Porphyromonas gingivalis lipopolysaccharide rapidly activates trigeminal sensory neurons and may contribute to pulpal pain.

AIM: To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved and to establish whether activation leads to secretion of the neuropeptide calcitonin gene-related peptide (CGRP) and/or the translocation of NF-κB. METHODOLOGY: Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2 days. The effect of P. gingivalis LPS (20 µg mL-1 ) on calcium signalling was assessed (by calcium imaging using Cal-520 AM) in comparison with the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100 µmol L-1 ), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1 µmol L-1 ) and high potassium (60 mmol L-1 KCl). TG cultures were pre-treated with either 1 µmol L-1 CLI-095 to block Toll-like receptor 4 (TLR4) signalling or with 3 µmol L-1 HC-030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF-κB was investigated using immunocytochemistry. Data were analysed by one-way analysis of variance, followed by Bonferroni's post hoc test as appropriate. RESULTS: Porphyromonas gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non-neuronal cells (P < 0.001 to P < 0.05). The effects on neurons appear to be mediated via TLR4- and TRPA1-dependent pathways. The responses were accompanied by an increased release of CGRP (P < 0.001) and by NF-κB nuclear translocation (P < 0.01). CONCLUSIONS: Porphyromonas gingivalis LPS directly activated trigeminal sensory neurons (via TLR4 and TRPA1 receptors) and non-neuronal cells, resulting in CGRP release and NF-κB nuclear translocation. This indicates that P. gingivalis can directly influence activity in trigeminal sensory neurons and this may contribute to acute and chronic inflammatory pain.

Mannose-6-phosphate facilitates early peripheral nerve regeneration in thy-1-YFP-H mice.

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)-ß 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 1.0mm 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.

Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis.

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. RESULTS: 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. CONCLUSIONS: 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.

Evidence for anti-inflammatory and putative analgesic effects of a monoclonal antibody to calcitonin gene-related peptide.

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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

A comparison between the effects of three potential scar-reducing agents applied at a site of sciatic nerve repair.

We have investigated the effect of three potential scar-reducing agents applied at a sciatic nerve repair site in C57-black-6 mice. Under anaesthesia the nerve was transected, repaired using four epineurial sutures, and 100 µl of either triamcinolone acetonide (1 mg/100 µl), an interleukin-10 peptide fragment (125 ng/100 µl or 500 ng/100 µl) or mannose-6-phosphate (M6P, 200 mM or 600 mM) was injected into and around the nerve. 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 2 mm distal or proximal to the repair site. The conduction velocity of the fastest components in the CAP was also calculated. The percentage area of collagen staining (PAS) at the repair site was analysed using Picrosirius Red and image analysis. Comparisons were made with a placebo group (100 µl of phosphate buffered saline) and sham-operated controls. The median CAP modulus ratio in the 600 mM M6P group was 0.44, which was significantly higher than in the placebo group (0.24, P=0.012: Kruskal-Wallis test). Conduction velocities were also faster in the 600 mM M6P group (median 30 m s(-1)) than in the placebo group (median 27.8 m s(-1); P=0.0197: Kruskal-Wallis test). None of the other treated groups were significantly different from the placebo, and all had significantly lower CAP ratios than the sham controls (P<0.05). All repair groups had a significantly higher PAS for collagen than sham controls. We conclude that the administration of 600 mM mannose-6-phosphate to a nerve repair site enhances axonal regeneration.

Pulpal expression of TRPV1 in molar incisor hypomineralisation.

