Anatomical Relationship Between the Lingual Nerve and Submandibular Duct.

ABSTRACT: The purpose of this study was to investigate the anatomical relationship between the lingual nerve and submandibular duct. This study included 1403 patients with submandibular or sublingual gland diseases who underwent intraoral removal of submandibular gland sialoliths, submandibular glands, or sublingual glands. Of all patients, 33 patients underwent bilateral surgeries. All surgeries were performed a single surgeon, and the anatomical relationship between the lingual nerve and submandibular duct was always identified intraoperatively and recorded in the operation recorded. The anatomical relationship was investigated based on the intraoperative findings. The lingual nerve which crosses above the submandibular duct was detected in 8 of 1436 sides (0.6%). There were 4 in the right sides and 4 in the left sides. The lingual nerve below the submandibular gland was seen in 99.4%. Although the lingual nerve crosses above the submandibular duct with a rarer incidence, surgeons should beware of injuring the lingual nerve during intraoral salivary gland surgery.

Perineural Invasion and Perineural Tumor Spread in Head and Neck Cancer.

Perineural invasion (PNI), the neoplastic invasion of nerves, is a common pathologic finding in head and neck cancer that is associated with poor clinical outcomes. PNI is a histologic finding of tumor cell infiltration and is distinct from perineural tumor spread (PNTS), which is macroscopic tumor involvement along a nerve extending from the primary tumor that is by definition more advanced, being radiologically or clinically apparent. Despite widespread acknowledgment of the prognostic significance of PNI and PNTS, the mechanisms underlying its pathogenesis remain largely unknown, and specific therapies targeting nerve invasion are lacking. The use of radiation therapy for PNI and PNTS can improve local control and reduce devastating failures at the skull base. However, the optimal volumes to be delineated with respect to targeting cranial nerve pathways are not well defined, and radiation can carry risks of major toxicity secondary to the location of adjacent critical structures. Here we examine the pathogenesis of these phenomena, analyze the role of radiation in PNI and PNTS, and propose guidelines for radiation treatment design based on the best available evidence and the authors' collective experience to advance understanding and therapy of this ominous cancer phenotype.

Parasympathectomy increases resting secretion of the submandibular gland in minipigs in the long term.

Parasympathectomy leads to retrogressive alteration and dysfunction of the submandibular gland (SMG) within 1 month, but its long-term effect is unclear. Excessive secretion is observed in half of the patients 4-6 months after SMG transplantation, which completely denervates the gland. Here, we investigated the long-term effect of parasympathectomy on the secretion of SMGs in minipigs. The results showed that the resting salivary secretion of SMGs decreased by 82.9% of that in control at 2 months after denervation, but increased by 156% at 6 months. Although experiencing an atrophic period, the denervated glands regained their normal morphology by 6 months. The expression of the function-related proteins, including muscarinic acetylcholine receptor (mAChR) 3, aquaporin 5 (AQP5), tight junction protein claudin-3, and claudin-4 was decreased at 2 months after denervation. Meanwhile, the protein expression of stem cell markers, including sex-determining region Y-box 2 and octamer-binding transcription factor 4, and the number of Ki67+ cells were significantly increased. However, at 6 months after denervation, the expression of mAChR3, AQP5, claudin-1, claudin-3, and claudin-4 was significantly raised, and the membrane distribution of these proteins was increased accordingly. The autonomic axonal area of the glands was reduced at 2 months after denervation but returned to the control level at 6 months, suggesting that reinnervation took place in the long term. In summary, parasympathectomy increases resting secretion of the SMGs in the long term with a possible mechanism involving improved transepithelial fluid transport. This finding may provide a new strategy for xerostomia treatment.

Accessory Submandibular Salivary Gland Forming a "Horseshoe" With the Main Submandibular Salivary Gland: A Unique Variation.

Presence of accessory submandibular salivary gland (ASSG) is an extremely rare variation. Knowledge of its relations could be very useful to oral and maxillofacial surgeons, head and neck surgeons, and radiologists. During dissection classes, an ASSG was noted between the mylohyoid and hyoglossus muscles. The main submandibular salivary gland had superficial and deep parts. The deep part was narrow and measured about 5 cm. The lingual nerve passed between the superficial and deep parts. The accessory submandibular gland was situated below and parallel to the deep part of SSG. It also measured 5 cm. The ASSG had its own duct, which joined the duct of main gland. The ASSG and the deep part of the SSG were united at the lateral border of geniohyoid muscle to give a characteristic "horseshoe" appearance. The ASSG overlapped both lingual and hypoglossal nerves.

Feasibility of endoscopic submandibular ganglion neurectomy for drooling.

