The Role of miRNAs in Neuropathic Pain.

Neuropathic pain is a debilitating condition affecting around 8% of the adult population in the UK. The pathophysiology is complex and involves a wide range of processes, including alteration of neuronal excitability and synaptic transmission, dysregulated intracellular signalling and activation of pro-inflammatory immune and glial cells. In the past 15 years, multiple miRNAs-small non-coding RNA-have emerged as regulators of neuropathic pain development. They act by binding to target mRNAs and preventing the translation into proteins. Due to their short sequence (around 22 nucleotides in length), they can have hundreds of targets and regulate several pathways. Several studies on animal models have highlighted numerous miRNAs that play a role in neuropathic pain development at various stages of the nociceptive pathways, including neuronal excitability, synaptic transmission, intracellular signalling and communication with non-neuronal cells. Studies on animal models do not always translate in the clinic; fewer studies on miRNAs have been performed involving human subjects with neuropathic pain, with differing results depending on the specific aetiology underlying neuropathic pain. Further studies using human tissue and liquid samples (serum, plasma, saliva) will help highlight miRNAs that are relevant to neuropathic pain diagnosis or treatment, as biomarkers or potential drug targets.

Neural Crest-Derived Stem Cells (NCSCs) Obtained from Dental-Related Stem Cells (DRSCs): A Literature Review on Current Knowledge and Directions toward Translational Applications.

Evidence from dental-related stem cells (DRSCs) suggests an enhanced potential for ectodermal lineage differentiation due to their neural crest origin. Growing evidence that DRSC cultures can produce cells with a neural crest-derived stem cell (NCSC)-like phenotype supports their potential for future therapeutic approaches for neurodegenerative diseases and nerve injuries. However, most of the evidence is limited to the characterization of DRSCs as NCSCs by detecting the expression of neural crest markers. Only a few studies have provided proof of concept of an improved neuro-glial differentiation or direct applicability in relevant models. In addition, a current problem is that several of the existing protocols do not meet manufacturing standards for transferability to a clinical scenario. This review describes the current protocols to obtain NCSCs from DRSCs and their characterization. Also, it provides important considerations from previous work where DRSCs were established and characterized as mesenchymal stromal cells but studied for their neuro-glial differentiation potential. The therapeutic advancement of DRSCs would depend on establishing protocols that can yield a neural crest-like phenotype efficiently, using appropriate manufacturing standards and testing them in relevant models of disease or injury. Achieving these conditions could then facilitate and validate the therapeutic potential of DRSC-NCSCs in regenerative therapies.

A Tuneable, Photocurable, Poly(Caprolactone)-Based Resin for Tissue Engineering-Synthesis, Characterisation and Use in Stereolithography.

Stereolithography is a useful additive manufacturing technique for the production of scaffolds for tissue engineering. Here we present a tuneable, easy-to-manufacture, photocurable resin for use in stereolithography, based on the widely used biomaterial, poly(caprolactone) (PCL). PCL triol was methacrylated to varying degrees and mixed with photoinitiator to produce a photocurable prepolymer resin, which cured under UV light to produce a cytocompatible material. This study demonstrates that poly(caprolactone) methacrylate (PCLMA) can be produced with a range of mechanical properties and degradation rates. By increasing the degree of methacrylation (DM) of the prepolymer, the Young's modulus of the crosslinked PCLMA could be varied from 0.12-3.51 MPa. The accelerated degradation rate was also reduced from complete degradation in 17 days to non-significant degradation in 21 days. The additive manufacturing capabilities of the resin were demonstrated by the production of a variety of different 3D structures using micro-stereolithography. Here, ß-carotene was used as a novel, cytocompatible photoabsorber and enabled the production of complex geometries by giving control over cure depth. The PCLMA presented here offers an attractive, tuneable biomaterial for the production of tissue engineering scaffolds for a wide range of applications.

Chemokines and Pain in the Trigeminal System.

Chemotactic cytokines or chemokines are a large family of secreted proteins able to induce chemotaxis. Chemokines are categorized according to their primary amino acid sequence, and in particular their cysteine residues that form disulphide bonds to maintain the structure: CC, CXC, CX3C, and XC, in which X represents variable amino acids. Among their many roles, chemokines are known to be key players in pain modulation in the peripheral and central nervous systems. Thus, they are promising candidates for novel therapeutics that could replace current, often ineffective treatments. The spinal and trigeminal systems are intrinsically different beyond their anatomical location, and it has been suggested that there are also differences in their sensory mechanisms. Hence, understanding the different mechanisms involved in pain modulation for each system could aid in developing appropriate pharmacological alternatives. Here, we aim to describe the current landscape of chemokines that have been studied specifically with regard to trigeminal pain. Searching PubMed and Google Scholar, we identified 30 reports describing chemokines in animal models of trigeminal pain, and 15 reports describing chemokines involved in human pain associated with the trigeminal system. This review highlights the chemokines studied to date at different levels of the trigeminal system, their cellular localization and, where available, their role in a variety of animal pain models.

