Two novel bis-azomethines derived from quinoxaline-2-carbaldehyde.

The Schiff base compounds N,N'-bis[(E)-quinoxalin-2-ylmethylidene]propane-1,3-diamine, C(21)H(18)N(6), (I), and N,N'-bis[(E)-quinoxalin-2-ylmethylidene]butane-1,4-diamine, C(22)H(20)N(6), (II), crystallize in the monoclinic crystal system. These molecules have crystallographically imposed symmetry. Compound (I) is located on a crystallographic twofold axis and (II) is located on an inversion centre. The molecular conformations of these crystal structures are stabilized by aromatic pi-pi stacking interactions.

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.

Effects of capsaicin and menthol on oral thermal sensory thresholds.

OBJECTIVE: To determine the long-term effect of capsaicin and short-term effect of menthol on oral thermal thresholds. DESIGN: The thresholds for cold detection (CDT), warm detection (WDT), cold pain (CPT) and warm pain (WPT) were determined in 11 regular chilli-eaters (capsaicin group) and 11 control subjects that were closely matched for age, gender and ethnicity. The effect of menthol was determined by asking all 22 participants to suck a lozenge containing 0.52% menthol for 5min. RESULTS: An ANOVA revealed a significant difference between the capsaicin and control groups (P=0.014), with the greatest difference in the WDT (capsaicin group 4.7+/-2.7[S.D.] degrees C; control group 2.3+/-2.2 degrees C). Immediately after sucking a menthol lozenge there was a significant rise in the CDT (2.2+/-1.1 degrees C to 5.9+/-6.2 degrees C; P<0.01) and WDT (3.6+/-2.7 degrees C to 7.6+/-4.4 degrees C; P<0.001). CONCLUSIONS: The consumption of foods containing capsaicin and menthol significantly alters thermal sensory thresholds in the oral cavity. Dietary habits should therefore be taken into account when intra-oral thermal thresholds are determined.

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.

Spontaneous and mechanically evoked afferent activity originating from myelinated fibres in ferret inferior alveolar nerve neuromas.

The inferior alveolar nerve is a predominantly sensory branch of the trigeminal nerve which runs within a bony canal, and is frequently damaged in patients. A small proportion of these patients develop neuropathic pain, and this may result from neural activity generated at the injury site. To investigate this abnormality we have used electrophysiological techniques in an animal model to determine the level of spontaneous activity and mechanical sensitivity of myelinated fibres ending in a neuroma of the inferior alveolar nerve. In 20 anaesthetised adult male ferrets the left inferior alveolar nerve was ligated in the region of the third premolar tooth, cut distally, and recovery permitted for periods of 3-113 days prior to making single unit recordings from the nerve central to the injury. The proportion of units which were spontaneously active ranged from 0% to 26%, with discharge rates 0.3-12.9 Hz. Discharge in response to mechanical stimulation of the neuroma was found in 0-36% of the units. Both spontaneous activity and mechanical sensitivity were significantly higher after shorter recovery periods and the majority of the spontaneously active units was also mechanically sensitive. These data reveal that the inferior alveolar nerve responds to injury in a similar way to some other peripheral nerves, and the neural activity generated at the injury site may play a role in the development of dysaesthesia.

Afferent activity from myelinated inferior alveolar nerve fibers in ferrets after constriction or section and regeneration.

To investigate possible peripheral mechanisms for post-injury sensory disorders in the trigeminal system, we have made electrophysiological recordings from myelinated fibres in the inferior alveolar nerve (IAN) which have previously sustained an injury. In earlier experiments we have shown that axons in ligature-induced neuromas of the IAN develop spontaneous activity and mechanical sensitivity. The present study has investigated these responses after two different types of injury. In 24 anaesthetised adult male ferrets the left IAN was either chronically constricted by four loose chromic gut ligatures (12 animals) or sectioned and regeneration permitted (12 animals). After recovery periods of 3 days, 1, 3, 6, 12 or 24 weeks, single unit recordings were made from the nerve proximal to the injury site. The proportion of units which were spontaneously active ranged from 0% to 19% after constriction injury and from 0% to 10% after nerve section and regeneration. Both groups revealed a marked variability between individual animals at similar time periods. Mechanical sensitivity was found in 0-42% of units after constriction and 0-25% of units after nerve section; both groups showed a significant negative correlation between mechanical sensitivity and recovery period. None of the fibres which had regained peripheral receptive fields was either spontaneously active or mechanically sensitive. There was no significant difference between the levels of spontaneous activity or mechanical sensitivity in the two groups or that previously found in ligature-induced neuromas. Thus we conclude that widely differing types of peripheral nerve injury are capable of initiating similar raised levels of afferent activity in myelinated inferior alveolar nerve fibres.