We performed endoscopic transoral neurectomy of the submandibular and sublingual glands to treat drooling. We bilaterally operated two adult cases with treatment-resistant drooling. In these patients, conventional treatment had failed. Repeated botilinum toxin type A (BOTOX®, Abdi Ibrahim Pharmaceutical Company, Istanbul, Turkey) injections had been effective but were becoming less so. The patients benefited from surgery in that their saliva scores decreased. No issue emerged over 6 months of follow-up. Endoscopic transoral neurectomy of the submandibular and sublingual glands reduces saliva production and allows management of drooling in treatment-resistant patients. Laryngoscope, 127:1604-1607, 2017.

Parasympathectomy increases resting salivary secretion in normal and irradiated submandibular glands of rats.

Fluid and ion secretion from the submandibular gland (SMG) is mainly regulated by parasympathetic nerves. This study evaluated the effect of parasympathectomy on salivary secretion from normal and irradiated rat SMGs from 1 to 24 wk after denervation. Although stimulated salivary secretion was significantly lower in denervated SMGs compared with contralateral self-controls, the resting salivary flow rates were markedly higher in the denervated SMGs at 1, 12, and 24 wk after denervation. The levels of muscarinic acetylcholine M1 and M3 receptors, as well as of aquaporin 5, were up-regulated. Notably, although irradiated SMGs showed significantly lower resting and stimulated salivary secretion rates than non-irradiated SMGs, the resting salivary secretion rates of the irradiated and denervated SMGs were markedly higher than seen in the irradiated self-control SMGs at 1, 12, and 24 wk after parasympathectomy, and were even higher than seen in the non-irradiated sham-operated rats. The expression of M1 and M3 receptors was similarly elevated. Taken together, our results suggest that parasympathetic denervation increases resting salivary secretion of both normal and irradiated SMGs. This approach might provide a potential modality for relieving radiation-induced xerostomia, which is a common complication following treatment of head and neck cancer.

Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

[Topographic features of lingual nerve and its relationship with other anatomical structures in maxillolingual groove].

By surgical interventions in maxillolingual groove area one should consider anatomical variations and topography of vessels, glands ducts and lingual nerve to prevent their injury. At the Department of Operative Surgery and Topographic Anatomy of the First Moscow State Medical University named after I.M. Sechenov we carried out anatomical study on cadavers (men and women, n=30).The study revealed topographical features of the lingual nerve and its relationship to other anatomical structures in the maxillolingual groove. It was found out that at the level of the second molar (96%) lingual nerve "crosses" duct of submandibular salivary gland, at the level of the third molar lingual nerve is located under the duct and lateral to it, closer to the inner surface of the body of the mandible. At the level of the first molar lingual nerve is located above and medial to Wharton duct and passes along sublingual-lingual muscles (m.hyoglossus).

Transoral submandibular ganglion neurectomy: an anatomical feasibility study.

BACKGROUND: The management of sialorrhea can be difficult for both the patient and the clinician. Current management includes behavioral modification, anticholinergics, botulinum injections, and a variety of surgical options, which all have demonstrated some efficacy. As minimally invasive procedures flourish, we explore the feasibility of highly selective transoral submandibular neurectomy (TOSN) for the management of sialorrhea. METHODS: Ten human cadaver dissections of the floor of mouth were performed bilaterally, for a total of 20 separate cases. An intraoral technique for highly selective, submandibular ganglion neurectomy is demonstrated. RESULTS: A transoral submandibular ganglion neurectomy was performed in 10 cadavers (20 neurectomies) easily and reliably, without injury to the submandibular duct or the main trunk of the lingual nerve. CONCLUSION: Transoral submandibular neurectomy is an attractive addition to the armamentarium of surgical options for the treatment of medically intractable sialorrhea. Further study in selected patients would need to be performed to demonstrate clinical feasibility.

Transient activation of hedgehog pathway rescued irradiation-induced hyposalivation by preserving salivary stem/progenitor cells and parasympathetic innervation.

PURPOSE: To examine the effects and mechanisms of transient activation of the Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer. EXPERIMENTAL DESIGN: Mouse salivary glands and cultured human salivary epithelial cells were irradiated by a single 15-Gy dose. The Hedgehog pathway was transiently activated in mouse salivary glands, by briefly overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), parasympathetic innervation, and expression of related genes were examined by flow cytometry, salisphere assay, immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA. RESULTS: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the Bmi1 and Chrm1-HB-EGF pathways. The preservation of parasympathetic innervation was associated with the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation. CONCLUSIONS: These findings suggest that transient activation of the Hedgehog pathway has the potential to restore salivary gland function after irradiation-induced dysfunction.

Functional salivary gland regeneration by transplantation of a bioengineered organ germ.