Establishment and neural differentiation of neural crest-derived stem cells from human dental pulp in serum-free conditions.

The potential of obtaining cell cultures with neural crest resemblance (neural crest-derived stem cells [NCSCs]) from dental-related tissues, including human dental pulp cells (hDPCs), has been discussed in the literature. However, most reports include the use of serum-rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum-free, and also under sphere-aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest-related markers. Culturing hDPCs as neurospheres (hDPC-NCSCs) resulted in an increased expression of neural crest-related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural-like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1, and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum-free medium, as NCSC that differentiate into neural-like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs.

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits.

Entubulating devices to repair peripheral nerve injuries are limited in their effectiveness particularly for critical gap injuries. Current clinically used nerve guidance conduits are often simple tubes, far stiffer than that of the native tissue. This study assesses the use of poly(glycerol sebacate methacrylate) (PGSm), a photocurable formulation of the soft biodegradable material, PGS, for peripheral nerve repair. The material was synthesized, the degradation rate and mechanical properties of material were assessed and nerve guidance conduits were structured via stereolithography. In vitro cell studies confirmed PGSm as a supporting substrate for both neuronal and glial cell growth. Ex vivo studies highlight the ability of the cells from a dissociated dorsal root ganglion to grow out and align along the internal topographical grooves of printed nerve guide conduits. In vivo results in a mouse common fibular nerve injury model show regeneration of axons through the PGSm conduit into the distal stump after 21 days. After conduit repair levels of spinal cord glial activation (an indicator for neuropathic pain development) were equivalent to those seen following graft repair. In conclusion, results indicate that PGSm can be structured via additive manufacturing into functional NGCs. This study opens the route of personalized conduit manufacture for nerve injury repair. STATEMENT OF SIGNIFICANCE: This study describes the use of photocurable of Poly(Glycerol Sebacate) (PGS) for light-based additive manufacturing of Nerve Guidance Conduits (NGCs). PGS is a promising flexible biomaterial for soft tissue engineering, and in particular for nerve repair. Its mechanical properties and degradation rate are within the desirable range for use in neuronal applications. The nerve regeneration supported by the PGS NGCs is similar to an autologous nerve transplant, the current gold standard. A second assessment of regeneration is the activation of glial cells within the spinal cord of the tested animals which reveals no significant increase in neuropathic pain by using the NGCs. This study highlights the successful use of a biodegradable additive manufactured NGC for peripheral nerve repair.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability.

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

Nerve guides manufactured from photocurable polymers to aid peripheral nerve repair.

The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (µSL) setup that incorporated a 405 nm laser source to produce 3D constructs with ∼ 50 µm resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter × 5 mm length with a wall thickness of 250 µm were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.

Temporal mismatch between pain behaviour, skin Nerve Growth factor and intra-epidermal nerve fibre density in trigeminal neuropathic pain.

BACKGROUND: The neurotrophin Nerve Growth factor (NGF) is known to influence the phenotype of mature nociceptors, for example by altering synthesis of neuropeptides, and changes in NGF levels have been implicated in the pathophysiology of chronic pain conditions such as neuropathic pain. We have tested the hypothesis that after partial nerve injury, NGF accumulates within the skin and causes 'pro-nociceptive' phenotypic changes in the remaining population of sensory nerve fibres, which could underpin the development of neuropathic pain. RESULTS: Eleven days after chronic constriction injury of the rat mental nerve the intra-epidermal nerve fibre density of the chin skin from had reduced from 11.6 ± 4.9 fibres/mm to 1.0 ± 0.4 fibres/mm; this slowly recovered to 2.4 ± 2.0 fibres/mm on day 14 and 4.0 ± 0.8 fibres/mm on day 21. Cold hyperalgesia in the ipsilateral lower lip was detectable 11 days after chronic constriction injury, although at this time skin [NGF] did not differ between sides. At 14 days post-injury, there was a significantly greater [NGF] ipsilaterally compared to contralaterally (ipsilateral = 111 ± 23 pg/mg, contralateral = 69 ± 13 pg/mg), but there was no behavioural evidence of neuropathic pain at this time-point. By 21 days post-injury, skin [NGF] was elevated bilaterally and there was a significant increase in the proportion of TrkA-positive (the high-affinity NGF receptor) intra-epidermal nerve fibres that were immunolabelled for the neuropeptide Calcitonin Gene-related peptide. CONCLUSIONS: The temporal mismatch in behaviour, skin [NGF] and phenotypic changes in sensory nerve fibres indicate that increased [NGF] does not cause hyperalgesia after partial mental nerve injury, although it may contribute to the altered neurochemistry of cutaneous nerve fibres.