AIM: This was to compare the pulpal expression of the transient receptor potential ion channel (TRPV1), a noxious heat receptor, in sound and hypomineralised human first permanent molars. The rationale for the investigation was to gain further insight into pulpal changes in hypomineralised teeth and the possible biological mechanisms underlying thermal hypersensitivity. STUDY DESIGN: This was a laboratory study using a quantitative immuncocytochemical approach. METHODS: The experimental material comprised 17 sound and 18 hypomineralised molars (10 with intact enamel and 8 with enamel loss), obtained from children requiring dental extractions under general anaesthesia. Coronal pulps were removed and processed for indirect immunofluorescence using antibodies raised against TRPV1 and either the general neuronal marker, protein gene-product 9.5 or alpha smooth muscle actin in conjunction with Ulex europaeus agglutinin 1 lectin to fully label the pulp vasculature. Computerised image analysis was used to quantify the expression of TRPV1 in both pulpal nerves and blood vessels within different regions of the pulp including the pulp horn, subodontoblastic plexus and mid-coronal region. RESULTS: Mean neuronal and vascular TRPV1 expression was significantly greater in some pulpal regions of hypomineralised teeth (both with and without enamel loss) than for sound samples (p<0.05, ANOVA). CONCLUSIONS: Increased TRPV1 expression within the pulps of hypomineralised teeth may be indicative of an underlying pulpal inflammation and may help to explain the heat sensitivity experienced by some patients with this condition. However, future lines of enquiry should seek to correlate patient symptoms and responses to controlled hot and cold stimuli with pulpal expression of a variety of thermal receptors to gain further insight into dental pain mechanisms.

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.

Immunocytochemical investigation of immune cells within human primary and permanent tooth pulp.

AIM: The aim of this study was to determine whether there are any differences in the number and distribution of immune cells within human primary and permanent tooth pulp, both in health and disease. DESIGN: The research took the form of a quantitative immunocytochemical study. One hundred and twenty-four mandibular first permanent molars and second primary molars were obtained from children requiring dental extractions under general anaesthesia. Following exodontia, 10-microm-thick frozen pulp sections were processed for indirect immunofluorescence. Triple-labelling regimes were employed using combinations of the following: (1) protein gene product 9.5, a general neuronal marker; (2) leucocyte common antigen (LCA); and (3) Ulex europaeus I lectin, a marker of vascular endothelium. Image analysis was then used to determine the percentage area of immunostaining for LCA. RESULTS: Leucocytes were significantly more abundant in the pulp horn and mid-coronal region of intact and carious primary teeth, as compared to permanent teeth (P < 0.05, anova). Both dentitions demonstrated the presence of well-localized inflammatory cell infiltrates and marked aborization of pulpal nerves in areas of dense leucocyte accumulation. CONCLUSIONS: Primary and permanent tooth pulps appear to have a similar potential to mount inflammatory responses to gross caries The management of the compromised primary tooth pulp needs to be reappraised in the light of these findings.

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.

Nerve injury-induced pain in the trigeminal system.

This article reviews some recent findings on peripheral mechanisms related to the development of oro-facial pain after trigeminal nerve injury. Chronic injury-induced oro-facial pain is not in itself a life-threatening condition, but patients suffering from this disorder undoubtedly have a reduced quality of life. The vast majority of the work on pain mechanisms has been carried out in spinal nerve systems. Those studies have provided great insight into mechanisms of neuropathic spinal pain, and much of the data from them is obviously relevant to studies of trigeminal pain. However, it is now clear that the pathophysiology of the trigeminal nerve (a cranial nerve) is in many ways different to that found in spinal nerves. Whereas some of the changes seen in animal models of trigeminal nerve injury mimic those occurring after spinal nerve injury (e.g., the development of spontaneous activity from the damaged axons), others are different, such as the time-course of the spontaneous activity, some of the neuropeptide changes in the trigeminal ganglion, and the lack of sprouting of sympathetic terminals in the ganglion. Recent findings provide new insights that help our understanding of the etiology of chronic injury-induced oro-facial pain. Future investigations will hopefully explain how data gained from these studies relate to clinical pain experience in man and should enable the rapid development of new therapeutic regimes.

Neuropeptide expression in the ferret trigeminal ganglion following ligation of the inferior alveolar nerve.

