The sodium channel isoform transition at developing nodes of Ranvier in the peripheral nervous system: dependence on a Genetic program and myelination-induced cluster formation.

Among sodium channel isoforms, Nav 1.6 is selectively expressed at nodes of Ranvier in both the CNS and the PNS. However, non-Nav 1.6 isoforms such as Nav 1.2 are also present at the CNS nodes in early development but gradually diminish later. It has been proposed that myelination is part of a glia-neuron signaling mechanism that produces this change in nodal isoform expression. The present study used isoform-specific antibodies to demonstrate that, in the PNS, four other neuronal sodium channel isoforms were also clustered at nodes in early development but eventually disappeared during maturation. To study possible roles of myelination in such transitions, we investigated the nodal expression of selected isoforms in the sciatic nerve of the transgenic mouse Oct6(ΔSCE/ßgeo) , whose PNS myelination is delayed in the first postnatal week but eventually resumes. We found that delayed myelination retarded the formation of nodal channel clusters and altered the expression-elimination patterns of sodium channel isoforms, resulting in significantly reduced expression levels of non-Nav 1.6 isoforms in such delayed nodes. However, delayed myelination did not significantly affect the gene expression, protein synthesis, or axonal trafficking of any isoform studied. Rather, we found evidence for a developmentally programmed increase in neuronal Nav 1.6 expression with constant or decreasing neuronal expression of other isoforms that were unaffected by delayed myelination. Thus our results suggest that, in the developmental isoform switch of the PNS, myelination does not play a signaling role as that proposed for the CNS but rather serves only to form nodal clusters from existing isoform pools.

Vanilloids induce oral cancer apoptosis independent of TRPV1.

OBJECTIVE: To investigate the mechanisms of vanilloid cytotoxicity and anti-tumor effects in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Immunohistochemistry and qPCR analyses demonstrated expression of the TRP vanilloid type 1 (TRPV1) receptor in OSCC. Using cell proliferation assays, calcium imaging, and three mouse xenograft models, prototypical vanilloid agonist (capsaicin) and antagonist (capsazepine) were evaluated for cytotoxic and anti-tumor effects in OSCC. RESULTS: OSCC cell lines treated with capsaicin displayed significantly reduced cell viability. Pre-treatment with capsazepine failed to reverse these effects. Moreover, capsazepine alone was significantly cytotoxic to tumor cells, suggesting the mechanism-of-action is independent of TRPV1 activation. This was further confirmed by calcium imaging indicating that TRPV1 channels are not functional in the cell lines tested. We then examined whether the observed vanilloid cytotoxicity was due to the generation of reactive oxygen species (ROS) and subsequent apoptosis. Induction of ROS was confirmed by flow cytometry and reversed by co-treatment with the antioxidant N-acetyl-cysteine (NAC). NAC also significantly reversed vanilloid cytotoxicity in cell proliferation assays. Dose-dependent induction of apoptosis with capsazepine treatment was demonstrated by FACS analyses and c-PARP expression in treated cells. Our in vivo xenograft studies showed that intra-tumoral injections of capsazepine exhibited high effectiveness in suppressing tumor growth with no identifiable toxicities. CONCLUSIONS: These findings confirm TRPV1 channel expression in OSCC. However anti-tumor effects of vanilloids are independent of TRPV1 activation and are most likely due to ROS induction and subsequent apoptosis. Importantly, these studies demonstrate capsazepine is a potential therapeutic candidate for OSCC.

The role of sodium channels in chronic pain.

Here we review recent research into the mechanisms of chronic pain that has focused on neuronal sodium channels, a target of classic analgesic agents. We first discuss evidence that specific sodium channel isoforms are essential for the detection and conduction of normal acutely painful stimuli from nociceptors. We then review findings that show changes in sodium channel expression and localization in chronic inflammation and nerve injury in animal and human tissues. We conclude by discussing the role that myelination plays in organizing and maintaining sodium channel clusters at nodes of Ranvier in normal development and how inflammatory processes or nerve injury alter the characteristics of such clusters. Based on these findings, we suggest that chronic pain may in part result from partial demyelination of axons during chronic injury, which creates aberrant sodium channel clusters that serve as sites of ectopic sensitivity or spontaneous activity.

