Phenotypic change in trigeminal ganglion neurons associated with satellite cell activation via extracellular signal-regulated kinase phosphorylation is involved in lingual neuropathic pain.

Iatrogenic trigeminal nerve injuries remain a common and complex clinical problem. Satellite glial cell (SGC) activation, associated phosphorylation of extracellular signal-regulated kinase (ERK), and neuropeptide expression in the trigeminal ganglion (TG) are known to be involved in trigeminal neuropathic pain related to trigeminal nerve injury. However, the involvement of these molecules in orofacial neuropathic pain mechanisms is still unknown. Phosphorylation of ERK1/2 in lingual nerve crush (LNC) rats was observed in SGCs. To evaluate the role of neuron-SGC interactions under neuropathic pain, calcitonin gene-related peptide (CGRP)-immunoreactive (IR), phosphorylated ERK1/2 (pERK1/2)-IR and glial fibrillary acidic protein (GFAP)-IR cells in the TG were studied in LNC rats. The number of CGRP-IR neurons and neurons encircled with pERK1/2-IR SGCs was significantly larger in LNC rats compared with sham rats. The percentage of large-sized CGRP-IR neurons was significantly higher in LNC rats. The number of CGRP-IR neurons, neurons encircled with pERK1/2-IR SGCs, and neurons encircled with GFAP-IR SGCs was decreased following CGRP receptor blocker CGRP8-37 or mitogen-activated protein kinase/ERK kinase 1 inhibitor PD98059 administration into the TG after LNC. Reduced thresholds to mechanical and heat stimulation to the tongue in LNC rats were also significantly recovered following CGRP8-37 or PD98059 administration. The present findings suggest that CGRP released from TG neurons activates SGCs through ERK1/2 phosphorylation and TG neuronal activity is enhanced, resulting in the tongue hypersensitivity associated with lingual nerve injury. The phenotypic switching of large myelinated TG neurons expressing CGRP may account for the pathogenesis of tongue neuropathic pain.

Differences in control of parasympathetic vasodilation between submandibular and sublingual glands in the rat.

We examined blood flow in the submandibular gland (SMGBF) and sublingual gland (SLGBF) during electrical stimulation of the central cut end of the lingual nerve (LN) in the urethane-anesthetized rats using a laser speckle imaging flow meter. LN stimulation elicited intensity- and frequency-dependent SMGBF and SLGBF increases, and the magnitude of the SMGBF increase was higher than that of the SLGBF increase. The increase in both glands was significantly inhibited by intravenous administration of the autonomic cholinergic ganglion blocker hexamethonium. The antimuscarinic agent atropine markedly inhibited the SMGBF increase and partly inhibited the SLGBF increase. The atropine-resistant SLGBF increase was significantly inhibited by infusion of vasoactive intestinal peptide (VIP) receptor antagonist, although administration of VIP receptor antagonist alone had no effect. The recovery time to the basal blood flow level was shorter after LN stimulation than after administration of VIP. However, the recovery time after LN stimulation was significantly delayed by administration of atropine in a dose-dependent manner to the same level as after administration of VIP. Our results indicate that 1) LN stimulation elicits both a parasympathetic SMGBF increase mainly evoked by cholinergic fibers and a parasympathetic SLGBF increase evoked by cholinergic and noncholinergic fibers, and 2) VIP-ergic mechanisms are involved in the noncholinergic SLGBF increase and are activated when muscarinic mechanisms are deactivated.

Correlation of Nav1.8 and Nav1.9 sodium channel expression with neuropathic pain in human subjects with lingual nerve neuromas.

BACKGROUND: Voltage-gated sodium channels Nav1.8 and Nav1.9 are expressed preferentially in small diameter sensory neurons, and are thought to play a role in the generation of ectopic activity in neuronal cell bodies and/or their axons following peripheral nerve injury. The expression of Nav1.8 and Nav1.9 has been quantified in human lingual nerves that have been previously injured inadvertently during lower third molar removal, and any correlation between the expression of these ion channels and the presence or absence of dysaesthesia investigated. RESULTS: Immunohistochemical processing and quantitative image analysis revealed that Nav1.8 and Nav1.9 were expressed in human lingual nerve neuromas from patients with or without symptoms of dysaesthesia. The level of Nav1.8 expression was significantly higher in patients reporting pain compared with no pain, and a significant positive correlation was observed between levels of Nav1.8 expression and VAS scores for the symptom of tingling. No significant differences were recorded in the level of expression of Nav1.9 between patients with or without pain. CONCLUSIONS: These results demonstrate that Nav1.8 and Nav1.9 are present in human lingual nerve neuromas, with significant correlations between the level of expression of Nav1.8 and symptoms of pain. These data provide further evidence that changes in expression of Nav1.8 are important in the development and/or maintenance of nerve injury-induced pain, and suggest that Nav1.8 may be a potential therapeutic target.

