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.

Effect of High Fat and Fructo-Oligosaccharide Consumption on Immunoglobulin A in Saliva and Salivary Glands in Rats.

Consumption of indigestible dietary fiber increases immunoglobulin A (IgA) levels in saliva. The purpose of this study is to clarify the synergistic effect of the intake of a high amount of fats and indigestible dietary fiber on IgA levels in saliva and submandibular glands (SMG). Seven-week-old Wistar rats were fed a low-fat (60 g/kg) fiberless diet, low-fat fructo-oligosaccharide (FOS, 30 g/kg) diet, high-fat (220 g/kg) fiberless diet, or high-fat FOS diet for 70 days. The IgA flow rate of saliva (IgA FR-saliva) was higher in the low-fat FOS group than in the other groups (p < 0.05). Furthermore, the concentration of tyrosine hydroxylase (a marker of sympathetic nerve activation) in the SMG was higher in the low-fat FOS group (p < 0.05) and positively correlated with the IgA FR-saliva (rs = 0.68. p < 0.0001. n = 32) in comparison to that in the other groups. These findings suggest that during low-fat FOS intake, salivary IgA levels may increase through sympathetic nerve activation.

Orexin A and B in the rat superior salivatory nucleus.

Orexin (OX), which regulates sleep and wakefulness and feeding behaviors has 2 isoforms, orexin-A and -B (OXA and OXB). In this study, the distribution of OXA and OXB was examined in the rat superior salivatory nucleus (SSN) using retrograde tracing and immunohistochemical and methods. OXA- and OXB-immunoreactive (-ir) nerve fibers were seen throughout the SSN. These nerve fibers surrounded SSN neurons retrogradely labeled with Fast blue (FB) from the corda-lingual nerve. FB-positive neurons had pericellular OXA- (47.5%) and OXB-ir (49.0%) nerve fibers. Immunohistochemistry for OX receptors also demonstrated the presence of OX1R and OX2R in FB-positive SSN neurons. The majority of FB-positive SSN neurons contained OX1R- (69.7%) or OX2R-immunoreactivity (57.8%). These neurons had small and medium-sized cell bodies. In addition, half of FB-positive SSN neurons which were immunoreactive for OX1R (47.0%) and OX2R (52.2%) had pericellular OXA- and OXB-ir nerve fibers, respectively. Co-expression of OX1R- and OX2R was common in FB-positive SSN neurons. The present study suggests a possibility that OXs regulate the activity of SSN neurons through OX receptors.

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.

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.

Autonomic reinnervation and functional regeneration in autologous transplanted submandibular glands in patients with severe keratoconjunctivitis sicca.

Autologous submandibular gland (SMG) transplantation has been proved to ameliorate the discomforts in patients with severe keratoconjunctivitis sicca. The transplanted glands underwent a hypofunctional period and then restored secretion spontaneously. This study aims to investigate whether autonomic nerves reinnervate the grafts and contribute to the functional recovery, and further determine the origin of these nerves. Parts of the transplanted SMGs were collected from the epiphora patients, and a rabbit SMG transplantation model was established to fulfill the serial observation on the transplanted glands with time. The results showed that autonomic nerves distributed in the transplanted SMGs and parasympathetic ganglionic cells were observed in the stroma of the glands. Low-dense and unevenly distributed cholinergic axons, severe acinar atrophy and fibrosis were visible in the patients' glands 4-6 months post-transplantation, whereas the cholinergic axon density and acinar area were increased with time. The acinar area or the secretory flow rate of the transplanted glands was statistically correlated with the cholinergic axon density in the rabbit model, respectively. Meanwhile, large cholinergic nerve trunks were found to locate in the temporal fascia lower to the gland, and sympathetic plexus concomitant with the arteries was observed both in the adjacent fascia and in the stroma of the glands. In summary, the transplanted SMGs are reinnervated by autonomic nerves and the cholinergic nerves play a role in the morphological and functional restoration of the glands. Moreover, these autonomic nerves might originate from the auriculotemporal nerve and the sympathetic plexus around the supplying arteries.