Unmyelinated nerve fibers in the human dental pulp express markers for myelinated fibers and show sodium channel accumulations.

BACKGROUND: The dental pulp is a common source of pain and is used to study peripheral inflammatory pain mechanisms. Results show most fibers are unmyelinated, yet recent findings in experimental animals suggest many pulpal afferents originate from fibers that are myelinated at more proximal locations. Here we use the human dental pulp and confocal microscopy to examine the staining relationships of neurofilament heavy (NFH), a protein commonly expressed in myelinated afferents, with other markers to test the possibility that unmyelinated pulpal afferents originate from myelinated axons. Other staining relationships studied included myelin basic protein (MBP), protein gene product (PGP) 9.5 to identify all nerve fibers, tyrosine hydroxylase (TH) to identify sympathetic fibers, contactin-associated protein (caspr) to identify nodal sites, S-100 to identify Schwann cells and sodium channels (NaChs). RESULTS: Results show NFH expression in most PGP9.5 fibers except those with TH and include the broad expression of NFH in axons lacking MBP. Fibers with NFH and MBP show NaCh clusters at nodal sites as expected, but surprisingly, NaCh accumulations are also seen in unmyelinated fibers with NFH, and in fibers with NFH that lack Schwann cell associations. CONCLUSIONS: The expression of NFH in most axons suggests a myelinated origin for many pulpal afferents, while the presence of NaCh clusters in unmyelinated fibers suggests an inherent capacity for the unmyelinated segments of myelinated fibers to form NaCh accumulations. These findings have broad implications on the use of dental pulp to study pain mechanisms and suggest possible novel mechanisms responsible for NaCh cluster formation and neuronal excitability.

Sodium channel expression and localization at demyelinated sites in painful human dental pulp.

UNLABELLED: The expression of sodium channels (NaCh(s)) change after inflammatory and nerve lesions, and this change has been implicated in the generation of pain states. Here we examine NaCh expression within nerve fibers from normal and painful extracted human teeth with special emphasis on their localization within large accumulations, like those seen at nodes of Ranvier. Pulpal tissue sections from normal wisdom teeth and from teeth with large carious lesions associated with severe and spontaneous pain were double-stained with pan-specific NaCh antibody and caspr (paranodal protein used to visualize nodes of Ranvier) antibody, while additional sections were triple-stained with NaCh, caspr and myelin basic protein (MBP) antibodies. Z-series of images were obtained with the confocal microscope and evaluated with NIH ImageJ software to quantify the density and size of NaCh accumulations, and to characterize NaCh localization at caspr-identified typical and atypical nodal sites. Although the results showed variability in the overall density and size of NaCh accumulations in painful samples, a common finding included the remodeling of NaChs at atypical nodal sites. This remodeling of NaChs included prominent NaCh expression within nerve regions that showed a selective loss of MBP staining in a pattern consistent with a demyelinating process. PERSPECTIVE: This study identifies the remodeling of NaChs at demyelinated sites within the painful human dental pulp and suggests that the contribution of NaChs to spontaneous pulpal pain generation may be dependant not only on total NaCh density but may also be related to NaCh expression at atypical nodal sites.

Tetrodotoxin-resistant voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 are expressed in the retina.

