[Show abstract][Hide abstract] ABSTRACT: The formation of scar tissue following nerve injury has been shown to adversely affect nerve regeneration and evidence suggests that mannose-6-phosphate (M6P), a potential scar reducing agent that affects transforming growth factor (TGF)-β activation, may enhance nerve regeneration. In this study we utilized thy-1-YFP-H mice – a transgenic strain expressing yellow fluorescent protein (YFP) within a subset of axons – to enable visual analysis of axons regenerating through a nerve graft. Using this strain of mouse we have developed analysis techniques to visualize and quantify regeneration of individual axons across the injury site following the application of either M6P or vehicle to the site of nerve injury.
No significant differences were found in the proportion of axons regenerating through the graft between M6P- and vehicle-treated grafts at any point along the graft length. Maximal sprouting occurred at 1.0 mm from the proximal graft ending in both groups. The maximum change in sprouting levels for both treatment groups occurred between the graft start and 0.5-mm interval for both treatment groups. The difference between repair groups was significant at this point with a greater increase seen in the vehicle group than the M6P group. The average length of axons regenerating across the initial graft entry was significantly shorter in M6P- than in vehicle-treated grafts, indicating that they encountered less impedance.
Application of M6P appears to reduce the disruption of regenerating axons and may therefore facilitate quicker recovery; this is likely to result from altered scar tissue formation in M6P grafts in the early stages of recovery. This study also establishes the usefulness of our methods of analysis using the thy-1-YFP-H mouse strain to visualize and quantify regeneration at the level of the individual axon.
[Show abstract][Hide abstract] ABSTRACT: Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc.
[Show abstract][Hide abstract] ABSTRACT: 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.
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 post-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.
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.
[Show abstract][Hide abstract] ABSTRACT: Background
Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc.
Chronic inflammation alone induced bilateral expression of pERK and pp38 in Vc, but did not induce Fos expression. Stimulation of both non-inflamed and inflamed pulps significantly increased pERK and pp38 bilaterally; expression was greatest in inflamed, stimulated animals, and was similar following 10-min and 60-min stimulation. Stimulation for 60 min, but not 10 min, induced Fos in ipsilateral Vc; Fos expression was significantly greater in inflamed, stimulated animals. pERK was present in both neurons and astrocytes; pp38 was present in neurons and other non-neuronal, non-astrocytic cell types.
This study provides the first demonstration that chronic inflammation of tooth pulp induces persistent bilateral activation of ERK and p38 within Vc, and that this activation is further increased by acute stimulation. This altered activity in intracellular signaling is likely to be linked to the sensitization that is seen in our animal model and in patients with pulpitis. Our data indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In our model, localization of pERK and pp38 within specific cell types differs from that seen following acute stimulation. This may indicate specific roles for different cell types in the induction and maintenance of pulpitic and other types of pain.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Objective: To examine relationships between expression of neuropeptides in the human dental pulp and caries status, pain history, residual dentine thickness (RDT) and response to thermal testing, including latency, temperature and intensity of sensation.
Method: Teeth were collected from children undergoing routine exodontia under general anaesthetic, and were assessed for caries status and pain history using a visual analogue scale (VAS). The teeth underwent quantitative thermal testing using a newly devised protocol, which included recording of latency and temperature at response and intensity of the sensation using a VAS. Following extraction teeth were split, placed in Zamboni’s fixative and RDT measured. Pulps were then removed, frozen, cryosectioned and mounted on slides. Immunofluoresence was used to visualise neuronal tissue (using PGP9.5), substance P (SP) and calcitonin gene related peptide (CGRP). Correlations were sought between expression of the neuropeptides and the above clinical parameters. Results were analysed using mixed model and Pearson’s correlation, with statistical significance set at 0.05.
Result: Expression of SP and CGRP was greater in carious painful teeth compared to carious non-painful and non-carious teeth. There was a negative correlation between expression of SP and CGRP and the RDT. Expression of CGRP was greater in those teeth associated with a patient response to thermal testing and those teeth associated with a shorter latency period between initiation of testing and a sensation being felt. However, expression of CGRP was not associated with temperature at response, intensity of sensation or VAS for previous pain. Expression of SP was not associated with any of the thermal testing variables or VAS for previously reported pain.
