Overexpression of artemin in the tongue increases expression of TRPV1 and TRPA1 in trigeminal afferents and causes oral sensitivity to capsaicin and mustard oil.

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, supports a subpopulation of trigeminal sensory neurons through activation of the Ret/GFRalpha3 receptor tyrosine kinase complex. In a previous study we showed that artemin is increased in inflamed skin of wildtype mice and that transgenic overexpression of artemin in skin increases TRPV1 and TRPA1 expression in dorsal root ganglia neurons. In this study we examined how transgenic overexpression of artemin in tongue epithelium affects the anatomy, gene expression and calcium handling properties of trigeminal sensory afferents. At the RNA level, trigeminal ganglia of artemin overexpresser mice (ART-OEs) had an 81% increase in GFRalpha3, a 190% increase in TRPV1 and a 403% increase in TRPA1 compared to wildtype (WT) controls. Myelinated and unmyelinated fibers of the lingual nerve were increased in diameter, as was the density of GFRalpha3 and TRPV1-positive innervation to the dorsal anterior tongue and fungiform papilla. Retrograde labeling of trigeminal afferents by WGA injection into the tip of the tongue showed an increased percentage of GFRalpha3, TRPV1 and isolectin B4 afferents in ART-OE mice. ART-OE afferents had larger calcium transients in response to ligands of TRPV1 (capsaicin) and TRPA1 (mustard oil). Behavioral sensitivity was also exhibited by ART-OE mice to capsaicin and mustard oil, measured using a two-choice drinking test. These results suggest a potential role for artemin-responsive GFRalpha3/TRPV1/TRPA1 sensory afferents in mediating sensitivity associated with tissue injury, chemical sensitivity or disease states such as burning mouth syndrome.

Close apposition and exposure of non-myelinated axons in traumatic neuromas of the human lingual nerve.

Peripheral nerve injury is sometimes followed by the development of persistent painful sensory disorders, such as dysaesthesia. The aetiology of these disorders is not clear, but abnormal behaviour of damaged axons at the injury site is likely to be involved. In this study, we quantified some ultrastructural characteristics that may be related to the development of abnormal spontaneous activity, sympathetic interactions, and fibre-to-fibre crosstalk. Using electron microscopy, we have determined the frequency and extent of axonal exposure and close apposition among non-myelinated axons from 34 traumatic neuromas of the human lingual nerve. These specimens were removed at the time of microsurgical nerve repair, and the presence or absence of symptoms of dysaesthesia was determined pre-operatively. Comparisons were also made with eight normal control lingual nerve specimens obtained from patients undergoing organ donor retrieval. More non-myelinated axons showed signs of axonal exposure in traumatic neuromas (26%) than in controls (5%), and exposure was higher in nerve-end neuromas (31%) than in neuromas-in-continuity (22%). In addition, the proportion of the non-myelinated axolemma that was exposed was significantly higher in neuromas (32%) than in controls (21%). The frequency of close apposition between neighbouring non-myelinated axons was also higher in neuromas (11%) than in controls (0.35%). The majority of axons showing signs of exposure or close apposition had diameters <1 microm. These ultrastructural changes may account for some of the altered electrophysiological properties of axons within neuromas. However, no significant correlations were found between these ultrastructural characteristics and the patients' reported symptoms of dysaesthesia.

Lingual deficits in neurotrophin double knockout mice.

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF(-/-) and wild-type mice. Taste papillae morphology was severely distorted in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF(-/-) and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF(-/-) and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF(-/-) xNT-3(-/-) mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.

[Suggestive evidences for a microanatomical relationship between mast cells and nerve in the tongue of the toad Bufo marinus demonstrated by means of high resolution light microscopy]. / Evidencias que sugieren una asociación microanatómica mastocito-nervio en la lengua del sapo Bufo marinus demostradas mediante microscopia de luz de alta resolucion.

Mast cells are connective tissue cells, present in all vertebrates and characterized by the metachromatic stain of their granules. Nowadays mast cells have been recognized as a potent cellular source of multiple cytokines, suggesting an important role in immunoregulation and host defense. These cells have been described as preferentially located around blood vessels but more recently close spatial relationship between mast cells and nerves has been reported mostly in mammalian species. The microanatomy of nerve tissue and associated mast cells in the toad Bufo marinus tongue have been studied here by means of high resolution light microscopy. Mast cell population was identified by the metachromatic staining of their cytoplasm granules in Epon embedded semithin sections counterstained with Toluidine Blue and Azure A. Numerous mast cells were observed scattered throughout the submucosal region, adjacent and/or within of nerve bundles and nerve ganglia, near skeletal muscle fibers and adjacent of blood vessels. Additionally, mast cells adjacent to single but conspicuous myelinated nerve fibers were seen under endothelia of lymphatic vessels wall and this is apparently a unreported event. Results suggest nerve-mast cell associations are functionally important in the toad tongue.

Quantitative ultrastructure of slowly adapting lingual afferent terminals in the principal and oral nuclei in the cat.