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.

The effect of a monoclonal antibody to calcitonin-gene related peptide (CGRP) on injury-induced ectopic discharge following lingual nerve injury.

The development of ectopic neural discharge at a site of peripheral nerve injury is thought to contribute to the initiation of sensory disturbances and pain. We have previously shown that this discharge can be initiated or increased by the neuropeptide calcitonin gene-related peptide (CGRP). We have now studied a potential therapeutic approach to reducing the discharge by evaluating the effect of a systemically administered monoclonal antibody to CGRP on injury-induced activity in the lingual nerve. In 16 anaesthetised adult ferrets the left lingual nerve was sectioned. One day after the injury, the animals received a subcutaneous injection of either a monoclonal antibody to CGRP or a vehicle control. Three days after the injury, under a second anaesthetic, single-unit electrophysiological recordings were made from central to the injury site (469 and 391 units were analysed in antibody and vehicle groups, respectively), and the proportion of units that were spontaneously active was determined. In the vehicle-treated animals 6.4±2.7 [SEM]% of the units were spontaneously active, with conduction velocities of 8.8-40.8m/s and discharge frequencies of 0.03-2.7Hz. In the monoclonal antibody-treated animals 5.7±2.0% of the units were spontaneously active, with conduction velocities of 13.9-38.8m/s and discharge frequencies of 0.07-1.8Hz. There was no significant difference between these two groups (for spontaneous activity and conduction velocity: p>0.05, Student's t-test; for discharge frequency: p>0.05, Mann-Whitney test), suggesting that the spontaneous activity initiated by a nerve injury cannot be modulated by administration of a monoclonal antibody to CGRP.

Histomorphometric changes in repaired mouse sciatic nerves are unaffected by the application of a scar-reducing agent.

Microsurgical repair of transected peripheral nerves is compromised by the formation of scar tissue and the development of a neuroma, thereby limiting the success of regeneration. The aim of this study was to quantify histomorphometrically the structural changes in neural tissue that result from repair, and determine the effect of mannose-6-phosphate (M6P), a scar-reducing agent previously shown to enhance regeneration. In anaesthetised C57-black-6 mice, the left sciatic nerve was sectioned and repaired using four epineurial sutures. Either 100 µL of 600 mm M6P (five animals) or 100 µL of phosphate-buffered saline (placebo controls, five animals) was injected into and around the nerve repair site. A further group acted as sham-operated controls. After recovery for 6 weeks, the nerve was harvested for analysis using light and electron microscopy. Analysis revealed that when compared with sham controls, myelinated axons had smaller diameters both proximal and distal to the repair. Myelinated axon counts, axonal density and size all decreased across the repair site. There were normal numbers and densities of non-myelinated axons both proximal and distal to the repair. However, there were more Remak bundles distal to the repair site, and fewer non-myelinated axons per Remak bundle. Application of M6P did not affect any of these parameters.

The effect of Mannose-6-Phosphate on recovery after sciatic nerve repair.

We have determined the effect of applying Mannose-6-Phosphate (M6P), a scar reducing agent, to a site of sciatic nerve repair. In anaesthetised C57-Black-6 mice, the left sciatic nerve was sectioned and repaired using 4 epineurial sutures. Either 100 µl of 600 mM Mannose-6-Phosphate (29 animals), or 100 µl of phosphate buffered saline as a placebo control (29 animals), was injected into and around the nerve repair site. A further group acted as sham-operated controls. After 6 or 12 weeks of recovery the extent of regeneration was assessed electrophysiologically and the percentage area of collagen staining at the repair site was analysed using picrosirius red and image analysis. Gait analysis was undertaken pre-operatively and at 1, 3, 6, 9 and 12 weeks postoperatively, to assess functional recovery. At 6 weeks the compound action potentials recorded from the regenerated nerves in the M6P group were significantly larger than in the placebo controls (P=0.015), and the conduction velocities were significantly faster (P=0.005), but there were no significant differences between these groups at 12 weeks. Gait analysis suggested better early functional recovery in the M6P group. In both repair groups there was a significant reduction in collagen staining between 6 and 12 weeks, suggestive of scar remodelling. We conclude that the normal scar remodelling process aids long term recovery in repaired nerves. Administration of 600 mM M6P to the nerve repair site enhances nerve regeneration and functional recovery in the early stages, and may lead to improved outcomes.

Fos expression induced by activation of NMDA and neurokinin-1 receptors in the trigeminal subnucleus caudalis in vitro: role of protein kinases.