Conclusion: Expression of the neuropeptides SP and CGRP is associated with the caries/pain status of the tooth and RDT. Expression of CGRP is associated with response to thermal testing and the latency period before a sensation is felt.
[Show abstract][Hide abstract] ABSTRACT: Objective: Peripheral nerve injuries requiring surgical repair, such as those following lower 3rd molar extraction, rarely regenerate to a satisfactory level.
The thy-1-YFP-H transgenic mouse strain is potentially useful for analysing nerve regeneration in experimental studies. A subset of nerve cells within the mice express yellow fluorescent protein along the entire length of their axons, allowing visual analysis of the regenerating axons to be performed.
Here we report the use of this strain in two studies of nerve regeneration; the first assessed the effects of a potential anti-scarring agent, mannose-6-phosphate, and the second assessed the ability of hollow poly(ethylene-glycol) nerve guide conduits (NGCs) to support nerve regeneration.
Method: Under anaesthesia the common fibular nerve in thy-1-YFP-H mice was exposed, sectioned and a gap created. In the mannose-6-phosphate study, this was repaired using a graft from a wild-type littermate soaked for 30mins in mannose-6-phosphate or vehicle. In the NGC study nerves were repaired using a NGC or wild-type graft. After recovery (2-weeks for mannose-6-phosphate study and 3-weeks for NGC study) mice were re-anaesthetised and the nerve fixed in situ with 4% formaldehyde. The repaired nerve was excised, mounted whole on a slide and coverslipped.
Result: In the mannose-6-phosphate study no significant differences were found between treated and untreated repairs in terms of overall and unique regenerating axon numbers. However, axonal disruption was significantly less in mannose-6-phosphate treated animals (p<0.05).
In the NGC study no significant quantitative differences were found between NGC and graft repairs, however, axons in NGC repairs displayed less organisation.
Conclusion: The visual analysis made possible by using the thy-1-YFP-H mouse strain has provided us with useful insights into axonal disruption and organisation at sites of nerve repair and regeneration. These insights would not have been possible using traditional analysis methods.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
The voltage-gated sodium channel Nav1.8, expressed predominantly in small- and medium-sized neurons, has been shown to increase following inflammation and may play a pivotal role in the development of chronic pain. The aim of the present study was to examine the expression of sodium channel subtype Nav1.8 in non-carious, carious non-painful and carious painful human permanent teeth, and identify any association between its expression, clinical symptoms and response to thermal stimulus.
Mandibular first permanent molar teeth were extracted from children under general anaesthesia, and categorised into three groups according to the degree of caries and reported symptoms of pain: non-carious (n=11), carious non-painful (n=11) and carious painful (n=11). The teeth were prepared and processed for indirect immunofluorescence using primary antibodies raised against Nav1.8 and PGP 9.5, a general neuronal marker. Antibody binding was visualized using fluorescent microscopy and image analysis employed to quantify the percentage area of PGP 9.5 immunoreactive tissue also labelled positively for Nav1.8 in three regions of the tooth pulp: the pulp horn, the subodontoblastic plexus and the mid-coronal region.
Immunoreactivity for Nav1.8 was present within the three sampled regions of the tooth pulps, in all three groups analysed, and dual labelling immunohistochemistry revealed Nav1.8 to be co-localised with PGP 9.5 labelled nerve fibres. Quantitative image analysis revealed that levels of Nav1.8 were significantly higher (ANOVA, p=0.013) in carious painful tooth pulps compared to carious non-painful specimens, in the pulp horn region. Furthermore in the patients reporting experiences of pain, there was a significant negative correlation between the level of expression of Nav1.8 and the Visual Analogue Score (VAS) recorded (p<0.005).
These data provide further evidence that changes in expression of Nav1.8 are important in the development of inflammatory-induced pain and may be a potential target for novel therapeutics for chronic pain.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
Inflammation at a site of nerve injury and/or repair can hinder axonal regeneration, leading to the development of a neuroma and the development of neuropathic pain. Recently it has become clear that activated spinal cord glia play a major role in the development of neuropathic pain, and it has been established that inhibition of glial activation by intrathecal administration of anti-inflammatory cytokines, can reduce or reverse neuropathic pain. We have investigated the hypothesis that application of a Tumour Necrosis Factor-α (TNF-α) inhibitor to a nerve repair site would reduce inflammation peripherally and reduce activation of microglia and astrocytes centrally.