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.

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.

Recession of sensory loss from the midline following trigeminal sensory root section: collateral sprouting from the normal side?

The extent of transmedian innervation of the lower lip, chin, tongue and teeth has been investigated in 20 patients who had undergone trigeminal sensory root section between 4 months and 9 years previously, and in 20 control patients whose inferior alveolar and lingual nerves had been anesthetized unilaterally. There was significantly greater transmedian cutaneous innervation in the surgical group. This may be explained by collateral sprouting of sensory nerves across the midline into the chronically denervated area.

Evidence against a central stimulus for collateral reinnervation of oro-facial tissues in the cat.

Experiments in adult cats have shown that when the inferior alveolar nerve is sectioned and regeneration prevented, there is collateral reinnervation from neighbouring ipsilateral and contralateral nerves. Trigeminal rhizotomy prevents nerve impulses from entering the central nervous system and causes central degeneration but leaves the trigeminal ganglion and peripheral nerve intact. This investigation was designed to determine whether these central changes provide the stimulus for collateral reinnervation. In 5 adult cats, under general anaesthesia, a unilateral trigeminal rhizotomy was carried out via a temporal craniotomy. Evidence for the development of collateral innervation was sought by testing, at 3-week intervals, for the return of the jaw opening reflex evoked by electrical stimulation of the lower teeth, skin, mucous membrane and tongue, on the side of the rhizotomy. In addition, 12 or 15 weeks after the rhizotomy, recordings were made from ipsilateral and contralateral branches of the trigeminal nerve to determine whether collateral fibres were present. There was little evidence for the return of reflex responses to stimulation of tissues on the side of the rhizotomy and at the terminal experiment recordings revealed only a few collateral fibres. These results show that trigeminal rhizotomy does not stimulate collateral innervation and so suggest that the stimulus for sensory nerve sprouting is peripheral rather than central.

Regenerating nerve fibres do not displace the collateral reinnervation of cat teeth.

Experiments in adult cats have shown that when the inferior alveolar nerve is sectioned and regeneration blocked, there is extensive collateral reinnervation from neighbouring nerves which reinnervate the denervated teeth. In the present experiments the fate of collateral nerve fibres supplying the teeth has been investigated following regeneration of the inferior alveolar nerve. In some experiments the inferior alveolar nerve was sectioned and regeneration temporarily blocked whilst in others the nerve was frozen but not sectioned, to allow a more complete restoration of normal properties after regeneration. The jaw opening reflex evoked by electrical stimulation of the left canine and incisor teeth was abolished by left inferior alveolar nerve section or freeze but returned within 3-9 weeks due to reinnervation by collateral nerve fibres. Regenerating inferior alveolar nerve fibres were allowed to reinnervate the teeth 12-15 weeks after the initial nerve injury. Twenty seven weeks after the initial nerve injury, pulpal nerve fibres supplying the teeth which had been denervated were present in the regenerated inferior alveolar nerve as well as the ipsilateral lingual and mylohyoid nerves and the contralateral inferior alveolar, lingual and mylohyoid nerves. Except for the ipsilateral inferior alveolar and lingual nerves, these nerves do not normally include pulpal fibres from these teeth. In these experiments, therefore, after inferior alveolar nerve section or freeze, the collateral reinnervation of tooth pulps was not withdrawn following the return of regenerating nerve fibres.

A peripheral stimulus initiates the collateral reinnervation of cat teeth.

The ability of trigeminal nerve fibres to sprout across the midline into the pulps of denervated contralateral teeth has been examined after removing their central terminals by rhizotomy. An extensive transmedian reinnervation developed from fibres on the side of the rhizotomy suggesting that the stimulus for collateral reinnervation must be sited in the tissues peripherally.