Chronic restraint stress in rats suppresses sweet and umami taste responses and lingual expression of T1R3 mRNA.

Effects of chronic restraint stress on the taste responses to five basic taste qualities were investigated electrophysiologically in the rat chorda tympani. In addition, the mRNA expression for T1R3, the common G-protein-coupled receptor (GPCR) for sweet and umami tastes, was studied quantitatively by RT-PCR after such stress. Rats were restrained in a small cylindrical restrainer made of steel wire for 8h daily for 14 successive days. The integrated responses to sweet and umami tastes, as recorded from the chorda tympani, were significantly suppressed after such stress, but the other three basic taste responses were unaffected. Expression of T1R3 mRNA in the fungiform papillae, as estimated by RT-PCR, was slightly reduced by the stress, and a quantitative real time RT-PCR study revealed a significant suppression of T1R3 mRNA expression in the stress group. These results suggest that the observed stress-induced changes in taste sensation could be caused by a peripheral disorder of the transduction mechanism in taste-receptor cells, involving in particular a stress-induced inhibition of T1R3 expression.

TRPA1 expression in human lingual nerve neuromas in patients with and without symptoms of dysaesthesia.

The TRPA1 receptor is a member of the ankyrin family and is found in both spinal and trigeminal neurones. There is evidence to suggest that this receptor may be a sensor of noxious thermal stimuli in normal animals. After nerve injury, TRPA1 shows increased expression in uninjured axons, and has been implicated in the development and maintenance of hyperalgesia. We examined expression of TRPA1 in lingual nerve neuromas and investigated any potential correlation with the presence or absence of symptoms of dysaesthesia. Thirteen neuroma-in-continuity specimens were obtained from patients undergoing repair of a lingual nerve that had previously been damaged during lower third molar removal. Visual analogue scales (VAS) were used to record the degree of pain, tingling and discomfort. Tissue was processed for indirect immunofluorescence and the percentage area of PGP 9.5-immunoreactive neuronal tissue also labelled for TRPA1 was quantified. No significant difference between levels of TRPA1 in neuromas from patients with or without symptoms of dysaesthesia and no relationship between TRPA1 expression and VAS scores for pain, tingling or discomfort were observed. TRPA1 expression and the time after initial injury that the specimen was obtained also showed no correlation. These data show that TRPA1 is expressed in lingual nerve neuromas, but, it appears that, at this site, TRPA1 does not play a principal role in the development of neuropathic pain.

Immunohistochemical analysis of the purinoceptor P2X7 in human lingual nerve neuromas.

AIMS: Recent evidence suggests that the purinoceptor P2X7 may be involved in the development of dysesthesia following nerve injury, therefore, the aim of the present study was to investigate whether a correlation exists between the level of P2X7 receptor expression in damaged human lingual nerves and the severity of the patients' symptoms. METHODS: Neuroma-in-continuity specimens were obtained from patients undergoing surgical repair of the damaged lingual nerve. Specimens were categorized preoperatively according to the presence or absence of dysesthesia, and visual analog scales scores were used to record the degree of pain, tingling, and discomfort. Indirect immunofluorescence using antibodies raised against S-100 (a Schwann cell marker) and P2X7 was employed to quantify the percentage area of S-100 positive cells that also expressed P2X7. RESULTS: P2X7 was found to be expressed in Schwann cells of lingual nerve neuromas. No significant difference was found between the level of P2X7 expression in patients with or without symptoms of dysesthesia, and no relationship was observed between P2X7 expression and VAS scores for pain, tingling, or discomfort. No correlation was found between P2X7 expression and the time between initial injury and nerve repair. CONCLUSION: These data show that P2X7 is expressed in human lingual nerve neuromas from patients with and without dysesthesia. It therefore appears that the level of P2X7 expression at the injury site may not be linked to the maintenance of neuropathic pain after lingual nerve injury.

P2X(3) expression is not altered by lingual nerve injury.

