The effects of ketogenic and chitosan-based diets on submandibular salivary gland in rat model: a comparative histological study.

OBJECTIVE: This study was carried out in the submandibular salivary glands (SSGs) of rats to demonstrate the effect of a ketogenic diet (KD) in comparison with dietary chitosan supplementation. METHOD: Eighteen albino rats were randomly divided into three equal groups of six animals each. Rats in Group I were fed a balanced diet and considered controls. Meanwhile, those of Groups II and III were fed a KD, a balanced diet with high molecular weight chitosan, respectively. After 45 days, rats were euthanized, and the SSGs were dissected carefully for staining with hematoxylin and eosin (H&E), alpha-smooth muscle actin (α-SMA) immunohistochemical staining, and Congo red special stain. Quantitative data from α-SMA staining and Congo red staining were statistically analyzed using one-way ANOVA followed by Tukey's multiple comparisons post hoc test. RESULTS: Regarding Congo red and α-SMA staining, one-way ANOVA revealed a significant difference between the three groups. For α-SMA staining and Congo red staining, Group II had the highest mean values of 91.41 ± 3.30 and 68.10 ± 5.04, respectively, while Group I had the lowest values of 56.13 ± 3.96 and 16.87 ± 2.19, respectively. Group III had mean values of 60.70 ± 3.55 for α-SMA and 19.50 ± 1.78 for Congo red. Tukey's multiple comparisons post hoc test revealed significant differences between groups I & II and between groups II & III (P < 0.05). Meanwhile, there was a nonsignificant difference between groups I and III (P > 0.05). CONCLUSION: A KD has a deleterious effect on rats' SSG whatever the test we used, and dietary chitosan supplementation ameliorates these damaging effects.

Comparing development and regeneration in the submandibular gland highlights distinct mechanisms.

A common question in organ regeneration is the extent to which regeneration recapitulates embryonic development. To investigate this concept, we compared the expression of two highly interlinked and essential genes for salivary gland development, Sox9 and Fgf10, during submandibular gland development, homeostasis and regeneration. Salivary gland duct ligation/deligation model was used as a regenerative model. Fgf10 and Sox9 expression changed during regeneration compared to homeostasis, suggesting that these key developmental genes play important roles during regeneration, however, significantly both displayed different patterns of expression in the regenerating gland compared to the developing gland. Regenerating glands, which during homeostasis had very few weakly expressing Sox9-positive cells in the striated/granular ducts, displayed elevated expression of Sox9 within these ducts. This pattern is in contrast to embryonic development, where Sox9 expression was absent in the proximally developing ducts. However, similar to the elevated expression at the distal tip of the epithelium in developing salivary glands, regenerating glands displayed elevated expression in a subpopulation of acinar cells, which during homeostasis expressed Sox9 at lower levels. A shift in expression of Fgf10 was observed from a widespread mesenchymal pattern during organogenesis to a more limited and predominantly epithelial pattern during homeostasis in the adult. This restricted expression in epithelial cells was maintained during regeneration, with no clear upregulation in the surrounding mesenchyme, as might be expected if regeneration recapitulated development. As both Fgf10 and Sox9 were upregulated in proximal ducts during regeneration, this suggests that the positive regulation of Sox9 by Fgf10, essential during development, is partially reawakened during regeneration using this model. Together these data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands.

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.

Negative effects of submandibular botulinum neurotoxin A injections on oral motor function in children with drooling due to central nervous system disorders.

AIM: The aims of this study were: (1) to determine the incidence and nature of adverse effects on oral motor function after first injections of botulinum neurotoxin A (BoNT-A) in submandibular glands for excessive drooling in children with central nervous system disorders; and (2) to identify independent predictors of these adverse effects. METHOD: A cohort study involved 209 children (123 males, 86 females, aged 4-27y, median 8y 4mo), who received submandibular BoNT-A injections for drooling. Adverse effects were categorized into swallowing, eating, drinking, articulation, and other problems. Univariable logistic regression was used to study differences in patients with and without adverse effects. Possible predictors were identified using multivariable logistic regression. RESULTS: Transient adverse effects occurred in 33% of the 209 BoNT-A treatments. Almost 80% of these were mild, versus 8.7% severe. Approximately 54% of the adverse effects spontaneously resolved within 4 weeks; 3% still existed after 32 weeks. A diagnosis of cerebral palsy, higher range of BoNT-A dosage, and a pre-treatment drooling quotient <18% were found to be independent predictors of adverse effects. INTERPRETATION: Before using submandibular BoNT-A injections for drooling, potential adverse effects should be discussed. Oral motor function needs to be monitored, because existing dysphagia may be worsened. The identified clinical predictors could be helpful to optimize patient selection.

