significantly higher in CNX rats (at day 7 after CNX). The heat and mechanical nocifensive behaviors were significantly depressed and the number of pERK-LI cells in Vc and C1-C2 following noxious and non-noxious mechanical stimulation of the face was also significantly decreased following i.t. administration of the astroglial inhibitor fluoroacetate.</p> <p>Conclusions</p> <p>The present findings have demonstrated that mechanical allodynia and thermal hyperalgesia occur in the lateral facial skin after CNX and also suggest that ERK phosphorylation of Vc and C1-C2 neurons and astroglial cell activation are involved in orofacial extraterritorial pain following cervical nerve injury.</p

Piezo1-pannexin-1-P2X3 axis in odontoblasts and neurons mediates sensory transduction in dentinal sensitivity

According to the “hydrodynamic theory,” dentinal pain or sensitivity is caused by dentinal fluid movement following the application of various stimuli to the dentin surface. Recent convergent evidence in Vitro has shown that plasma membrane deformation, mimicking dentinal fluid movement, activates mechanosensitive transient receptor potential (TRP)/Piezo channels in odontoblasts, with the Ca2+ signal eliciting the release of ATP from pannexin-1 (PANX-1). The released ATP activates the P2X3 receptor, which generates and propagates action potentials in the intradental Aδ afferent neurons. Thus, odontoblasts act as sensory receptor cells, and odontoblast-neuron signal communication established by the TRP/Piezo channel-PANX-1-P2X3 receptor complex may describe the mechanism of the sensory transduction sequence for dentinal sensitivity. To determine whether odontoblast-neuron communication and odontoblasts acting as sensory receptors are essential for generating dentinal pain, we evaluated nociceptive scores by analyzing behaviors evoked by dentinal sensitivity in conscious Wistar rats and Cre-mediated transgenic mouse models. In the dentin-exposed group, treatment with a bonding agent on the dentin surface, as well as systemic administration of A-317491 (P2X3 receptor antagonist), mefloquine and 10PANX (non-selective and selective PANX-1 antagonists), GsMTx-4 (selective Piezo1 channel antagonist), and HC-030031 (selective TRPA1 channel antagonist), but not HC-070 (selective TRPC5 channel antagonist), significantly reduced nociceptive scores following cold water (0.1 ml) stimulation of the exposed dentin surface of the incisors compared to the scores of rats without local or systemic treatment. When we applied cold water stimulation to the exposed dentin surface of the lower first molar, nociceptive scores in the rats with systemic administration of A-317491, 10PANX, and GsMTx-4 were significantly reduced compared to those in the rats without systemic treatment. Dentin-exposed mice, with somatic odontoblast-specific depletion, also showed significant reduction in the nociceptive scores compared to those of Cre-mediated transgenic mice, which did not show any type of cell deletion, including odontoblasts. In the odontoblast-eliminated mice, P2X3 receptor-positive A-neurons were morphologically intact. These results indicate that neurotransmission between odontoblasts and neurons mediated by the Piezo1/TRPA1-pannexin-1-P2X3 receptor axis is necessary for the development of dentinal pain. In addition, odontoblasts are necessary for sensory transduction to generate dentinal sensitivity as mechanosensory receptor cells

Microglia–Astrocyte Communication via C1q Contributes to Orofacial Neuropathic Pain Associated with Infraorbital Nerve Injury

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia&ndash;astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain

Microglia cause structural remodeling of noradrenergic axon in the trigeminal spinal subnucleus caudalis after infraorbital nerve injury in rats

The dysfunction of descending noradrenergic (NAergic) modulation in second-order neurons has long been observed in neuropathic pain. In clinical practice, antidepressants that increase noradrenaline levels in the synaptic cleft are used as first-line agents, although adequate analgesia has not been occasionally achieved. One of the hallmarks of neuropathic pain in the orofacial regions is microglial abnormalities in the trigeminal spinal subnucleus caudalis (Vc). However, until now, the direct interaction between descending NAergic system and Vc microglia in orofacial neuropathic pain has not been explored. We found that reactive microglia ingested the dopamine-β-hydroxylase (DβH)-positive fraction, NAergic fibers, in the Vc after infraorbital nerve injury (IONI). Major histocompatibility complex class I (MHC-I) was upregulated in Vc microglia after IONI. Interferon-γ (IFNγ) was de novo induced in trigeminal ganglion (TG) neurons following IONI, especially in C-fiber neurons, which conveyed to the central terminal of TG neurons. Gene silencing of IFNγ in the TG reduced MHC-I expression in the Vc after IONI. Intracisternal administration of exosomes from IFNγ-stimulated microglia elicited mechanical allodynia and a decrease in DβH in the Vc, which did not occur when exosomal MHC-I was knocked down. Similarly, in vivo MHC-I knockdown in Vc microglia attenuated the development of mechanical allodynia and a decrease in DβH in the Vc after IONI. These results show that microglia-derived MHC-I causes a decrease in NAergic fibers, culminating in orofacial neuropathic pain