We have investigated a possible role for the ATP receptor subunit P2X(3), in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine anaesthetised adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was applied to the nerve to allow identification of cell bodies in the trigeminal ganglion with axons in the injured nerve. Indirect immunofluorescence for P2X(3) and image analysis was used to quantify the percentage area of staining at the site of injury. Additionally, the proportion of fluorogold-positive cells that expressed P2X(3) was determined and compared with expression in non-fluorogold containing cells in another part of the ganglion. Comparisons were made with results from control animals that only received the tracer injection. After lingual nerve injury there was no significant change in P2X(3) expression at the site of nerve injury or within cell bodies linked to either injured (lingual) or uninjured (ophthalmic) axons, at any of the time periods investigated. Overall, this study suggests that P2X(3) expression at these sites is not involved in the development of neuropathic pain following lingual nerve injury.

Na(v)1.7 sodium channel expression in human lingual nerve neuromas.

OBJECTIVE: Peripheral branches of the trigeminal nerve are often damaged during the removal of lower third molar teeth, and a small proportion of patients who sustain an injury develop persistent chronic pain. The cause of the pain is not clear and there are no satisfactory methods of treatment. The aim of the present study was to examine the expression of the sodium channel subtype Na(v)1.7 in damaged human lingual nerves, and to identify any association between Na(v)1.7 expression and reported symptoms of dysaesthesia. METHODS: Eleven neuromas-in-continuity (NICs) and 11 nerve-end neuromas (NENs) were studied, and were all obtained at the time of surgical repair of the damaged lingual nerve. Specimens were categorised as being obtained from patients with symptoms or without symptoms, according to the degree of pain, tingling or discomfort that had been experienced. The tissue was prepared and processed for indirect immunofluorescence, and image analysis was used to quantify the percentage area of PGP 9.5-labelled tissue that also contained Na(v)1.7. RESULTS: The results demonstrated that sodium channel Na(v)1.7 was expressed in human lingual nerve neuromas. There was no direct relationship between the level of expression of Na(v)1.7 and the patients' symptoms of dysaesthesia. However, in NICs there was found to be an inverse correlation between Na(v)1.7 and macrophage expression, and in symptomatic NICs a direct correlation was found between Na(v)1.7 expression and axonal apposition. CONCLUSIONS: These data suggest that Na(v)1.7 expression alone does not play a primary role in initiating the painful symptoms of dysaesthesia. The development of neuropathic pain may involve complex interactions including changes in ultrastructure and ion channel density.

Changes in vanilloid receptor 1 (TRPV1) expression following lingual nerve injury.

We have investigated a possible role for vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was injected into the damaged nerve to identify associated cell bodies in the trigeminal ganglion. Three further ferrets, receiving only tracer injection, served as uninjured controls. Indirect immunofluorescence for TRPV1 and image analysis was used to quantify the percentage area of staining (PAS) of TRPV1 in the left and right lingual nerves. Additionally, the proportion of fluorogold positive and fluorogold negative cells expressing TRPV1 in the ganglion was determined. TRPV1 expression increased significantly at the injury site of damaged nerves 3 days after injury and this was matched by a reduction in the proportion of fluorogold positive cells expressing TRPV1 in the ganglion. At 3 weeks TRPV1 expression at the injury site was still high, while in the ganglion was significantly greater than in the controls. In the 3-month recovery group TRPV1 expression in both nerve fibres and ganglion cells, was not significantly different from controls and there were no changes in expression in the fluorogold negative cells in the ganglion at any time point studied. These data suggest that after injury there is an increase in the axonal transport of TRPV1 from the cell bodies to the damaged axons and this is followed by an increase in synthesis in the ganglion. These changes in expression may be involved in development of sensory disturbances or dysaesthesia after injury.

Vanilloid receptor 1 (TRPV1) expression in lingual nerve neuromas from patients with or without symptoms of burning pain.

The lingual nerve, a peripheral branch of the trigeminal nerve, can be damaged during the surgical removal of lower third molar teeth. This damage can lead to the development of dysaesthesia, with some patients complaining of burning pain. We investigated the hypothesis that vanilloid receptor 1 (TRPV1), a transducer of noxious heat stimuli, was involved in the development of this burning pain. Neuroma specimens were obtained from patients undergoing microsurgical repair of a damaged lingual nerve. Repair was undertaken where there was little evidence of spontaneous recovery, 7-41 months after the initial injury. Preoperatively the incidence of dysaesthesia was determined by reported symptoms and using visual analogue scales (VAS) for pain, tingling and discomfort. Nine neuromas were studied from patients with burning dysaesthesia and six from patients with a sensory deficit but no dysaesthesia. Indirect immunofluorescence for protein gene product (PGP) 9.5 and TRPV1 was used to quantify the percentage area of PGP 9.5 positive neuronal tissue that also expressed TRPV1. The results showed no significant difference between the mean percentage area of TRPV1 expression in neuromas from patients with or without burning dysaesthesia. Furthermore, there was no correlation between TRPV1 expression and the VAS scores for pain, tingling or discomfort. However, if data from all patients was pooled, there was a negative correlation between the level of TRPV1 expression and the time after initial injury. These data do not rule out involvement of TRPV1 in the aetiology of burning dysaesthesia following lingual nerve injury but suggest that TRPV1 at the injury site does not play a primary role.

