How to avoid iatrogenic lingual nerve injury in the retromolar area: an anatomical study of retromolar pad and lingual nerve.

PURPOSE: This study aimed to investigate the relationship between the retromolar gland and pad, and the relationship between the LN and retromolar gland/pad to establish a new landmark for avoiding LN injury. METHODS: Sixty-two lingual nerves from fresh-frozen cadavers were used for this study. The age of the specimens at the time of death ranged from 57 to 98 with a mean of 76.5 years. The mucous incision was made into the medial border of the retromolar pad and the submucosal tissue depth of the initial incision was bluntly dissected to expose the lingual nerve. When the LN was identified, the mucosa overlying the retromolar pad was removed to expose the retromolar gland to confirm if the retromolar pad corresponds to the retromolar gland. RESULTS: On all sides, the lingual nerve was found to course medial to the retromolar pad and inferior to the inferior border of the superior pharyngeal constrictor muscle to enter the sublingual space via the pterygomandibular space. The retromolar pad corresponded to the retromolar gland on all sides. This demonstrated that the retromolar pad is an overlying mucosa of the retromolar gland. No LN was found to travel through the retromolar gland. CONCLUSION: We suggest that the retromolar pad can be used as a new landmark for avoiding iatrogenic LN injury.

Acetylcholine synthesis, muscarinic receptor subtypes, neuropeptides and secretion of ferret salivary glands with special reference to the zygomatic gland.

Studies on salivary secretion are usually focused on parotid and submandibular glands. However, the film of mucin, that protects the oral structures and is responsible for the feeling of oral comfort, is produced by the submucosal glands. The submucosal zygomatic and molar glands are particularly large in carnivores such as the ferret. Comparisons between the mucous sublingual, zygomatic and molar glands, serous parotid and sero-mucous submandibular glands showed the acetylcholine synthesis, in terms of concentration, to be three to four times higher in the mucous glands than in the parotid and submandibular glands. Bromoacetylcholine inhibited 95-99% of the synthesis of acetylcholine in the incubates of the five types of glands, showing the acetylcholine synthesis to depend on the activity of choline acetyltransferase. The high acetylcholine synthesis in the zygomatic gland was of nervous origin, since cutting the buccal nerve, aiming at parasympathetic denervation, and allowing time for nerve degeneration, reduced the acetylcholine synthesising capacity of the gland by 95%. A similar reduction (96%) in the parotid gland followed upon the avulsion of the parasympathetic auriculo-temporal nerve. Zygomatic saliva was very viscous. The salivary flow rate in response to electrical stimulation (20 Hz) of the buccal nerve (zygomatic gland), expressed per gland weight, was one-third of that to stimulation of the auriculo-temporal nerve (parotid gland) or the chorda-lingual nerve (submandibular gland). As previously shown for the parotid and submandibular gland, a certain fraction (25%) of the parasympathetic secretory response of the zygomatic gland depended on non-adrenergic, non-cholinergic transmission mechanisms, probably involving substance P and vasoactive intestinal peptide and possibly calcitonin gene-related peptide. Particularly, high concentrations of vasoactive intestinal peptide were found in the sublingual and molar glands, and of substance P in the submandibular, zygomatic and molar glands; notably, the concentration of calcitonin gene-related peptide of the sublingual gland was not detectable. All five muscarinic receptor subtypes were detected in the five glands. The receptor protein profile, as judged by immunoblotting and semi-quantitative estimations, was about the same in the glands: high level of M3, low level of M2 and levels roughly in the same range of M1, M4 and M5. Compared to the parotid and submandibular glands, the M5 receptor level was particularly low in the mucin-secreting glands. The present study points out both similarities and dissimilarities between the five types of glands investigated. The zygomatic gland, in particular, appears to be a suitable model for future studies aiming at causing relief of dry mouth by local pharmacological treatment.