The reinnervation of the tongue and salivary glands after lingual nerve injuries in cats.

The recovery of fibres in the chorda tympani and lingual nerves has been investigated in cats following nerve injury by recording the receptor properties of gustatory, thermosensitive and mechanosensitive units and the return of vasomotor and secretomotor responses. The combined trunk of the chorda tympani and lingual nerves was either crushed (4 animals) or sectioned (3 animals) unilaterally and recovery allowed for 12 weeks. After nerve crush, integrated whole nerve activity recorded from the chorda tympani during stimulation of the tongue with gustatory or thermal stimuli revealed a response profile which was similar to controls. After nerve section little or no activity could be recorded. Recordings made from 52 single units in the chorda tympani after nerve crush revealed that the proportions of gustatory, thermosensitive and mechanosensitive units were similar to those of controls. The units had slower conduction velocities, responded less vigorously and to a narrower range of stimuli. Recordings made from 46 units in the chorda tympani after nerve section revealed very few gustatory or thermosensitive units, the majority were purely mechanosensitive and the decrease in conduction velocity was greater than after nerve crush. Electrical stimulation of efferent vasodilator fibres in both the chorda tympani and lingual nerves, evoked a temperature rise on the dorsal surface of the tongue. This effect was completely restored after nerve crush but was significantly smaller after nerve section. The flow rate of saliva from the submandibular salivary gland was not significantly changed by nerve crush but was significantly smaller after nerve section. There was no evidence for functional reinnervation of gustatory or secretomotor terminals by inappropriate fibre types.

Reinnervation of teeth, mucous membrane and skin following section of the inferior alveolar nerve in the cat.

The reinnervation of teeth, mucous membrane and skin has been investigated in the cat following section of the inferior alveolar nerve. Evidence for regeneration of sectioned fibres and for sprouting of unsectioned nerves supplying adjacent tissues (collateral sprouting) was sought. In some experiments the cut nerve ends were reapposed whilst in others the central stump was either covered with an acrylic cap or sealed inside a nylon tube. The jaw opening reflex evoked by electrical stimulation of canine tooth pulp was abolished by inferior alveolar nerve section but returned within 3-9 weeks with a raised threshold and increased latency. After re-apposition or acrylic capping, some sectioned nerves regenerated but, compared with normal, they had decreased conduction velocities, greater variation in their mechanoreceptor fields and produced smaller compound action potentials in the teeth. There was little evidence of collateral sprouting. The nylon tube completely blocked regeneration but the denervated tissues were reinnervated by collateral sprouting. Fibres supplying tooth pulp were present in the ipsilateral mylohyoid, the ipsilateral and contralateral lingual nerves and the contralateral inferior alveolar nerve. Except for the ipsilateral lingual nerve, these nerves do not normally include pulpal fibres. Partial reinnervation of skin and mucous membrane occurred and this was derived from the ipsilateral mylohyoid, lingual and buccal nerves and the contralateral inferior alveolar nerve.

Characteristics of periodontal mechanoreceptors reinnervated after freezing the inferior alveolar nerve.

Previous electrophysiological investigations have shown that reinnervated periodontal mechanoreceptors, 12 weeks after crushing or sectioning the inferior alveolar nerve, have altered discharge properties. These alterations may have resulted from the regeneration of axons along inappropriate endoneurial sheaths distal to the injury site and this possibility was investigated by comparing the properties of reinnervated mechanoreceptors after nerve crush with those after nerve freeze (after which endoneurial sheath guidance is not lost). In 4 cats, 67 periodontal mechanosensitive units supplying the lower canine were examined 12 weeks after freezing the inferior alveolar nerve. When compared with controls, they were found to have significantly smaller arcs of sensitivity, raised force thresholds, lower discharge frequencies and reduced dynamic indices. These characteristics were, however, similar to those of units examined after nerve crush. These results suggest that the alterations in receptor properties after nerve injury cannot be attributed to a mismatch between the regenerating axons and their receptors.

Reinnervation of the canine tooth pulp after section of the inferior alveolar nerve in the cat.