Previous studies have found changes in neuropeptide expression in trigeminal ganglion cells after inferior alveolar nerve (IAN) section. These changes may play a part in the persistent sensory abnormalities that can be experienced after trigeminal nerve injuries. Here, neuropeptide expression after IAN ligation was studied, as this type of injury is thought to be more likely to result in sensory disturbances. The neuropeptides investigated were substance P, calcitonin gene-related peptide, enkephalin (ENK), galanin (GAL), neuropeptide Y (NPY) and vasoactive intestinal polypeptide. In anaesthetised adult female ferrets the left IAN was sectioned and the central stump tightly ligated. Recovery was allowed for 3 days, 3 or 12 weeks before perfusion-fixation. In a second procedure, 1 week before perfusion, the IAN was exposed and an injection made central to the injury site, using a mixture of 4% Fluorogold and 4% Isolectin B4 conjugated to horseradish peroxidase, to identify cell bodies with axons in the inferior alveolar nerve and cells with unmyelinated axons within this population, respectively. Control experiments involved tracer injection alone. After harvesting the tissue, sagittal sections were taken from both the right and left ganglia and immunohistochemical staining used to reveal the presence of peptides and Isolectin B4 tracer. The results showed a significant decrease in GAL expression after injury and an increase in ENK and NPY expression. No significant differences were seen in the expression of the other peptides or in the proportion of lectin-positive cells at any time after injury. When compared with previous data, significant differences were found between peptide expression following nerve ligation and nerve section. These results reveal that the changes in neuropeptide expression in the trigeminal ganglion that follow IAN injury are dependent upon the type of injury. The extent to which changes in the central neuropeptide levels contribute to the development of sensory disorders remains to be established.

Innervation of human tooth pulp in relation to caries and dentition type.

The neural status of carious teeth, particularly those associated with a painful pulpitis, is largely unknown. This study sought to determine differences in the innervation density of human primary and permanent teeth and whether caries or painful pulpitis was associated with anatomical changes in pulpal innervation. Coronal pulps were removed from 120 primary and permanent molars with a known pain history. Teeth were categorized as intact, moderately carious, or grossly carious. Using indirect immunofluorescence, we labeled sections for the general neuronal marker, protein gene product 9.5. Using image analysis, we found permanent teeth to be significantly more densely innervated than primary teeth. While there was no significant correlation with reported pain experience, neural density in both dentitions increased significantly with caries. Analysis of these data suggests that caries-induced changes in neural density may be functionally more important in the regulation of pulpal inflammation and healing than in the processing and perception of dental pain.

Changes in neuropeptide expression in the trigeminal ganglion following inferior alveolar nerve section in the ferret.

Changes in neuropeptide expression in afferent nerve fibres may play a role in the persistent sensory abnormalities that can be experienced following trigeminal nerve injuries. We have therefore studied changes in the expression of the neuropeptides substance P, calcitonin gene-related peptide, enkephalin, galanin, neuropeptide Y and vasoactive intestinal polypeptide in the trigeminal ganglion following peripheral nerve injury. In anaesthetised adult female ferrets, the left inferior alveolar nerve was sectioned and recovery allowed for three days, three weeks or 12 weeks prior to perfusion-fixation. During a second procedure, one week prior to perfusion, the inferior alveolar nerve was exposed and an injection made central to the injury site using a mixture of 4 % Fluorogold and 4 % isolectin B4 conjugated to horseradish peroxidase to identify cell bodies with axons in the inferior alveolar nerve and cells with unmyelinated axons within this population, respectively. Control animals received tracer injection alone. After harvesting the tissue, sagittal sections were taken from both the right and left ganglia and immunohistochemical staining was used to reveal the presence of peptides and isolectin B4-horseradish peroxidase tracer. Within the Fluorogold-labelled population, cell counts revealed a significant reduction in the proportion of substance P-containing cells at three days (P = 0.0025), three weeks (P = 0.0094) and three months (P = 0.0149) after nerve section, and a significant reduction in the proportion of calcitonin gene-related peptide-containing cells at three days (P = 0.0003) and three weeks (P = 0.007). No significant changes were seen in the expression of the other peptides, or at other time periods. A significant reduction in the number of isolectin B4-horseradish peroxidase-positive cells (with unmyelinated axons) was seen at three days (P = 0.0025), three weeks (P = 0.0074) and three months after the injury (P = 0.0133). These results demonstrate a significant reduction in the expression of some neuropeptides in the early stages after inferior alveolar nerve section. Some of the results differ markedly from those reported previously in other systems, and may be related to the specific nerve studied, species variations or differences between spinal and trigeminal nerves.