Minor salivary glands morphology in xerostomia patients / Morfología de las Glándulas Salivales Menores en Pacientes Xerostomizados

Las glándulas salivales menores son encontradas distribuidas a través de la mucosa oral, especialmente en los labios y en la mucosa del paladar blando. Varios factores pueden causar xerostomía, donde las características histológicas de las glándulas salivales son también consideradas como factores para definir la etiología. Así, las biopsias de las glándulas salivales menores representan una herramienta fundamental para alcanzar los criterios diagnósticos requeridos en la clasificación de pacientes con síndrome de Sjõgren, ya que no representa riesgo para los pacientes. El objetivo de este estudio es determinar las características histológicas de las glándulas mencionadas, obtenidas de biopsias de pacientes con xerostomía y clasificar los aspectos histológicos de las glándulas en el síndrome de Sjõgren. Estudiamos 40 láminas de pacientes con xerostomía, cuyas glándulas salivales menores fueron sometidas a biopsia en el Servicio de Estomatología de la Santa Casa de São Paulo, Brasil. Se observaron las variaciones de su aspecto histológico, desde la normalidad hasta la presencia de focos inflamatorios, los cambios del tejido conjuntivo entre los acinos y conductos, como también el parénquima. En 15 casos, el infltrado de células inflamatorias invadió el foco, es decir, grupos de al menos 50 células inflamatorias alrededor de acinos o conductos, lo cual es un aspecto característico del síndrome de Sjõgren. Por lo tanto, el hallazgo de al menos un foco inflamatorio de 4 mm2 de tejido glandular, representa un buen criterio, aunque no es uno de los criterios a considerar cuando se trata de clasificar a los pacientes con el Síndrome de Sjõgren.

The minor salivary glands are found scattered throughout the oral mucosa, especially in the lips and soft palate mucosa. Several factors can cause xerostomia, whereas the salivary glands histological characteristics are also considered as factors for defining the etiology. Thus, the minor salivary glands biopsy represents an essential tool for attending the required diagnosis criterion in the classification of Sjögren's Syndrome patients, since it does not present risk for the patient. The objective of this study is to determine the histological description of the minor salivary glands obtained from the biopsies of xerostomia patients and to classify the minor salivary gland histological aspect as the Sjögren's Syndrome. Forty laminas of xerostomia patients that were submitted to minor salivary glands biopsy at the Santa Casa de Sao Paulo Stomatology ambulatory were retrospectively studied. The variation in the glands histological aspect was observed, from the normality up to the presence of inflammatory focus, replacing the conjunctive between acini and ducts, as well as the parenchyma. In 15 cases, the infiltrated inflammatory cells amounted to focus, that is to say, groups of at least 50 inflammatory cells around the acini or ducts, which is a characteristic aspect of the Sjögren's Syndrome. Therefore, the finding of at least one inflammatory focus of 4 mm2 of glandular tissue represents a set criterion, although, not the only one in order to classify this patient as having the Sjögren's Syndrome.

An anatomical study of the cutaneous branches of the mental nerve.

Minor surgical procedures to the inner (mucosal) aspect of the lower lip may occasionally cause numbness of the overlying skin. This study was designed to find, by means of dissection and computerized three-dimensional reconstruction, why surgical interference with nerve fibres in the deep aspect of the lip can cause neurological deficit in the superficial layers. Thirteen cadaveric lips were examined by dissection under a surgical microscope (9 lips) or serial sectioning and computerized three-dimensional reconstruction (4 lips). Muscle mass, minor labial salivary glands and nerve fibres were identified and traced. Three patterns of mental nerve distribution were seen on dissection and two on computerized reconstruction; these latter corresponded to two of the patterns seen on dissection. Fibres passing close to the labial minor salivary gland mass were seen to travel towards the superficial aspect of the lip, terminating in the dermis. It is clear that there is no safe anatomical space for minor surgical procedures to the inner (mucosal) aspect of the lower lip if avoidance of cutaneous numbness is an important consideration. However, we describe a technique that may minimize the possibility of cutaneous numbness.

[Changes of the nerve fibers innervating the minor salivary glands in Sjögren syndrome]. / A kis nyálmirigyek beidegzésében résztvevó idegelemek változása Sjögren-szindrómában.