The apices of lower canine teeth have been examined 9 weeks after unilateral section of the inferior alveolar nerve. The teeth were extensively reinnervated although there was considerable variation in the number of axons present. The myelinated axons were smaller than normal with thinner myelin sheaths. There were fewer axons per non-myelinated fibre (Remak bundle). The proportions of myelinated and non-myelinated fibres were normal.

The course of postganglionic sympathetic fibres distributed with the facial nerve in the cat.

Electrophysiological techniques have been used to determine whether sympathetic fibres are present in branches of the facial nerve. Compound action potentials could be recorded from all branches of the facial nerve during stimulation of the cervical sympathetic trunk. The course taken by the sympathetic fibres was determined by observing whether nerve sections at different sites blocked these responses. Two pathways were found: one group of fibres leaves the superior cervical ganglion in the internal carotid nerve, joins the auricular branch of the vagus nerve at the jugular foramen and passes via this nerve to the facial nerve, which it joins in the facial canal just central to the stylomastoid foramen. A second, apparently smaller group of fibres travels in the internal carotid nerve, crosses the roof of the tympanic bulla in the tympanic plexus, enters the middle cranial cavity through the foramen lacerum and passes with the greater superficial petrosal nerve to join the facial nerve at the geniculate ganglion.

The role of nerve growth factor in collateral reinnervation by cutaneous C-fibres in the rat.

We have investigated whether chronic nerve growth factor (NGF) depletion affects the development of transmedian collateral reinnervation by C-fibres. Using a dye-labelled plasma extravasation technique in rats, the extent of transmedian innervation of the skin by C-fibres in the inferior alveolar nerve (IAN) was determined 8-10 weeks after sectioning and preventing regeneration of the contralateral IAN. Another group of animals were immunised against NGF prior to the nerve section and a third group acted as unoperated controls. A small but significant transmedian collateral reinnervation by C-fibres developed after contralateral denervation alone, but was not found in the animals also immunised against NGF. These results suggest that NGF is essential for the development of collateral reinnervation from cutaneous C-fibres.

The effect of delayed nerve repair on the properties of regenerated fibres in the chorda tympani.

The characteristics of regenerated fibres in the chorda tympani have been investigated in cats after nerve section without repair or after section followed by nerve repair twelve weeks later. In the unrepaired group the animals were allowed to recover for twenty four weeks and after delayed repair there was a further recovery period of twelve or twenty four weeks. The properties of gustatory, thermosensitive and mechanosensitive units and the return of vasomotor and secretomotor responses were then investigated and data compared with that from normal controls and from animals which had undergone immediate nerve repair. After nerve section, integrated whole-nerve activity recorded from the chorda tympani during gustatory or thermal stimulation of the tongue was reduced when compared to controls, but there were only small differences between the repaired and unrepaired groups. Recordings made from single units in the chorda tympani revealed that more units were spontaneously active after repair (P < 0.05) and the gustatory units produced more impulses when stimulated (P < 0.005). Twelve weeks after delayed repair the units had slower conduction velocities than those in the unrepaired nerves (P < 0.001), but by twenty four weeks after repair they were significantly faster (P < 0.05). There was little difference in the level of recovery twelve weeks after immediate or delayed repair. We conclude that delayed nerve repair results in better recovery than leaving the nerve unrepaired and that a twelve week delay before repair has little effect.

A role for nerve growth factor in collateral reinnervation from sensory nerves in the guinea pig.

We have investigated whether chronic nerve growth factor (NGF) depletion affects the development of a transmedian collateral reinnervation. The extent of transmedian innervation of the skin supplied by the left inferior alveolar nerve (IAN) was determined either immediately, 2 days or 7-9 weeks after sectioning and preventing regeneration of the contralateral IAN and in another group of animals left to recover for 7-9 weeks but also autoimmunised against NGF. Transmedian innervation was measured by recording the area from which a jaw-opening reflex could be evoked and by recording activity in the left IAN during mechanical and electrical stimulation of the skin. Nerve recording during electrical stimulation revealed extensive transmedian collateral reinnervation 7-9 weeks after denervation but this was prevented by NGF autoimmunisation. No change in transmedian innervation could be detected in any of the groups by nerve recording during mechanical stimulation and reflex responses revealed changes in the anaesthetic area which could not be attributed to collateral reinnervation. These results suggest that NGF plays an important role in collateral reinnervation from high-threshold sensory nerves.