In 12 anaesthetized Sprague-Dawley rats the left sciatic nerve was transected and microsurgical repair undertaken using four epineural sutures. In 2 groups either 100µl of phosphate buffered saline (PBS) or 100µl TNF-α inhibitor were injected into the nerve repair site, and a third group acted as controls. After a 7 day recovery period, injured nerves and spinal cords were harvested. Immunohistochemistry and image analysis were used to quantity macrophages and leucocytes peripherally, and astrocytes and microglia within the central nervous system.
Astrocytic and microglial activation increased significantly following repair in the PBS group (p=0.018, p=0.048, Kruskal-Wallis test), but not in the TNF-α inhibitor group. Quantification of macrophage and leucocyte presence at the repair site showed a similar pattern.
These results show that TNF-α inhibition reduces inflammation at a site of peripheral nerve repair, and that peripheral application results in a reduction of neuropathic pain markers in the central nervous system. This suggests that TNF-α inhibition may have the potential to reduce neuropathic pain following peripheral nerve injury and/or repair.
Academy of Medical Sciences/Wellcome Trust
[Show abstract][Hide abstract] ABSTRACT: Background:
Calcitonin gene-related peptide (CGRP) is a powerful pro-inflammatory mediator thought to play a significant role in the development of inflammation and pain. We investigated the role of CGRP in trigeminal inflammatory pain by determining the ability of a monoclonal antibody to CGRP to modify central Fos expression in response to stimulation of the inflamed ferret tooth pulp. We also assessed the effect of the antibody on pulpal inflammation.
Ten adult ferrets were prepared under anaesthesia to allow stimulation of the upper and lower left canine pulps, recording from the digastric muscle and intravenous injections at subsequent experiments. In all animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. Four days later animals were treated intravenously with either CGRP antibody (n=5) or vehicle (n=5). After a further 2 days animals were re-anaesthetised and the tooth pulps stimulated at 10 times jaw-opening reflex threshold. Brainstems and tooth pulps were processed immunohistochemically for Fos and the common leucocyte marker CD45, respectively.
Fos was expressed in ipsilateral trigeminal subnuclei caudalis (Vc) and oralis (Vo). Significantly fewer Fos-positive nuclei were present within Vc of CGRP antibody-treated animals (p=0.003 vs vehicle-treated). Mean percentage area of staining for CD45 was significantly less in antibody-treated animals (p=0.04 vs vehicle-treated).
This is the first direct evidence that sequestration of CGRP has anti-inflammatory and putative analgesic effects. Previous studies using this Fos model have demonstrated that it is able to predict clinical analgesic efficacy. Thus these data indicate that this antibody may have analgesic effects in dental pain and other types of inflammatory-mediated transmission, and suggest that this is in part due to peripheral anti-inflammatory effects.
[Show abstract][Hide abstract] ABSTRACT: Currently there is a great interest in the development of nerve guide conduits to improve regeneration of injured peripheral nerves. In this study we tested simple polyethylene-glycol (PEG) conduit repair against graft repair in thy-1-YFP-H mice; a transgenic strain with a subset of axons labelled with yellow fluorescent protein.
Under anaesthesia the common fibular nerve in 10 mice was exposed and a 3mm section removed. The gap was repaired with PEG conduit or nerve graft taken from a wild-type littermate. After 3-weeks recovery mice were re-anaesthetised and the nerve fixed in situ with 4% formaldehyde. The repaired nerve was excised mounted whole on a slide and coverslipped using Vectashield. Images of nerves were acquired using Image ProPlus.
No significant differences were detected between the two groups in the three methods of analysis used. Both groups saw axon numbers of ~150% compared to the pre-repair axon numbers at 0.5mm into the repair, this number proceeded to fall at each subsequent interval to ~62% at the distal stump 4.0mm into the repair. The proportion of axons at the repair start represented at the distal stump was 34% in conduit repairs and 37% in. Axons increased in length by 16% in conduits and 22% grafts over the initial 1.5mm of the repair.