Neuropeptide expression following ligation of the ferret lingual nerve.

Previous studies on the ferret inferior alveolar nerve found a close association between the spontaneous neural activity generated at a site of nerve injury, and the accumulation of neuropeptides in the injured axons. More recent electrophysiological studies on the lingual nerve revealed high levels of spontaneous activity 3 days after injury, a decline at 3 weeks and a late rise at 3 months. In the present study we have used immunocytochemical techniques to see whether this time course of spontaneous activity is again paralleled by an accumulation of neuropeptides. In 20 anaesthetised adult ferrets the left lingual nerve was ligated and sectioned distally, and the animals left to recover for 3 days, 3 weeks or 3 months. The tissue was processed using indirect immunofluorescence and image analysis was used to quantify levels of the neuropeptides; calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), enkephalin (ENK) and neuropeptide Y (NPY). Immunoreactivity to all of the neuropeptides was present proximal to the ligature 3 days after injury, and these high levels of expression had decreased considerably by 3 weeks. By 3 months ENK and NPY expression had almost disappeared proximal to the ligature, but levels of CGRP, SP, VIP and GAL had increased slightly. This was also accompanied by an accumulation of all of the neuropeptides, except NPY, in the portion of nerve immediately distal to the ligature. This late accumulation of certain neuropeptides coincides with the increase in spontaneous activity seen in our previous electrophysiological studies and supports the suggestion that neuropeptides may play a role in the aetiology of sensory disorders after nerve injury.

Trigeminal nerve responses in the rat elicited by chemical stimulation of the tongue.

Epithelial and neural mechanisms underlying trigeminal chemoreception were investigated by recording lingual nerve responses to chemical stimulation of the tongue. The chloride salts, NaCl, KCl, NH4Cl, and CaCl2, each elicited distinctly different, integrated whole-nerve responses (thresholds, 0.5-2.0 M). Incubation of the tongue with lanthanum (2.5 mM), which reduces the permeability of epithelial tight junctions, reversibly attenuated these salts responses. It did not affect neural responses to mechanical and thermal stimuli. Incubation with other established transport inhibitors--amiloride, tetra-ethyl ammonium or tetrodotoxin, had no effect on the salt responses. Acetic and hydrochloric acids (thresholds, 0.1-1.0 M), and MgCl2 and BaCl2 (greater than or equal to 0.5 M), also elicited distinctive responses. Other salts, MgSO4, Na isethionate and LaCl3 (greater than or equal to 1.0 M), and also ethanol (4 M), capsaicin (100 mM), nicotine (29.6 mM) and dextrose (0.5-2.5 M), did not elicit responses. These results indicate that ions of selected salts can diffuse through the tight junctions of the lingual epithelium to activate the trigeminal nerve, and suggest that both the cation and the anion may be important in determining if the nerves are activated and the wave-form of the responses.

Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat: distribution, possible origins and functional implications.

The distribution of nerves containing vasoactive intestinal polypeptide(VIP)-immunoreactive material was examined in the cephalic arteries and cranial nerves of cats using an indirect immunofluorescence procedure on whole mounts. Perivascular VIP-immunoreactive nerves were widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-immunoreactive nerves were most abundant in the circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brainstem and ventral areas of the cerebral cortex. Nerves containing VIP-immunoreactive material were absent from distal portions of arteries supplying the posterior brainstem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-immunoreactive nerves had extracerebral origins probably from VIP-immunoreactive perikarya within microganglia in the cavernous plexus and external rete. Extracerebral perivascular VIP-immunoreactive nerves probably arose from VIP-immunoreactive perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. Therefore, it seems likely that each major segment of the cephalic circulation is supplied by local VIP-immunoreactive neurons. If the VIP-immunoreactive nerves cause vasodilation, they are well placed to allow redistribution of arterial blood flow within the head. During heat stress, neurogenic vasodilation of the appropriate beds would permit efficient cooling of cerebral blood, particularly that supplying the rostral brainstem and surrounding areas of the cerebral cortex.