Calcitonin gene-related peptide modifies the ectopic discharge from damaged nerve fibres in the ferret.

Previous studies in our laboratory have shown that the neuropeptide, calcitonin gene-related peptide (CGRP) accumulates at a site of inferior alveolar nerve injury at the time when high levels of spontaneous activity and mechanical sensitivity are recorded electrophysiologically. The present study was undertaken to determine whether or not the CGRP could be playing a role in initiating or modulating the neuronal activity. In 18 anaesthetised adult ferrets the left inferior alveolar nerve was sectioned and ligated and recovery permitted for 3 days. Under a second anaesthetic recordings were made from a fine nerve filament, containing up to four active or silent units, dissected from the nerve proximal to the injury. After recording activity for a 30 min control period, CGRP and then the CGRP antagonist (CGRP 8-37) were applied either by close-arterial injection or topically (10(-4) M, 0.2 ml). After each application activity was recorded for a 30 min period. Recordings were made from 52 units, of which 26 (50%) were spontaneously active and 30 (58%) were mechanically sensitive. The spontaneous activity in five units was increased by the application of CGRP, and the CGRP antagonist subsequently reduced the activity in two of these units. Activity was induced by CGRP in three previously silent units. Overall, activity was affected in 19% of the units studied. We conclude that CGRP present within a neuroma may initiate or modulate the level of ectopic discharge from some damaged nerve fibres and therefore may contribute to the sensory disturbances which follow nerve injury.

The 5-HT(3) receptor antagonist alosetron inhibits the colorectal distention induced depressor response and spinal c-fos expression in the anaesthetised rat.

BACKGROUND: Noxious intestinal distention elicits a reflex depressor response in the sodium pentobarbitone anaesthetised rat, which can be used as an index of visceral nociception. 5-HT(3) receptor antagonists inhibit this reflex. Repeated colorectal distention (CRD) induces Fos like immunoreactivity (Fos-LI) in the rat spinal cord. AIMS: To examine the effect of the 5-HT(3) receptor antagonist alosetron on the depressor response to CRD, and on Fos expression in the lumbosacral spinal cord. METHODS: Male rats were anaesthetised with sodium pentobarbitone, and mean arterial blood pressure monitored during repeated colorectal balloon inflation before and after treatment with alosetron or saline. Rats anaesthetised with urethane and treated with alosetron or saline underwent a repeated CRD paradigm, after which the lumbosacral spinal cord was removed and processed for visualisation of Fos-LI. RESULTS: CRD elicited reproducible, volume dependent falls in arterial blood pressure, and repeated distention-effect curves were constructed. Alosetron (1-100 microg/kg intravenously) inhibited the depressor response to CRD in a dose related manner, with an ID(50) value of 3.0 microg/kg. Following repeated CRD, numbers of Fos-LI neurones were significantly increased to 1246 (total in 12 sections at 120 microm intervals from L6 to S1) compared with 49 in sham distended animals. Pretreatment with alosetron (100 microg/kg) significantly reduced numbers of Fos-LI neurones to 479.8. CONCLUSION: The 5-HT(3) receptor antagonist alosetron inhibits the depressor response to CRD in a potent and dose dependent manner. It also inhibits CRD induced Fos-LI in the spinal cord. These results suggest that 5-HT(3) receptors are involved in visceral nociceptive transmission, perhaps located on primary afferent or spinal neurones.