Salivary gland hypofunction, also known as xerostomia, occurs as a result of radiation therapy for head cancer, Sjögren's syndrome or aging, and can cause a variety of critical oral health issues, including dental decay, bacterial infection, mastication dysfunction, swallowing dysfunction and reduced quality of life. Here we demonstrate the full functional regeneration of a salivary gland that reproduces the morphogenesis induced by reciprocal epithelial and mesenchymal interactions through the orthotopic transplantation of a bioengineered salivary gland germ as a regenerative organ replacement therapy. The bioengineered germ develops into a mature gland through acinar formations with a myoepithelium and innervation. The bioengineered submandibular gland produces saliva in response to the administration of pilocarpine and gustatory stimulation by citrate, protects against oral bacterial infection and restores normal swallowing in a salivary gland-defective mouse model. This study thus provides a proof-of-concept for bioengineered salivary gland regeneration as a potential treatment of xerostomia.

In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins.

A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.

Botox(®) to reduce drooling in a paediatric population with neurological impairments: a Phase I study.

BACKGROUND: The treatment of drooling in a paediatric population with neurological impairments is clinically challenging. Surgery is considered invasive, while behaviour modification techniques, correction of situational factors and oral-motor therapy do not always produce sustained improvement. In recent years the use of Botox® to decrease drooling has been investigated. AIMS: To review the clinical data from a Drooling Treatment Project for children with neurological impairments and to establish the validity of the drooling severity and frequency rating scales, establishing Phase I-level information about the therapeutic use of submandibular salivary gland injections of Botox® in various contexts. METHOD & PROCEDURES: A retrospective, explanatory design was used to review the data. Nine children, seven with cerebral palsy and two with operculum syndrome, ranging in age from 5 to 17 years (mean = 9;3 years) were included. Drooling was assessed by qualified speech-language therapists using drooling rating scales, in five different situations and at different time points pre- and post-Botox® injection up to 6 months. Quantitative and qualitative analyses were computed. Parents'/primary caregivers' perceptions of drooling and treatment with Botox® were also considered using an interview form and a quality of life questionnaire. OUTCOMES & RESULTS: Statistically significant reductions in drooling with large effect sizes were obtained in the communicating and general appearance situations. There was a difference in the pattern of response between the children with cerebral palsy and those with operculum syndrome. Discrepancies between the parents and the speech-language therapists regarding the context of drooling reduction were found. Most parents/primary caregivers felt their children's lives and their own had improved following the Botox® injection and would repeat the treatment. The drooling rating scales were a valid method to assess drooling in a clinical situation. CONCLUSIONS & IMPLICATIONS: In the clinical setting of the Drooling Treatment Project, the results indicated that the context in which drooling occurs is an important factor and suggested the value of considering the situational context when making drooling judgments. Further, there was a difference in the pattern of response between the children with cerebral palsy and those with operculum syndrome, suggesting that aetiology may be involved in the response to Botox®.

Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration.

An irreversible loss of salivary gland function often occurs in humans after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjögren's syndrome and in genetic syndromes affecting gland development. The permanent loss of gland function impairs the oral health of these patients and broadly affects their quality of life. The regeneration of functional salivary gland tissue is thus an important therapeutic goal for the field of regenerative medicine and will likely involve stem/progenitor cell biology and/or tissue engineering approaches. Recent reports demonstrate how both innervation of the salivary gland epithelium and certain growth factors influence progenitor cell growth during mouse salivary gland development. These advances in our understanding suggest that developmental mechanisms of mouse salivary gland development may provide a paradigm for postnatal regeneration of both mice and human salivary glands. Herein, we will discuss the developmental mechanisms that influence progenitor cell biology and the implications for salivary gland regeneration.

The changes in submandibular gland size and function following chorda tympani section.