INTRODUCTION: A large number of nerve fibres containing different neuropeptides/transmitters are also found in the salivary glands. The number and the distribution of nerve fibres is altered in many diseases, including in Sjögren's syndrome. AIM: Therefore in the present study the distribution and precise localisation of the nerve fibres containing the frequently observed neuropeptides were studied in the minor salivary glands. METHODS: Vasoactive intestinal polypeptide, neuropeptide Y, substance P, calcitonin gene-related peptide, somatostatin, nitric oxide synthase and tyrosine beta-hydroxylase antibodies were used as primary antisera, and then by the aid of avidin-biotin-peroxidase complex method the immunoreactive fibers in human labial glands (control and with Sjögren's syndrome) and in minor glands of the root of the rat's tongue were detected. RESULTS: Large number of vasoactive intestinal polypeptide and nitric oxide synthase immunoreactive nerve fibres were seen around the acini. The neuropeptide Y and tyrosine beta-hydroxylase positive nerve fibres were mainly found around the blood vessels. Some of the IR fibers were also found around the excretory ducts. In the biopsy of patients with Sjögren's syndrome, the acini were destroyed and only few excretory ducts were seen. The number of the nerve fibres was significantly decreased and many degenerated fibres were also observed among the acini. The electron-microscopic examinations showed that the immunoreactive nerve fibres were in close association to the secretory cells, to the smooth muscle cells of blood vessels and to the immunocells. The synaptic gap between the nerve fibres and the target cells were 40-200 nm. CONCLUSIONS: On the bases of the distribution of the different transmitters containing nerve fibres and their relationship to effector cells, the authors suppose that these transmitters control the function of the gland and regulate the blood flow. The close association to immunocells and decreasing the nerve fibres in Sjögren's syndrome imply that they may have also a role in the neuroimmunologic processes.

Innervation pattern and Ca2+ signalling in labial salivary glands of healthy individuals and patients with primary Sjögren's syndrome (pSS).

We have characterised the innervation pattern and intracellular Ca2+-signalling in labial salivary glands (LSG) of 16 patients with primary Sjögren's syndrome (pSS) and 27 healthy controls. Numerous immunoreactive nerve fibers (IRF) containing vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were found around acini, ducts and blood vessels. Substance P (SP)-, neuropeptide Y-, tyrosine hydroxylase- and nitric oxide synthase-IRF were mainly surrounding ducts and blood vessels. The majority of pSS patients had inflamed LSG and the presence of focal lymphocytic infiltrates (FI) were more frequent and pronounced as compared with healthy controls. In areas with normal or diffusely inflamed LSG tissue, pSS patients demonstrated the same distribution of IRF as healthy controls with similar histology. However, IRF were absent in central areas of FI both in pSS and age-matched healthy controls. Although all pSS patients had hyposalivation, stimulation with acetylcholine, norepinephrine, phenylephrine, isoproterenol, VIP, PACAP, SP, adenosine 5'-triphosphate and uridine 5'-triphosphate induced the same increase in the intracellular free Ca2+ concentration in LSG acini from both pSS patients and healthy controls, indicating the presence of functional receptor systems in vitro.

Phenotypes in canalicular adenoma of human minor salivary glands reflect the interplay of altered secretory product, absent neuro-effector relationships and the diversity of the microenvironment.

AIMS: Uncertainty about the factors influencing phenotypes in salivary canalicular adenoma prompted the present investigation. METHODS AND RESULTS: Specimens of canalicular adenoma from 15 patients were examined with the use of histology, histochemistry for protein, mucosubstances and pigments, nerve staining and immunocytochemistry for cytoskeleton components. The tumours consisted largely of simple cells lining tubules that were occasionally cystic or branching and budding, and were set in loose, vascular and often haemorrhagic stroma. Other phenotypes recognized were mucous cells, apocrine-like cells, pigmented cells, microliths and stromal macrophages, detected in 26.6%, 20%, 33.3%, 20% and 53. 3% of the patients, respectively. Simple cells showed moderate levels of -SH groups and strong immunoreactivity for 'simple' epithelial phenotype cytokeratin. The simple cells lining cystic tubules showed additional immunoreactivity for 'stratified' epithelial phenotype cytokeratin, possibly an adaptation to mechanical pressure. Lumina showed variable levels of neutral and carboxylated glycoproteins, and chondroitin sulphate. Stroma showed high levels of chondroitin sulphate and hyaluronic acid. Mucous cells showed high levels of -SS- groups and nonsulphated glycoproteins. Apocrine-like cells contained lipofuscin. Pigmented cells contained haemosiderin, possibly a consequence of localized iron overload. Microliths contained mucosubstances. Macrophages often contained lipofuscin. No nerves were found in relation to the tumours. CONCLUSIONS: The results suggest that, contrary to popular belief, phenotypes in canalicular adenoma do not reflect histogenetic concepts but rather may derive from the interplay between an altered secretory product, consisting of glycosaminoglycan and an immature form of glycoprotein, the lack of neuro-effector relationships and the different microenvironments throughout the tumour.