Substance P expression in human tooth pulp in relation to caries and pain experience.

The neuropeptide substance P (SP) is found within a subpopulation of nociceptive afferent nerve fibres and has been shown to be upregulated in a variety of sites following peripheral inflammation. The aim of this study was to investigate the expression of SP within human teeth, both in health and disease, and to seek a correlation between reported pain history and SP expression within pulpal nerves. Coronal pulps were removed from 62 permanent mandibular molars with a known pain history. Teeth were categorised as intact, moderately or grossly carious. Using indirect immunofluorescence, sections were double-labelled for the general neuronal marker protein gene product 9.5 (PGP 9.5) and for SP. Image analysis was then used to quantify the percentage area of PGP 9.5-labelled tissue which was also labelled for SP. Throughout the pulp, the expression of SP was found to be significantly increased with the progression of caries. Furthermore, SP expression was significantly greater in grossly carious painful specimens than in grossly carious asymptomatic specimens. These data would suggest that the expression of SP within pulpal nerves undergoes dynamic changes following caries, which may have an important clinical significance in terms of inflammation and pain experience.

Sympathetic nerve sprouting fails to occur in the trigeminal ganglion after peripheral nerve injury in the rat.

Peripheral nerve injury induces sprouting of sympathetic nerve fibers in dorsal root ganglia after spinal nerve injury. In the present study, we sought to determine the extent of intraganglionic noradrenergic sprouting in the trigeminal system. The inferior alveolar nerve, a major branch of the mandibular division, or the infraorbital nerve of the maxillary division was either ligated or chronically constricted in Sprague-Dawley rats and recovery permitted for either 2-3 or 6-9 weeks. In some animals both nerves were injured. Using immunohistochemistry with tyrosine hydroxylase antibodies, we found no signs of sympathetic nerve fiber sprouting in the trigeminal ganglion after injury. In contrast, sciatic nerve injury in rat littermates induced a widespread autonomic nerve outgrowth in affected DRGs. Thus, sensory ganglion sympathetic nerve sprouting does not seem to be a general outcome of PNS injury, but is restricted to certain specific locations. Sympathetic nerve fiber networks that surround primary sensory neurons have been suggested to form a structural basis for interactions between the sympathetic and sensory nervous systems after PNS injury. Such interactions, sometimes resulting in paraesthesia or dysaesthesia in patients, appear to be less common in territories innervated by the trigeminal nerve than in spinal nerve regions. The lack of injury-induced intraganglionic sympathetic sprouting in the trigeminal ganglion may help to explain this observation.

Immunocytochemical and electron-microscopic features of tooth pulp innervation in hereditary sensory and autonomic neuropathy.

Characteristics of the pulpal innervation in teeth obtained from a 4-year-old Asian boy with hereditary sensory and autonomic neuropathy, type II (HSAN) were investigated. Four minimally carious primary teeth were split longitudinally and prepared for either fluorescent immunocytochemistry or electron microscopy. The occurrence and distribution of specific neuropeptides were determined by the use of antisera to calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP). The overall innervation of the pulps was visualized using antiserum to protein gene product 9.5; an antiserum to dopamine beta-hydroxylase was used to identify postganglionic sympathetic fibres. Pulpal innervation in HSAN was notably different from that of normal teeth: in comparison with the controls, HSAN teeth had an overall marked reduction in pulpal innervation with an absence of large nerve bundles and the subodontoblastic plexus. CGRP- and SP-immunoreactivity was absent in HSAN specimens and VIP-immunoreactivity was reduced. However, NPY-immunoreactivity appeared to be increased within certain regions of the pulp/dentine complex. In addition, there was evidence of NPY-immunoreactive fibres extending into dentine, a feature not seen in the controls. Electron microscopy revealed an absence of myelinated nerve fibres and a paucity of unmyelinated fibres. CGRP and SP have a well-established role in nociceptive processing and their absence in the HSAN teeth would seem to correspond with the clinical presentation of marked peripheral sensory deficit, characteristic of this condition. An up-regulation of NPY-immunoreactivity has previously been reported in animal teeth following nerve injury and a similar mechanism may have stimulated increased NPY expression in HSAN teeth, but the functional significance of its presence within dentinal nerves is not known.