Swallowing Function After Continuous Neuromuscular Electrical Stimulation of the Submandibular Region Evaluated by High-Resolution Manometry.

Although neuromuscular electrical stimulation (NMES) is increasingly used in dysphagia therapy, patient responses to NMES are inconsistent and conflicting results have been reported. This, together with a lack of information about the effects of NEMS on the swallowing process, has led to an ongoing debate about its impact on swallowing function. In order to address this, we set out to (i) collect baseline information on the physiological effects of NMES on the complex pharyngeal phase of swallowing and (ii) to compare two different stimulation protocols. In doing so, we provide information useful for evaluating the therapeutic effectiveness of NMES on the swallowing process. In a prospective study, 29 healthy participants performed water swallows after receiving continuous NMES for 10 min. The stimulus was applied in the submandibular region using one of two different stimulation protocols: low-frequency stimulation (LFS) and mid-frequency stimulation (MFS). Swallowing parameters of the pharynx and UES were measured using high-resolution manometry. Maximum tongue base pressure increased by 8.4% following stimulation with the MFS protocol. Changes in UES function were not found. LFS stimulation did not result in any significant changes in the parameters examined. The MFS protocol enhances tongue base retraction during swallowing in healthy volunteers. The magnitude of the effect, however, was small, possibly due to the ability of healthy subjects to compensate for external influences, such as NMES, and may actually prove to be much greater in patients with diminished tongue base retraction. Thus, further studies are needed to determine whether a similar effect is also achievable in dysphagic patients with impaired bolus propulsion, possibly allowing MFS stimulation of the tongue base region to be used as an additional treatment tool.

Digestive and Nondigestive Functions of Rodents' Salivary Glands.

Major salivary glands play a role not only in digestion, but also in regulation of other functions in rodents. In this review, we analyzed and summarized the data about the rodents' parotid, submandibular and sublingual salivary glands functions, which is not limited to the production of saliva and action of its hydrolytic enzymes on food in the oral cavity. In recent decades significantly expanded understanding of major salivary glands nondigestive functions. They are involved in excretion of metabolic products, maintaining fluid and electrolyte balance. Special attention has been paid to the characteristics of specific (parotin, sialorphin, etc.) and nonspecific (epidermal growth factor, nerve growth factor, kallikrein, etc.) active substances of the major salivary glands and their involvement in wound healing, mineral metabolism, regulation of hematopoiesis and immunity system. Summarized and analyzed major salivary glands endocrine function in the organs and systems. Available literature data suggest: the structure of the major salivary glands, as well as the synthesis and secretion of a number of biologically active substances are controlled by sex hormones. In turn, these biologically active factors of the salivary glands, as epidermal growth factor, and parotin, sialorphin, whose expression is regulated by androgens, have an impact on the morphological and functional state of the gonads. Thus, major salivary glands operate a wide range of functions and involved in the regulation of sexual behavior of reproductive function and maintaining homeostasis in the body.

Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1.

The final stage in exocrine secretion involves translocation of vesicles from their storage areas to the apical membrane. We show that actin-coated secretory vesicles of the exocrine pancreas travel this distance over bundles of specialized actin cables emanating from the apical plasma membrane. These bundles are stable structures that require constant G-actin incorporation and are distinct from the actin web that surrounds the exocrine lumen. The murine mammalian Diaphanous-related formin 1 (mDia1) was identified as a generator of these cables. The active form of mDia1 localized to the apical membrane, and introduction of an active form of mDia1 led to a marked increase in bundle density along the lumen perimeter. Compromising formation of the cables does not prevent secretion, but results in disorganized trafficking and fusion between secretory vesicles. Similar apical secretory tracks were also found in the submandibular salivary glands. Together with previous results that identified a role for Diaphanous in apical secretion in tubular organs of Drosophila, the role of Diaphanous formins at the final stages of secretion appears to be highly conserved.

Two-stage autotransplantation of human submandibular gland: a novel approach to treat postradiogenic xerostomia.

Xerostomia is a persistent side effect of radiotherapy (RT), which severely reduces the quality of life of the patients affected. Besides drug treatment and new irradiation strategies, surgical procedures aim for tissue protection of the submandibular gland. Using a new surgical approach, the submandibular gland was autotransplanted in 6 patients to the patient's forearm for the period of RT and reimplanted into the floor of the mouth 2-3 months after completion of RT. Saxon's test was performed during different time points to evaluate patient's saliva production. Furthermore patients had to answer EORTC QLQ-HN35 questionnaire and visual analog scale. Following this two-stage autotransplantation, xerostomia in the patients was markedly reduced due to improved saliva production of the reimplanted gland. Whether this promising novel approach is a reliable treatment option for RT patients in general should be evaluated in further studies.