In conclusion, PEG conduits are able to support nerve regeneration across a 3mm gap at similar levels to nerve grafts. These results will help to assess the impact of adding internal structure and/or exogenous regeneration enhancing molecules in future conduit studies.
[Show abstract][Hide abstract] ABSTRACT: Aim:
The formation of scar tissue following nerve injury has been shown to adversely affect nerve regeneration. The aim of this study was to utilise the fluorescent labelling of axons in thy-1-YFP-H mice to analyse the effects of mannose-6-phosphate (M6P), a potential scar reducing agent that affects TGF-β activation, on nerve regeneration following injury.
Under anaesthesia (isoflurane 4%), the common fibular (CF) nerve of twenty 9–11 week-old thy-1-YFP-H mice was exposed and a small section of nerve removed to create a gap (2.5 mm) between the proximal and distal ends. A graft taken from a wild-type (WT) littermate, pre-soaked for 30 minutes in a solution of M6P (n=10) or vehicle (PBS; n=10), was positioned in this gap and fixed in place using fibrin glue.
After 2 weeks' recovery, the animals were re-anaesthetised (fluanisone 0.8 ml/kg and midazolam 4 mg/kg, ip) and the nerve fixed in situ (4% paraformaldehyde, 30 min). The nerve and graft were removed, positioned on a microscope slide and coverslipped using Vectashield.
Images were taken from the nerve using fluorescent structured illumination microscopy and reconstructed using Adobe Photoshop. These were used to calculate the sprouting index at 0.5-mm intervals along the nerve, to assess the proportion of axons at the start of the graft that had successfully regenerated through the graft, and the lengths of axons regenerating through the graft.
No significant differences in axon sprouting were found between M6P and vehicle grafts at any point along the graft length, with maximal sprouting occurring at 1.0mm post-graft in both groups (169.4% ± 19.8% [SEM] for M6P; 177.2% ± 26.9% for vehicle). There were also no significant differences in the proportion of axons regenerating through the graft in either group (23.8% ± 2.9% for M6P; 21.9% ± 3.7% for vehicle). However, the average length of axons regenerating through the graft was significantly different, with M6P treated graft axons reaching 3.5mm from the graft starting point along a shorter path than vehicle treated graft axons (increases of 9.33% ± 0.58% SEM and 12.27% ± 0.60% SEM for M6P and vehicle grafts respectively, p<0.05, Mann-Whitney U test).
Application of M6P appears to reduce the disruption of regenerating axons and may therefore facilitate quicker recovery; this is likely to result from reduced scar tissue formation in M6P grafts in the early stages of recovery. However the overall recovery does not appear to be improved as similar proportions of axons in M6P and vehicle grafts were found to have successfully regenerated through the graft.
Supported by Renovo Group plc and the MRC.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2011 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: In this study we have determined the amount of Nerve Growth Factor (NGF) and the innervation density of the glabrous hindpaw skin of diabetic rats (n=4) and controls (n=3). The proportion of intra-epidermal nerve fibres (IENF) expressing the high affinity NGF receptor (trkA) and calcitonin gene-related peptide (CGRP) were also determined. Four weeks after induction of diabetes by intraperitoneal streptozotocin injection skin was analyzed for: (i) NGF content using ELISA and (ii) the innervation density of peptidergic afferents that also expressed trkA using immunocytochemistry. NGF levels were approximately three-fold higher in diabetic skin compared to controls (diabetic: 134.7±24.0 (SD) pgml(-1), control: 42.7±21.5pgml(-1), p=0.002). As expected there was a significant reduction in IENF density in diabetic skin (2.7±1.3 fibresmm(-1)) compared to controls (6.9±1.5 fibresmm(-1); p=0.01). In diabetic rats there was no significant difference in the proportion of trkA-labelled IENF (diabetic 74±21%; control 83±15%, p=0.6), but significantly more trkA-positive IENF were also labelled by CGRP antibodies in diabetic skin compared to controls (diabetic 89±22%; control 38±2%, p=0.03). These data suggest that in diabetes the upregulation of cutaneous NGF may 'over-troph' the surviving axons, increasing CGRP labelling, which may be important in the aetiology of painful diabetic neuropathy.