Immunocytochemical localisation of neuropeptide-containing nerve fibres in human labial glands.

Different neuropeptide-containing nerve fibers (vasoactive intestinal polypeptide, substance P, neuropeptide Y) and nitric oxide synthase (NOS) positive nerve fibers were investigated to clarify their role in the function of human labial glands using immunohisto- and immunocytochemical techniques. The distribution pattern of all immunoreactive nerve fibers was similar both in the control and in the Sjögren's syndrome specimens. A large number of thin varicose vasoactive intestinal polypeptide and NOS positive nerve fibers were seen around or in close contact with the acini. Some of the immunoreactive nerve fibers were associated with the salivary ducts and blood vessels. Substance P and neuropeptide Y immunoreactive nerve fibers were located mainly around the blood vessels. Immunocytochemistry demonstrated that some of the positive nerve fibers were in direct contact with the acini, blood vessels and with the lymphocytes. The gap between the membranes of immunoreactive nerve terminals and the target cells was 40 to 200 nm. The number of the nerve terminals in Sjögren's syndrome specimens was decreased and some degenerated axons were also found. These results suggest that these neuropeptides and nitric oxide might act as a neurotransmitter in the regulation of secretion and blood flow in the labial glands. These fibers might also alter the neuroimmunological processes, because the investigated neuropeptides are known to be immunoregulators.

Neutral endopeptidase (EC 3.4.24.11) in labial salivary glands in healthy controls and in patients with Sjögren's syndrome.

OBJECTIVE: Neuropeptides from nerve fibres can cause neurogenic inflammation. The potency of these peptides in vitro has led to the hypothesis that enzyme degradative systems are operative in vivo to limit their action. To consider this question neutral endopeptidase (NEP) in labial salivary glands in patients with Sjögren's syndrome was studied. METHODS: Synthesis of NEP mRNA in situ in labial salivary glands was studied using the reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical staining was used to localise the NEP enzyme protein and its neuropeptide substrates and fluorophotometry to measure the corresponding enzyme activities in saliva. RESULTS: NEP was found in nerve fibres and in perivascular, periductal, and periacinar axon terminal varicosities. Double labelling of PGP 9.5 and NEP confirmed this neuronal localisation of NEP. Although some fibroblast-like cells and occasional intravascular neutrophils were NEP positive, NEP mRNA was not found in labial salivary glands. Patients with Sjögren's syndrome and healthy controls did not have nerves containing NEP or neuropeptides (vasoactive intestinal peptide, substance P, or calcitonin gene related peptide (CGRP)) in lymphocyte foci. Salivary NEP activity was not decreased in patients compared with controls. CONCLUSION: NEP in labial salivary glands is almost totally of neuronal origin and plays a part in proteolytic modulation of neuropeptides in salivary glands and saliva. These regulatory interactions seem to be altered in focal lymphocyte accumulations in Sjögren's syndrome.

The reinnervation of the tongue and salivary glands after two methods of lingual nerve repair in the cat.

The recovery of fibres in the chorda tympani after repair by epineurial suture or entubulation was investigated. The combined trunk of the chorda tympani and lingual branch of the trigeminal nerve was sectioned unilaterally, repaired using either epineurial sutures or entubulation, and allowed to recover for 12 weeks. The properties of gustatory, thermosensitive and mechanosensitive units, and the return of vasomotor and secretomotor responses were then investigated. After repair by epineurial suture, integrated whole-nerve activity recorded from the chorda tympani during stimulation of the tongue with gustatory or thermal stimuli was reduced in all areas of the tongue when compared to controls. After entubulation repair, little or no activity could be recorded. Recordings made from 57 single units in the chorda tympani after repair by epineurial suture revealed a greater proportion of purely mechanosensitive units and fewer gustatory units than in the controls. Fewer units were spontaneously active, they had lower maximum discharge frequencies, and produced fewer impulses when stimulated. Recordings made from 61 single units after repair by entubulation revealed receptor characteristics with greater differences from controls than after epineurial suture and there was only one gustatory and one thermosensitive unit. Vasomotor responses were completely restored after repair by epineurial suture, but some responses were smaller after entubulation repair. Secretomotor responses were significantly smaller after both methods of repair and there was no difference between the two groups.