Neuropeptide immunoreactivity in ligature-induced neuromas of the inferior alveolar nerve in the ferret.

Injury to branches of the trigeminal nerve can sometimes result in persistent dysaesthesia. In an attempt to understand the aetiology of this condition we are currently investigating changes which occur at the injury site. In the present study we have examined the expression of seven neuropeptides, all of which have been implicated in nociceptive transmission, or have previously been shown to have altered expression following nerve injury. In 20 adult ferrets the inferior alveolar nerve was sectioned and ligated, and recovery permitted for 3 days, 8 days, 3 weeks, 6 weeks or 12 weeks. Longitudinal sections of the neuromas were processed using immunohistochemical techniques to quantify the expression of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, galanin, somatostatin, enkephalin and neuropeptide Y. After 3 days, all of the neuropeptides were expressed at the injury site. In the neuromas examined after longer recovery periods these levels of expression had declined and were similar to those found in the contralateral controls. This initial high level, followed by a decline, parallels the incidence of ectopic neural activity recorded electrophysiologically in the same model. It is, therefore, possible that the accumulation of neuropeptides at the injury site may play a role in the initiation or modulation of ectopic neural activity.

Fos expression in the ferret trigeminal nuclear complex following tooth pulp stimulation.

The aim of this study was to establish which regions of the trigeminal nucleus are activated by tooth pulp stimulation in the normal ferret. The distribution of Fos-like immunoreactivity was examined following electrical stimulation of the tooth pulp in the awake and anaesthetized ferret. Stimulus-specific labelling was found in subnuclei caudalis and oralis of the trigeminal spinal nucleus. Three groups of chronically prepared animals; conscious, anaesthetized (alphaxolone/alphadolone) and anaesthetized-paralysed (alphaxolone/alphadolone with gallamine triethiodide), received electrical stimuli to both the upper and lower left canine teeth (1 Hz train of 3 x 0.5 ms at 200 Hz) at an amplitude of 10 times the threshold of the jaw opening reflex. Three control groups were treated identically except no stimulus was given. In stimulated anaesthetized and anaesthetized-paralysed animals, Fos-positive profiles were seen in laminae I and II of subnucleus caudalis and in the medial part of subnucleus oralis. There was no labelling evident in subnucleus interpolaris or the main sensory nucleus, or contralaterally in any of the subnuclei. In all conscious stimulated animals there was additional bilateral Fos-positive labelling, mainly in the deeper laminae of subnucleus caudalis. This bilateral labelling was not stimulus-specific as it was also seen in conscious non-stimulated animals. After correction for this bilateral labelling no significant difference was found between conscious, anaesthetized and anaesthetized-paralysed groups of stimulated animals or between the different groups of control animals. These results support the concept that the rostral parts of the trigeminal spinal nucleus are involved in processing of nociceptive input. They also demonstrate that light alphaxolone/alphadolone anaesthesia has no effect on stimulus-specific Fos expression following tooth pulp stimulation. The second aim of this study was to develop a clearly defined model for future studies in which Fos expression is no different to that seen in the conscious state. As in the conscious animal, labelling not associated with the stimulus is difficult to distinguish from stimulus specific labelling, further studies using this model of trigeminal nociceptive pathways would be best carried out in lightly anaesthetized animals.