Aging-Related Phenotypic Conversion of Medullary Microglia Enhances Intraoral Incisional Pain Sensitivity

Activated microglia involved in the development of orofacial pain hypersensitivity have two major polarization states. The aim of this study was to assess the involvement of the aging-related phenotypic conversion of medullary microglia in the enhancement of intraoral pain sensitivity using senescence-accelerated mice (SAM)-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) mice. Mechanical head-withdrawal threshold (MHWT) was measured for 21 days post palatal mucosal incision. The number of CD11c-immunoreactive (IR) cells [affective microglia (M1)] and CD163-IR cells [protective microglia (M2)], and tumor-necrosis-factor-&alpha; (TNF-&alpha;)-IR M1 and interleukin (IL)-10-IR M2 were analyzed via immunohistochemistry on days 3 and 11 following incision. The decrease in MHWT observed following incision was enhanced in SAMP8 mice. M1 levels and the number of TNF-&alpha;-IR M1 were increased on day 3 in SAMP8 mice compared with those in SAMR1 mice. On day 11, M1 and M2 activation was observed in both groups, whereas IL-10-IR M2 levels were attenuated in SAMP8 mice, and the number of TNF-&alpha;-IR M1 cells increased, compared to those in SAMR1 mice. These results suggest that the mechanical allodynia observed following intraoral injury is potentiated and sustained in SAMP8 mice due to enhancement of TNF-&alpha; signaling, M1 activation, and an attenuation of M2 activation accompanying IL-10 release

Hepcidin expression in the trigeminal ganglion and the oral mucosa in an oral ulcerative mucositis rat model.

Severe intraoral pain induces difficulty in eating and speaking, leading to a decline in the quality of life. However, the molecular mechanisms underlying intraoral pain remain unclear. Here, we investigated gene modulation in the trigeminal ganglion and intraoral pain-related behavior in a rat model of acetic acid-induced oral ulcerative mucositis. Oral ulceration was observed on day 2 after acetic acid treatment to the oral mucosa of male Wistar rats, causing spontaneous pain and mechanical allodynia. Deoxyribonucleic acid microarray analysis of trigeminal ganglion tissue indicated that Hamp (a hepcidin gene that regulates cellular iron transport) was the most upregulated gene. In the oral ulcerative mucositis model, the upregulation of Hamp was also induced in the ulcer region but not in the liver, with no increase in hepcidin levels in the plasma and saliva, indicating that hepcidin was produced locally in the ulcer region in the model. Systemic antibiotic pretreatment did not increase the mRNA levels of Hamp in the trigeminal ganglion and ulcer regions. Hepcidin injection into the oral mucosa enhanced neuronal excitability in response to noxious mechanical stimulation of the oral mucosa in trigeminal spinal subnucleus interpolaris/caudalis neurons. These results imply that oral ulcerative mucositis induces oral mucosal pain because of infectious inflammation of the ulcerative area and potentiates Hamp, which represents anti-bacterial and anti-peptidase gene expression in the ulcer region and trigeminal ganglion. The regulation of cellular iron transport by hepcidin is likely involved in oral ulcerative mucositis-induced pain

Analgesic Mechanisms of Steroid Ointment against Oral Ulcerative Mucositis in a Rat Model

Despite the long history of use of steroid ointments for oral mucositis, the analgesic mechanism has not been fully elucidated. In this study, we examined the effects of triamcinolone acetonide (Tmc) on oral ulcerative mucositis-induced pain in conscious rats by our proprietary assay system. Based on evaluations of the physical properties and retention periods in the oral mucosa of human volunteers and rats, we selected TRAFUL® ointment as a long-lasting base. In oral ulcerative mucositis model rats, TRAFUL® with Tmc suppressed cyclooxygenase-dependent inflammatory responses with upregulations of glucocorticoid receptor-induced anti-inflammatory genes and inhibited spontaneous nociceptive behavior. When an ointment with a shorter residual period was used, the effects of Tmc were not elicited or were induced to a lesser extent. Importantly, TRAFUL® with Tmc also improved oral ulcerative mucositis-induced mechanical allodynia, which has been reported to be independent of cyclooxygenase. Ca2+ imaging in dissociated trigeminal ganglion neurons showed that long-term preincubation with Tmc inhibited the hypertonic stimulation-induced Ca2+ response. These results suggest that the representative steroid Tmc suppresses oral ulcerative mucositis-induced pain by general anti-inflammatory actions and inhibits mechanical sensitivity in peripheral nerves. For drug delivery, long-lasting ointments such as TRAFUL® are needed to sufficiently induce the therapeutic effects