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.

Electrophysiological study on sensory nerve activity from the submandibular salivary gland in rats.

To evaluate the role of afferent information from the salivary gland, we analyzed the neural activity of the sensory nerve innervating the submandibular gland in anesthetized rats. The sensory nerves running through the parasympathetic nerve supply responded to mechanical pressure applied to the surface of the main duct and the body of the gland, whilst those in the sympathetic nerve supply responded only to the body of the gland. The sensory nerves in the sympathetic and parasympathetic nerve routes responded to pressure in the duct system produced by a retrograde injection of saline into the main duct. The threshold pressure for production of afferent discharges was higher than the maximum secretory pressure evoked by electrical stimulation of the parasympathetic secretory nerve. The retrograde ductal injection of drugs related to the inflammatory process (capsaicin and bradykinin) evoked intense multi-unit discharges in the sensory nerves of both routes. The sensory nerve in the sympathetic route was responsive to ligation of the artery to the gland. These results suggest that sensory nerves in the sympathetic and parasympathetic routes mainly conduct noxious information, and that those in the sympathetic route are responsive to ischemia and may control blood flow of the gland.

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.

Altered autophagy and sympathetic innervation in salivary glands from high-fat diet mice.

OBJECTIVE: to investigate the effects of a high fat diet (HFD) on salivary glands in vivo, in a mouse model. In particular, whether it will induce the appearance of fat cells in salivary glands, alterations related to autophagy, mTOR pathway and sympathetic innervation. DESIGN: 27 adult female ICR mice were separated in six groups. Three groups fed with (HFD) containing 55% fat, for one, two and three month and another three groups fed with normal diet (2.7% of fat), for the same time periods. The submandibular glands and liver were dissected and part homogenized for protein analyses and part fixed in formalin for histological analyses. RESULTS: After three months the HFD fed mice total body weight fold change increased compared to controls. The Oil Red O staining showed no fat cells deposit in salivary gland however a large increase was observed in liver after three months of HFD. Adiponectin levels were significantly decreased in the HFD group after three months. The group fed with HFD for three months showed increased conversion of the LC3 autophagy marker in salivary gland. mTOR showed no activation regarding the time point studied. Tyrosine hydroxylase significantly decreased after two and three month of HFD. CONCLUSION: HFD caused several changes after three months however the earliest change was noticed after two months regarding sympathetic innervation. This suggests neural alteration may drive other diet induced changes in salivary glands. These early changes may be the starting point for longer term alterations of salivary glands with alterations in diet.

[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).

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.

Pre- and Post-synaptic Effects of Botulinum Toxin A on Submandibular Glands.

Intraglandular injection of botulinum toxin type A (BoNT/A) is an effective treatment for sialorrhea. Despite numerous experimental and clinical studies on inhibition of saliva section by BoNT/A, the proteolysis of synaptosomal-associated protein 25 (SNAP-25) following BoNT/A treatment has not yet been confirmed in the salivary gland after injection of BoNT/A. More important, it is not known whether BoNT/A exerts a direct effect in acinar cells. Here, we show that injection of BoNT/A into the rat submandibular gland (SMG) decreased salivary flow in a dose-dependent manner; the inhibitory effect lasted at least 4 wk, and salivary flow recovered to normal levels by 12 wk. During the inhibitory period, SMG neurons and synapses expressed lower levels of full-length SNAP-25, and cleavage of SNAP-25 was observed, as indicated by detection of reduced molecular weight SNAP-25 using Western blotting. In addition, the water channel aquaporin 5 (AQP5) was downregulated and abnormally distributed in rat SMG after injection of BoNT/A. The direct effects of BoNT/A on AQP5 expression and distribution were assessed in vitro to exclude the influence of BoNT/A-induced inhibitory neurotransmission. In stable GFP-AQP5-transfected SMG-C6 cells, treatment with BoNT/A reduced the cell surface protein level of AQP5 in a dose- and time-dependent manner without affecting total AQP5 protein expression. Cell surface biotinylation and immunofluorescence demonstrated translocation of AQP5 from the membrane to the cytoplasm, which was confirmed by decreased levels of AQP5 protein in the membrane fraction and increased levels in the cytoplasmic fraction, suggestive of AQP5 redistribution. Taken together, these results indicated that BoNT/A reversibly decreased saliva secretion in rat SMGs through not only the presynaptic SNAP-25 cleavage but also the postsynaptic AQP5 redistribution. These data provide the first evidence for a direct effect of BoNT/A on the salivary gland.