[Activation of transient receptor potential vanilloid subtype 1 serves as a novel pathway to modulate secretion in submandibular gland].

Our work focused on the studies on the expression and function of transient receptor potential vanilloid subtype 1 (TRPV1) in the submandibular gland. By using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence, our data demonstrated the expression and distribution characteristics of TRPV1 in rabbit and human submandibular glands, as well as rat submandibular gland cell line SMG-C6. Furthermore, the possible intracellular signal molecules involved in the TRPV1-modulated saliva secretion were explored. Activation of TRPV1 increased the intracellular Ca(2+) concentration, upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediate water secretion through transcellular pathway, and led to AQP5 redistribution. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the TRPV1-regulated AQP5 content. Besides, TRPV1 activation also modulated the expression, distribution, and function of tight junction protein, and increased paracellular permeability. ERK1/2 and myosin light chain 2 (MLC2) were responsible for the regulation of TRPV1on tight junction properties. Taken together, our work suggested that TRPV1 was a potential target to promote saliva secretion, and activation of TRPV1 might provide a new and safe therapeutic strategy to ameliorate submandibular gland hypofunction.

Pineal gland as an endocrine gravitational lunasensor: manifestation of moon-phase dependent morphological changes in mice.

We found that some morphological properties of the pineal gland and submandibular salivary gland of mice are significantly distinct at the new and full moon. We suppose that the differences are initiated by the displacements of the electron-dense concretions in the secretory vesicles of pinealocytes. This presumably occurs under the influence of the gravitational field, which periodically changes during different phases of the moon. It seems that the pinealocyte is both an endocrine and gravisensory cell. A periodic secretion of the pineal gland probably stimulates, in a lunaphasic mode, the neuroendocrine system that, in turn, periodically exerts influence on different organs of the body. The observed effect probably serves, within the lifelong clock of a brain, to control development and aging in time.

Rat submandibular gland perfusion method for clarifying inhibitory regulation of GABAA receptor.

GABA is an inhibitory transmitter found in rat salivary gland. However, the inhibitory potential of GABA on salivary secretion is unclear. Using an in vivo cannulation method, intraperitoneal administration of GABA was ineffective in the absence of gabaculine, a GABA transaminase inhibitor, on pilocarpine-induced salivary secretion, suggesting that GABA was rendered metabolically inactive before reaching the salivary gland. We hypothesized that the action of a drug on the salivary glands could be measured directly using a submandibular gland perfusion system. The submandibular gland artery, veins, and duct were cannulated in situ so that physiological functions such as innervation would not be compromised. Hank's balanced salt solution (pH 7.4) was perfused at a rate of 0.5 ml/min together with 1 µM carbachol (CCh) over a 5-min period every 30 min. Amount of secreted saliva showed no change to the recurrent addition of CCh to the perfusate. GABA or muscimol dose-dependently inhibited CCh-induced salivary secretion. This effect was blocked by bicuculline, a GABA(A)-receptor (GABA(A)-R) antagonist, and enhanced by clonazepam, a central-type benzodiazepine-receptor agonist. These results suggest that salivary secretion is suppressed by GABA(A)-R in rat salivary gland and that the perfusion method used was effective in clarifying inhibitory regulation of GABA(A)-R.

Activation of transient receptor potential vanilloid subtype 1 increases expression and permeability of tight junction in normal and hyposecretory submandibular gland.

Tight junction (TJ) is an important structure that regulates material transport through the paracellular pathway across the epithelium, but its significance in salivary physiology and pathogenesis of salivary dysfunctional diseases is not fully understood. We previously demonstrated that a functional transient receptor potential vanilloid subtype 1 (TRPV1) expresses in submandibular gland (SMG). However, association of TRPV1-induced saliva secretion with TJ remains unknown. Here we explored the effect of TRPV1 activation on expression and function of TJ of rabbit SMG in vitro and in vivo. RT-PCR and western blot analysis revealed that capsaicin upregulated expression of zonula occludin-1 (ZO-1), claudin (Cldn)-3, and -11, but not Cldn-1, -2, -4, -5, and -7 in cultured SMG cells. Capsaicin also increased the entering of 4 kDa FITC-dextran into the acinar lumen, induced redistribution of cytoskeleton F-actin under confocal microscope, and these effects were abolished by preincubation of capsazepine, a TRPV1 antagonist, indicating that activation of TRPV1 increases expression and permeability of TJ in SMG. Additionally, in a hyposecretory model induced by rabbit SMG transplantation, the expression of ZO-1, Cldn-3, and -11 was decreased, whereas other TJs remained unaltered. The structure of TJ was impaired and the width of apical TJs was reduced under transmission electron microscope, concomitant with diminished immunofluorescence of F-actin in peri-apicolateral region, indicating impaired TJ expression and decreased paracellular permeability in the transplanted SMG. Moreover, topical capsaicin cream increased secretion, decreased TJ structural injury, reversed TJ expression levels, and protected F-actin morphology from disarrangement in transplanted SMGs. These data provide the first evidence to demonstrate that TJ components, particularly ZO-1, Cldn-3, and -11 have important roles in secretion of SMG under both physiological and pathophysiological conditions. The injury in TJ integrity was involved in the hypofunctional SMGs, and TRPV1 might be a potential target to improve saliva secretion through modulating expression and function of TJs.