Activity-induced neuronal plasticity is partly facilitated by the expression of the immediate-early gene c-fos and the resulting transcription factor Fos. Expression of Fos is associated with nociceptive afferent activation, but a detailed stimulation-transcription pathway for Fos expression has not yet been determined in the trigeminal system. This study utilized a novel in vitro model to determine whether Fos expression can be induced in trigeminal subnucleus caudalis by NMDA or neurokinin-1 receptor activation, and whether inhibition of intracellular kinases has any effect on Fos expression induced by activation of these receptors. Brainstems of male Wistar rats were excised and maintained in artificial cerebrospinal fluid at 37°C. NMDA or the specific neurokinin-1 receptor agonist [Sar(9),Met(O(2))(11)]-SP was applied. These agonists were subsequently tested in the presence of the protein kinase A inhibitor Rp-cAMP or protein kinase C inhibitor chelerythrine chloride. In all experiments the sodium channel blocker tetrodotoxin was used to prevent indirect neuronal activation. Brainstems were processed immunocytochemically for Fos expression, and positive cells were counted in the trigeminal subnucleus caudalis. NMDA and [Sar(9),Met(O(2))(11)]-SP significantly increased Fos expression, but these increases could be prevented by chelerythrine chloride. Rp-cAMP had no effect on Fos induced by NMDA but caused a significant reduction in Fos induced by [Sar(9),Met(O(2))(11)]-SP. These data demonstrate that in trigeminal subnucleus caudalis activation of either NK1 or NMDA receptors alone induces Fos expression; protein kinases A and C are involved in NK1R-induced Fos while protein kinase A is not required for NMDA receptor-induced Fos.

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.

Changes in proteinase-activated receptor 2 expression in the human tooth pulp in relation to caries and pain.

AIMS: To investigate the presence of proteinase-activated receptor 2 (PAR2) in the human tooth pulp and to determine whether there are any changes in receptor expression with caries and pain. METHODS: Forty-four mandibular first permanent molars were collected from children (n = 36, mean age 9.96 years +/- 2.11) requiring dental extractions under general anesthesia. Teeth were categorized as either intact (n = 22) or carious (n = 22). Carious teeth were further subdivided into asymptomatic (n = 10) and painful (n = 12). The coronal pulp was removed and processed for indirect immunofluorescence by using antibodies raised against PAR2 and double labeled with either a neuronal marker (protein gene product 9.5) or both a smooth muscle cell (aSMA) and endothelial (UEIL) marker, in order to examine PAR2 presence in both neuronal and vascular tissue. In addition, hemotoxylin and eosin staining was performed to identify pulpal fibroblasts. RESULTS: PAR2 expression was found to be present in pulpal nerve fibers, vascular tissue, and pulpal fibroblasts. PAR2 neuronal expression was not affected by the presence of caries (P > .05) but was significantly less in carious painful teeth than in carious asymptomatic teeth (P < .05). No changes in vascular PAR2 expression were found (P > .05); however, the number of PAR2-labeled fibroblast-like cells per mm2 was significantly greater in carious teeth (P < .05). CONCLUSION: These findings indicate that PAR2 receptors and changes in their level of expression may have relevance and clinical importance in nociception.

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.

Effect of SB-750364, a specific TRPV1 receptor antagonist, on injury-induced ectopic discharge in the lingual nerve.

Abnormal neural activity generated at a site of nerve injury is thought to contribute to the development of dysaesthesia. Vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, may be involved in the initiation of this abnormal activity and could provide a useful therapeutic target. We investigated the effect of a specific TRPV1 antagonist (SB-750364) on injury-induced discharge in the lingual nerve. In 12 anaesthetised adult ferrets the left lingual nerve was sectioned and animals were allowed to recover for 3-7 days. In terminal experiments under general anaesthesia, the nerve was re-exposed and electrophysiological recordings made from spontaneously active axons in fine filaments dissected from the nerve central to both the injury site and the junction with the chorda tympani. SB-750364 was infused via the cephalic vein in order to achieve three increasing but stable systemic blood levels of the compound (0.3, 1.0 and 3.0 microM). Twenty-eight spontaneously active units were studied, with discharge frequencies ranging from 0.02 to 4.9 Hz. There was a significant reduction in spontaneous activity in 17 units (61%) at 1.0 microM or less of SB-750364 (p<0.01; Friedman test with Dunn's multiple comparisons). A further 4 units (14%) showed a significant reduction in activity at 3.0 microM (p<0.01). In the remaining 7 units (25%) the discharge was unaffected (p>0.05). These data show that the TRPV1 antagonist SB-750364 can reduce the level of spontaneous activity initiated in some axons following lingual nerve injury.