Submandibular parasympathetic gangliogenesis requires sprouty-dependent Wnt signals from epithelial progenitors.

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found that deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation, and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons, and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus, K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance.

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.

Reinnervated nerves contribute to the secretion function and regeneration of denervated submandibular glands in rabbits.

This study aimed to investigate the contribution of redistributed nerves in the secretory function and regeneration of a denervated submandibular gland (SMG). The postganglionic parasympathetic and sympathetic denervated SMGs of rabbits were wrapped in polyester or acellular dermal matrices to block nerve regeneration either partially or completely. Submandibular glands were removed 4, 8, 16, and 24 wk after the operation and examined histologically. Furthermore, the aquaporin-5 (AQP5), muscarinic-3 (M3), and ß1-adrenergic receptors were evaluated by immunofluorescence and western blot analysis. After denervation, salivary flow was decreased and acinar cells were atrophic, and the expression levels of the M3, ß1-adrenergic, and AQP5 receptors were decreased. However, both impaired secretion function and atrophic parenchyma were gradually ameliorated with the growing redistribution of parasympathetic and sympathetic nerves. Apoptosis was markedly inhibited and expression of the M3, ß1-adrenergic, and AQP5 receptors was increased after reinnervation. In contrast, SMGs without reinnervated nerves maintained hyposecretion and atrophic parenchyma. In conclusion, reinnervated nerves in a rabbit's denervated SMG played an important role in the secretion function and regeneration of SMGs via up-regulation of the expression of neurotransmitter receptors and AQP5.

DLK1 regulates branching morphogenesis and parasympathetic innervation of salivary glands through inhibition of NOTCH signalling.

BACKGROUND INFORMATION: Delta-like proteins 1 and 2 (DLK1, 2) are NOTCH receptor ligands containing epidermal growth factor-like repeats, which regulate NOTCH signalling. We investigated the role of DLK and the NOTCH pathway in the morphogenesis of the submandibular salivary glands (SMGs), using in vitro organotypic cultures. RESULTS: DLK1 and 2 were present in all stages of SMG morphogenesis, where DLK1 inhibited both NOTCH activity and SMG branching. The addition of NOTCH inhibitory agents, either soluble DLK1 (sDLK1) or N-[N-(3, 5-difluorophenacetyl-L-alanyl]-S-phenylglycine t-buthyl ester (DAPT), to the SMG culture medium did not affect the rate of cell proliferation, but induced a strong reduction in SMG branching, increased epithelial apoptosis, and impaired innervation of the epithelial end buds by local parasympathetic ganglion neurons. SMG innervation could be restored by the acetylcholine analog carbachol (CCh), which also rescued cytokeratin 5 (CK5(+))-expressing epithelial progenitor cells. Despite this, CCh failed to restore normal branching morphogenesis in the presence of either sDLK1 or DAPT. However, it improved recovery of branching morphogenesis in SMGs, once DLK1 or DAPT were removed from the medium. CONCLUSIONS: Our data suggest that DLK1 regulates SMGs morphogenesis and parasympathetic nerve fibre outgrowth through inhibition of NOTCH signalling.

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.