Voltage-gated sodium channels (VGSCs) are one of the fundamental building blocks of electrically excitable cells in the nervous system. These channels are responsible for the generation of action potentials that are required for the communication of neuronal signals over long distances within a cell. VGSCs are encoded by a family of nine genes whose products have widely varying biophysical properties. In this study, we have detected the expression of two atypical VGSCs (Na(v)1.8 and Na(v)1.9) in the retina. Compared with more common VGSCs, Na(v)1.8 and Na(v)1.9 have unusual biophysical and pharmacological properties, including persistent sodium currents and resistance to the canonical sodium channel blocker tetrodotoxin (TTX). Our molecular biological and immunohistochemical data derived from mouse (Mus musculus) retina demonstrate expression of Na(v)1.8 by retinal amacrine and ganglion cells, whereas Na(v)1.9 is expressed by photoreceptors and Müller glia. The fact that these channels exist in the central nervous system (CNS) and exhibit robust TTX resistance requires a re-evaluation of prior physiological, pharmacological, and developmental data in the visual system, in which the diversity of VGSCs has been previously underestimated.

Nav1.7 expression is increased in painful human dental pulp.

BACKGROUND: Animal studies and a few human studies have shown a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth as a model system to study peripheral pain mechanisms and here examine the expression of the Nav1.7 NaCh isoform in normal and painful samples. Pulpal sections were labeled with antibodies against: 1) Nav1.7, N52 and PGP9.5, and 2) Nav1.7, caspr (a paranodal protein used to identify nodes of Ranvier), and myelin basic protein (MBP), and a z-series of optically-sectioned images were obtained with the confocal microscope. Nav1.7-immunofluorescence was quantified in N52/PGP9.5-identified nerve fibers with NIH ImageJ software, while Nav1.7 expression in myelinated fibers at caspr-identified nodal sites was evaluated and further characterized as either typical or atypical as based on caspr-relationships. RESULTS: Results show a significant increase in nerve area with Nav1.7 expression within coronal and radicular fiber bundles and increased expression at typical and atypical caspr-identified nodal sites in painful samples. Painful samples also showed an augmentation of Nav1.7 within localized areas that lacked MBP, including those associated with atypical caspr-identified sites, thus identifying NaCh remodeling within demyelinating axons as the basis for a possible pulpal pain mechanism. CONCLUSION: This study identifies the increased axonal expression and augmentation of Nav1.7 at intact and remodeling/demyelinating nodes within the painful human dental pulp where these changes may contribute to constant, increased evoked and spontaneous pain responses that characterize the pain associated with toothache.

[Effects of dentine phosphoprotein on remineralization of demineralized dentine].

OBJECTIVE: To investigate the relationship between dentine phosphoprotein (DPP) and remineralization of demineralized dentine. METHODS: (1) Soluble DPP was extracted with 1 mol/L NaCl from demineralized dentine and was evaluated. (2) Soluble DPP was removed with 0.1 mol/L NaCl or was not removed from demineralized dentine sections in human tooth roots. Then all sections were subjected to remineralization treatment, and remineralization degrees were compared by atomic absorption spectrum, SEM and microradiography. RESULTS: (1) Soluble DPP was extracted with 1 mol/L NaCl. (2) Removal of soluble DPP resulted in significantly lower calcium concentration in remineralization solution (P < 0.01), less mean light-absorbed value in demineralized dentin sections by microradiography (P < 0.01). CONCLUSIONS: Soluble DPP may have an inhibiting effect on remineralization of demineralized dentine, this study suggests that the remove of soluble DPP from root caries lesions may enhance their remineralization potential.

[The effect of dentin phosphoprotein on inducing mineralization].

OBJECTIVE: To investigate the effect of dentin phosphoprotein (DPP) in inducing dentinal mineralization. METHODS: Human DPP was combined with EAH-Sepharose 4B beads and its function of inducing mineralization was studied in mineralization system in-vitro. The mineral formed on the surface of the beads was analyzed by scanning electron microscopy (SEM) and the structure was analyzed by X-ray diffraction and plasma emission spectrum. RESULTS: There was mineral formed on the beads with combined DPP and the mineral was calcium phosphates whose ratio of calcium to phosphate was 1.33. The diffractogram of the formed mineral was more similar to hydroxyapatite than to other calcium phosphates. CONCLUSION: When tightly combined with certain support substance, human DPP can induce mineralization.