Postnatal development of von Ebner's glands: accumulation of a protein of the lipocalin superfamily in taste papillae of rat tongue.

Antibodies produced against rat von Ebner's gland (VEG) protein, a recently characterized member of a lipophilic ligand carrier protein family, detect this protein immunocytochemically in von Ebner's gland acini and show that it is present at high concentrations in the clefts of circumvallate and foliate papillae. During embryonic development, von Ebner's gland anlagen are innervated (as shown immunocytochemically using neuronal specific antibodies) as early as embryonic day 20, before lateral glandular outgrowth and VEG protein can be observed. Expression of the VEG protein as determined by in situ hybridization and immunocytochemistry begins at postnatal day-2 cells in differentiating and branching off from von Ebner's gland ducts, and sharply increases with further enlargement and maturation of the gland. The close temporal correlation of von Ebner's gland innervation and VEG protein expression with papilla innervation and taste-bud development suggests a functional relationship of both structures. VEG protein might control access of lipophilic sapid molecules, such as bitter substances, to the gustatory receptors.

Rat sublingual salivary glands: secretory changes on parasympathetic or sympathetic nerve stimulation and a reappraisal of the adrenergic innervation of striated ducts.

Sublingual glands were examined by light and electron microscopy after stimulating the parasympathetic nerve (5 Hz continuously) or the sympathetic nerve (50 Hz in bursts 1 s every 10 s) and compared with contralateral, unstimulated, normal glands from each animal. Parasympathetic stimulation caused secretion of mucin from the tubulo-acini and possibly a small amount of degranulation from the demilunes but no changes were detected in the striated ducts. Sympathetic stimulation, on the other hand, had no effect on the tubulo-acini or demilunes but caused a surprisingly extensive degranulation of the striated ducts plus loss of glycogen from their cells. Reassessment of the adrenergic innervation in the glands was therefore undertaken, by means of catecholamine fluorescence. This identified a regular association between adrenergic nerves and the striated ducts not only in sublingual but also in submandibular glands; features that have not previously been recognized. There was, however, only a sparse adrenergic innervation of the other parenchymal elements in the sublingual glands.

The significance of perineural spread in adenoid cystic carcinoma of the major and minor salivary glands.

Patient survival, local recurrence and distant metastasis were studied in relation to the pathological finding of perineural spread in 37 patients with adenoid cystic carcinoma of the major and minor salivary glands. All patients underwent a combined surgical and radiotherapeutical treatment. The overall incidence of perineural invasion in primary resection specimens was 52.6%. The 5-year actuarial survival rate for patients with perineural invasion was significantly lower (p less than 0.001) than for those without (36.9% versus 93.8%). In 26 patients with resection margins free of tumour, recurrences developed in 9/11 (81.8%) of the patients with perineural invasion as opposed to 4/15 (26.7%) of the patients without perineural invasion (p = 0.005). In the same group with resection margins free of tumour, distant metastasis developed after the primary treatment in 4/10 (40.0%) of the patients with perineural invasion, while none of the 14 patients without perineural invasion experienced distant metastasis (p less than 0.0002). The incidence of perineural invasion increased with a higher stage of the primary tumour.

Responses of inferior salivatory neurons to stimulation of trigeminal sensory branches.

A total of 84 single inferior salivatory neurons was identified by antidromic stimulation of the tympanic nerve. Their responsiveness was tested to stimulation of the ipsilateral infraorbital, lingual, and inferior alveolar nerves in urethane-chloralose-anesthetized cats. The conduction velocities of preganglionic fibers of inferior salivatory neurons ranged from 2.2 to 9.1 m/s, and 54% of those neurons responded with spikes to stimulation of at least one of the infraorbital, lingual, or inferior alveolar nerves (responsive type neurons). The latencies of spike responses to stimulation of the trigeminal sensory branches ranged from 4.0 to 21.0 ms, which were shorter than those of superior salivatory neurons. Impulses of both A-beta and A-delta afferent fibers of the trigeminal nerve were found to be effective for activation of inferior salivatory neurons. The convergence of excitatory inputs from more than one sensory nerve was found in most of the responsive type neurons (73%).