To investigate the effects of chorda tympani section on submandibular gland size and function in the early (postoperative day 7) and late (postoperative month 6) postoperative period by ultrasonography, scintigraphy, and biochemical analysis of the saliva patients with unilateral chronic otitis media. One-hundred and thirty patients (46 males and 84 females) who were ≥16 years of age and diagnosed with unilateral chronic otitis media and for whom type 1 tympanoplasty was indicated in 1st Outpatient Clinic of Kartal Dr. Lutfi Kirdar Training and Research Hospital between August 2004 and February 2007 were enrolled in the study. Of 130 patients, 102 patients who were eligible and gave written approval were included in the study. However, of these patients 99 underwent type 1 tympanoplasty and 3 had a canal down mastoidectomy. In 99 patients, chorda tympani nerves of 16 were cut, but 3 patients had to be excluded due to allergic reactions. Before the operation, bilateral submandibular gland ultrasonography was performed on all patients and the anterior-posterior length, the frontal lateral-medial width (transverse), and the paramandibular depth of both submandibular glands were measured. In scintigraphic examinations, perfusion index (PI), uptake ratio and excretion fraction were measured. Then, in biochemical analysis of the saliva the levels of sodium, potassium, chloride, calcium, magnesium, amylase, and the values of pH and density were assessed by the saliva collection through Wharton duct. In the statistical comparison of operated and healthy side of the patients with respect to these parameters Mann-Whitney U test, and in intragroup analysis Wilcoxon test was used. The volume of the submandibular gland of the operated side was significantly lower compared to the healthy side in postoperative month 6 (P < 0.05). According to the baseline volume of the submandibular gland of the healthy side, the increase in postoperative day 7 and month 6 were found to be statistically insignificant (P > 0.05). According to the baseline PI value, the decreases in the PI value in the postoperative day 7 and postoperative month 6 were statistically significant (P < 0.01). The uptake ratio of the patients was lower in the postoperative day 7 and month 6 than those at the baseline; however, the difference was statistically insignificant (P > 0.05). The excretion fraction values in the postoperative day 7 were significantly lower than the baseline values (P < 0.05), whereas the decrease in the EF values in the postoperative month 6 were statistically insignificant (P > 0.05). In conclusion, the present study was the first in the literature in which three parameters of assessment, such as ultrasonography, scintigraphy, and biochemical analysis, were used to determine the changes in submandibular gland size and function following the chorda tympani section. After chorda tympani section, the volume of submandibular gland decreased in the late postoperative period. Moreover, chorda tympani section led to decrease in the saliva secretion and the PI value of the patients in the early and late postoperative period.

Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.

The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during organogenesis are poorly understood. We report that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number and morphogenesis of keratin 5-positive epithelial progenitor cells. These effects were rescued with an acetylcholine analog. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and epidermal growth factor receptor, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Parasympathetic innervation maintained the epithelial progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration.

Roles of lysosomal proteolytic systems in AQP5 degradation in the submandibular gland of rats following chorda tympani parasympathetic denervation.

Chorda tympani denervation (CTD) of rats was earlier shown to result in loss of submandibular gland (SMG) weight (at only 1 wk) and in continued reduction in aquaporin 5 (AQP5) protein expression (until 4 wk), without affecting its mRNA synthesis (Li X, Azlina A, Karabasil MR, Purwanti N, Hasegawa T, Yao C, Akamatsu T, Hosoi K. Am J Physiol Gastrointest Liver Physiol 295: G112-G123, 2008). The present study indicated that despite elevation of bax, a proapoptosis protein, by CTD, the operation also increased the level of bcl-2, an antiapoptosis protein, in the SMG. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay) showed no increase in the number of apoptotic cells in the SMG. CTD, however, induced strongly and transiently (at 1-3 days) the protein expression of LC3B-II, a marker protein of autophagosomes, suggesting that the reduction in the gland weight was due to onset of autophagy by CTD. Upon CTD, Lamp2, a lysosomal marker, gradually increased in amount, reaching a peak at the 14th day. Immunohistochemical analysis revealed an increase in the number of lysosome-like structures positive for both AQP5 and Lamp2 in the acinar cells of the SMG after CTD; similar changes were observed also for AQP5 and LC3Bs. These data suggest that AQP5 in the SMG entered autophagosomes and/or lysosomes for degradation upon CTD. In vitro AQP5-degrading activity was found in the SMG extracts, and such activity was shown to be increased by CTD. Inhibitor experiments implied cathepsins B and L to be candidate enzymes for this degradation under normal and CTD conditions, respectively.

Nerve cell bodies and small ganglia in the connective tissue stroma of human submandibular glands.

The objective of the study was to investigate the presence and distribution of nerve cell bodies and small ganglia in the stroma of human submandibular gland. A retrospective immunohistochemical study in 13 human submandibular glands, fixed in neutral buffered formalin and embedded in paraffin wax, was undertaken. Six glands were excised in the course of radical neck dissection for oral squamous cell carcinoma and were disease-free, six showed sialadenitis, and one was involved by tuberculosis. Primary antibodies applied were neuron specific enolase, synaptophysin, and glial fibrilliary acidic protein. Neuron specific enolase and synaptophysin positive nerve cell bodies and small ganglia were found in 8/13 and 13/13 glands, respectively. They were found in the interlobular connective tissue stroma of human SMG, in close association to salivary parenchymal cells and blood vessels, and some of them were incorporated in GFAP positive peripheral nerves. To our knowledge, nerve cell bodies and small ganglia have been described only in the connective tissue stroma of autotransplanted human SMG and their functional importance is not clear.