Previous studies provide evidence that a structure/function correlation exists in the cytoarchitectonically different zones of the trigeminal sensory nuclei. To extend this relationship, we examined the ultrastructural features of trigeminal primary afferent neurons in the cat dorsal principal nucleus (Vpd) and the rostrodorsomedial oral nucleus (Vo.r) using intra-axonal labeling with horseradish peroxidase and morphometric analyses. All labeled boutons contained round synaptic vesicles. Eighty-two percent of the boutons in the Vo.r and 99% of the boutons in the Vpd were presynaptic to nonprimary dendrites. The remaining boutons in the Vo.r were presynaptic to somata (8%) or primary dendrites (10%). The average number of postsynaptic profiles per labeled bouton did not differ in the Vpd and Vo.r. Most labeled boutons in the two nuclei were postsynaptic to unlabeled axon terminals with pleomorphic vesicles (p-ending). The number of p-endings per labeled bouton was higher in the Vpd than Vo.r A morphometric analysis indicated that labeled bouton volume and apposed surface area were larger in the Vpd than Vo.r while active zone area and vesicle number did not differ. All these parameters were larger than those of p-endings in each nucleus. In both labeled boutons and p-endings, the parameters were positively correlated with bouton size. These results suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by presynaptic mechanisms in the Vpd than in the Vo.r, while the efficacy of transmission at primary afferent synapses does not differ.

A quantitative morphological comparison of cat lingual nerve repair using epineurial sutures or entubulation.

Since lingual nerves may be transected during a variety of oral surgical procedures, including third molar removal, we have investigated two possible methods of repair. Quantitative morphological observations were made on feline chorda tympani and lingual nerves proximal and distal to transection injuries repaired either by epineurial suturing or by insertion of the cut ends into a perforated silicon tube. Proximal to the repair, the most prominent difference was an increase in the number of myelinated axons in the lingual nerve following epineurial suturing but not entubulation. Proximal to the repair site, the number of nonmyelinated axons increased in comparison with controls in both chorda tympani and lingual nerves after both procedures, though the difference was statistically significant only in the lingual nerve proximal to entubulation. Distal to the injury, both types of repair showed a reduction in the number, size, and sheath thickness of myelinated axons in comparison with unoperated controls, but the difference in numbers was statistically signIficant only distal to repair by entubulation. The number of non-myelinated axons distal to the repair sites was much higher than that in controls, the difference being greater distal to entubulation repair. There were more axons per Remak bundle distal to entubulation repair than to epineurial suturing, suggesting, perhaps, that fewer axons would ultimately become myelinated. Though the morphological differences between the two repair techniques are not as striking as the parallel electrophysiological differences reported previously (Smith and Robinson, 1995a,b), they are consistent with them and support the conclusion that, for transected lingual and chorda tympani nerves, epineurial suturing is the preferred approach.

Persistence and calcium-dependent ATPase staining of denervated fungiform taste buds in the hamster.

Some fungiform taste buds in the hamster have been previously shown to persist for indefinite periods when deprived of their gustatory, chorda tympani (CT), innervation or both their CT and their trigeminal, lingual nerve, innervation (CT-L). The properties and numbers of persisting fungiform taste buds were examined 1 or 3 weeks after permanent CT or combined CT-L nerve cuts. The purpose was to reveal the status of taste buds at a time (3 weeks) when regenerating nerve fibres would normally be expected to reinnervate the epithelium. Denervated taste buds retain many normal characteristics including the pattern of histochemical staining for ectocalcium-dependent ATPase (Ca-ATPase). Taste-bud cells (including basal cells) have an intensely Ca-ATPase stained core surrounded by lightly stained peripheral cells. The Ca-ATPase stain was used to help identify and to define the size of the taste-bud core in denervated taste buds. Following CT-L or CT denervation most taste buds persisted; however the size of the taste-bud core was dramatically reduced. Fungiform taste buds differed in size based on their location in one of three tongue regions. The percentage decrease in size after denervation was also region specific and about the same for CT-L or CT cuts, suggesting that trigeminal fibres have no trophic effect on taste buds. However, trigeminal denervation caused a reduction in the number of persisting taste buds relative to CT denervation alone, which may be due to damage because of the loss of somatosensation.

Collagen fibrillar networks as skeletal frameworks: a demonstration by cell-maceration/scanning electron microscope method.

A cell-maceration/scanning electron microscope (SEM) method was employed to demonstrate the arrangement of the collagen fibrillar network of various tissues. Immersion of fixed tissues in NaOH (25 degrees C) for 3-7 days, followed by rinsing in distilled water successfully removed the cellular elements, exposing collagen fibrils which were identified as such by transmission electron microscopy in their natural locations. SEM observations of the preparations are able to demonstrate the three-dimensional architecture of collagen fibrils much more precisely than other methods, including the silver impregnation method. Collagen fibrils, forming sheaths for housing individual cardiac myocytes, fused together, thus ensuring an equal stretch of contiguous myocytes and preventing the slippage of adjacent cells. Individual skeletal muscle fibers and nerve fibers were ensheathed by the meshwork of collagen fibrils running in two opposite helices. Such structures seem to play an important role in resisting the stretching impetus. At the epithelial-connective tissue junction of the tongue and fingertip skin, interwoven collagen fibrils formed numerous microridges which probably provide a broad anchorage for the epithelium. In the intestinal mucosa, the collagen fibrillar network immediately below the basal laminae of the villous epithelium possessed heterogeneous pores. As the collagen fibrillar network shows morphological features specific to individual organs and tissues, it is suggested that such formations not only constitute the skeletal framework but also provide those cells which are housed there with a microenvironment suitable for their activities.