No preview · Article · Oct 2011 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: Objectives: Nerve injury represents a significant clinical problem, giving rise to substantial disability including chronic pain. Animal models have been developed to investigate and improve nerve regeneration. Many of these employ electrophysiological or behavioural approaches; however, our objective is to develop an in vivo model of peripheral nerve injury and repair that allows visual analysis of axonal regeneration, using genetically modified mice (thy-1-YFP-H [thy-1]) which express yellow fluorescent protein (YFP) in a subset of axons.
Methods: Under anaesthesia (isoflurane 4%), the common fibular (CF) nerve of 9–11 week-old thy-1 mice was exposed and a small section of nerve removed to create a gap (2.5 mm) between the proximal and distal ends. A graft taken from a wild-type (WT) littermate was positioned in this gap and fixed in place using fibrin glue.
After 2 weeks’ recovery, the animals were re-anaesthetised (fluanisone 0.8 ml/kg and midazolam 4 mg/kg, ip) and the nerve fixed in situ (4% paraformaldehyde, 30 min). The nerve and graft were removed, positioned on a microscope slide and coverslipped using Vectashield.
Images were taken from the nerve using fluorescent structured illumination microscopy and reconstructed using Adobe Photoshop. These were used to calculate the sprouting index at 0.5-mm intervals along the nerve and to assess the distance travelled through the graft by each axon.
Results: The CF nerve had a mean pre-graft YFP-axon count of 40 (± 1.46 SEM). The sprouting index was maximal at 1 mm (mean value 2.29 ± 0.09). Individual axons could be traced through the graft and the percentage of axons reaching each 0.5-mm interval could be calculated.
Conclusions: This method allows visual analysis of in vivo axon regeneration through a graft. Future studies will use this technique to assess the effects of molecules that may enhance nerve regeneration.
Funded by MRC
[Show abstract][Hide abstract] ABSTRACT: Objectives: Previous experiments in our laboratory using a rat partial mental nerve injury model have shown that nerve growth factor (NGF) acting on spared nerve fibres may be important in the development of neuropathic pain. In the current study we have investigated whether this mechanism is also important in neuropathic pain caused by partial nerve injury to the sciatic nerve, a non-trigeminal mixed nerve.
Methods: In 4 Sprague Dawley rats, the left sciatic nerve was exposed and constricted with four 4/0 chromic gut sutures. Behavioural testing was undertaken 10, 8, 6, 3 and 1 day pre-operatively and 1, 4, 6, 8, 11, 13 and 14 days post-operatively. The uninjured contralateral side served as a control. The mean latencies of foot withdrawal to cold (acetone) and hot (radiant heat) stimuli were determined (n=5 per session). Fifty percent withdrawal thresholds to graded mechanical stimuli (0.2 26g) were also determined using von Frey hairs. Fourteen days post-injury, glabrous hindpaw skin was harvested under terminal anaesthesia. An ELISA was used to assay the NGF content of skin that was snap frozen in liquid nitrogen.
Results: Following partial nerve injury, there were behavioural changes in paw withdrawal consistent with thermal and mechanical hyperalgesia that occurred within 14 days of the injury. Preliminary results show higher NGF levels in ipsilateral footpads compared to contralateral.
Conclusion: Preliminary data suggest that a constriction injury to the rat sciatic nerve results in thermal and mechanical hypersensitivity and that there are increased levels of NGF in ipsilateral footpad skin compared to contralateral. These results suggest a possible role for NGF in the aetiology of neuropathic pain after a partial nerve injury.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Accidental damage to branches of the trigeminal nerve during lower third molar removal can cause neuropathic pain. We hypothesized that neuropathic pain may arise due to an increased concentration of neurotrophins in the periphery acting on spared afferents. This study compared the concentration of nerve growth factor (NGF) in the oral mucosa of patients with inferior alveolar nerve (IAN) injuries (all of whom had symptoms of neuropathic pain) with that of control specimens from healthy volunteers.
Methods: Mucosal biopsies were taken from 15 patients undergoing repair of a traumatically injured IAN under general anaesthesia. Pre-operatively we had undertaken pain histories and quantitative sensory testing. Control tissue was obtained following an IAN block, from 5 age and sex matched volunteers who had no history of a previous trigeminal nerve injury. In all cases tissue was taken from from the mucosa of the lower lip, midway between the angle of the mouth and the midline. Care was taken to ensure that the biopsies did not contain any underlying muscle. The harvested tissue was snap frozen and stored at -80oC. The tissue was then homogenized and aliquots assayed for NGF by ELISA.
Results: The concentration of NGF was increased in the mucosa of patients with an IAN injury compared to control (injured=1.210.17[SEM]pg ml-1, control=0.540.04 pg ml-1, P=0.03, independent t-test).
Conclusion: These data suggest that IAN injury associated with neuropathic pain is linked with an increased concentration of NGF in the peripheral mucosa. These results support our hypothesis that an increase of NGF in the periphery may have an important role in the development of neuropathic pain.
[Show abstract][Hide abstract] ABSTRACT: Objectives: The objective of this study was to undertake quantitative sensory testing on teeth prior to extraction using a novel thermal testing protocol. The samples will be used to investigate correlations in pulpal expression of the thermosensitive transient receptor potential channels melastatin 8 (TRPM8) and ankyrin 1 (TRPA1) with caries status, pain status and responses to thermal testing.
Methods: Children attending for routine extractions were recruited to the study. Subjects completed a pain history that included a VAS score. Teeth to be extracted were tested using dedicated thermal testing equipment (Medoc, Israel). An intra-oral thermal probe was placed on the tooth at a baseline temperature of 20C and reduced to 0C maintaining at this temperature for 30 seconds before returning to baseline. Subjects pressed a button that aborted the test if they felt any sensation associated with the stimulus. The duration of the thermal stimulus and temperature evoking the sensation were recorded. A VAS score was also completed to indicate the intensity of the sensation evoked by the thermal stimulus.
Results: Forty-three samples were collected in total: 11 non-carious; 21 carious non-painful; 11 carious painful. Non-carious teeth responded least frequently (18.2%), carious non-painful teeth responded in 42.9% of samples and carious painful teeth responded most frequently (81.8%). The latency period was highest for the non-carious teeth (26s) followed by the carious non-painful teeth (16.2s), whilst the carious painful teeth responded quickest (13.1s). The carious painful teeth also responded at a higher temperature (5C) compared to the carious non-painful (3.7C) and non-carious teeth (0.95C). Average VAS scores were also higher for the carious painful teeth.
Conclusion: The thermal testing protocol used in this study is an effective method for determining pulpal response to thermal stimuli. Carious painful teeth appear more likely to respond to cold thermal testing using the study's protocols.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Recovery following peripheral nerve repair is variable and never complete. Previous research has shown that scar formation at a site of peripheral nerve injury can impede axonal regeneration and limit recovery. We have investigated whether the application of a Tumour Necrosis Factora antagonist (Etanercerpt, Pfizer) to a nerve repair site would permit enhanced regeneration.
Methods: Regeneration of the sciatic nerve was assessed in C57black6 mice. Under general anaesthesia (Fentanyl/Fluanisone 0.8ml/kg [Janssen] & Midazolam 4mg/kg [Roche], i.p.) the nerve was sectioned and repaired using 4 epineurial sutures. In one group (10 animals/group) Etanercept (15μg in 100μl of water carrier) was injected into and around the proximal and distal nerve stumps using a microdialysis cannula. A second group had 100μl of water injected alone, and these acted as controls. After 6 weeks the extent of regeneration was assessed electrophysiologically by determining the ratio of the compound action potential (CAP) modulus evoked by electrical stimulation of the nerve 2mm distal and proximal to the repair site, and the conduction velocities of the fastest fibres.
Results: The median CAP ratio in the Etanercept group was 0.68, which was significantly higher than the control group (0.37, p<0.005, Unpaired t-test). Conduction velocities of fibres distal to the repair were not significantly different between groups (p>0.05).
Conclusion: Our method and dosage for administration of TNFa antagonist Etanercept at a time of nerve repair significantly improves functional regeneration of sectioned axons in the mouse sciatic nerve.
Supported by the Royal College of Surgeons of Edinburgh (RCS 125796)