TRPV4 activation in mouse submandibular gland modulates Ca2+ influx and salivation.

Transient receptor potential vanilloid subtype 4 (TRPV4) is a ligand-gated nonselective cation channel that participates in the transduction of mechanical and osmotic stimuli in different tissues. TRPV4 is activated by endogenous arachidonic acid metabolites, 4α-phorbol-12,13 didecanoate, GSK1016790A, moderate heat, and mechanical stress. TRPV4 is expressed in the salivary glands, but its expression pattern and function are poorly understood. The aim of this study was to evaluate the functional role of TRPV4 channels in the mouse submandibular gland. Using RT-PCR and Western blot analysis, we detected expression of TRPV4 message and protein, respectively, in the submandibular gland. Immunolocalization studies showed that TRPV4 targeted to the basolateral membrane of mouse submandibular gland acinar cells. Pharmacological TRPV4 activation using the selective agonist GSK1016790A caused Ca(2+) influx in isolated acinar cells in a basal-to-apical wave. Consistent with these observations, GSK1016790A elicited salivation in the perfused submandibular gland that was dependent on extracellular Ca(2+). In summary, we report that activation of TRPV4 channels induced Ca(2+) influx and salivation and, thus, may contribute a novel nonadrenergic, noncholinergic secretion pathway in the mouse submandibular gland.

Histamine modulates salivary secretion and diminishes the progression of periodontal disease in rat experimental periodontitis.

OBJECTIVE: We have recently reported that experimental periodontitis (EP) reduced methacholine-induced submandibular gland (SMG) salivary secretion. The aim of the present study was to determine whether histamine could prevent SMG impairment produced by EP. MATERIALS AND METHODS: Bilateral EP was induced for 2 weeks and histamine treatment (0.1 mg/kg subcutaneously) was started 5 days before the end of the experimental period in male rats. The histamine effects on periodontitis-altered functional and histological parameters of SMG and on periodontal bone loss were evaluated. RESULTS: Histamine treatment partially reversed the methacholine-induced salivation reduction produced by EP while preventing SMG histological damage. Histamine's effect on SMG was associated with an increased proliferation rate (2.2 ± 0.3 vs. 0.2 ± 0.2 proliferative cells per field, P < 0.001). Furthermore, histamine completely prevented enhanced EP-induced apoptosis (1.0 ± 0.4 vs. 60.9 ± 4.6 apoptotic cells per field, P < 0.001). The protective effect exerted by histamine on SMG functionality is associated with attenuation of lingual and vestibular bone loss (0.66 ± 0.04 vs. 0.97 ± 0.06 mm; P < 0.001). CONCLUSIONS: Histamine is able to reduce periodontitis-induced damage to SMG and bone structure.

Relationship between aquaporin-5 expression and saliva flow in streptozotocin-induced diabetic mice?

OBJECTIVE: To investigate the expression and distribution of AQP5 in submandibular acinar cells from sham- and streptozotocin (STZ)-treated mice in relation to the salivary flow. METHODS: Mice were sham or STZ injected. Distribution of AQP5 subcellular expression in submandibular glands was determined by immunohistochemistry. AQP5 labelling indices (LI), reflecting AQP5 subcellular distribution, were determined in acinar cells. Western blotting was performed to determine the expression of AQP5 in submandibular glands. Blood glycaemia and osmolality and saliva flow rates were also determined. RESULTS: AQP5 immunoreactivity was primarily located at the apical and apical-basolateral membranes of submandibular gland acinar cells from sham- and STZ-treated mice. No significant differences in AQP5 protein levels were observed between sham- and STZ-treated mice. Compared to sham-treated mice, STZ-treated mice had significant increased glycaemia, while no significant differences in blood osmolality were observed. Saliva flow rate was significantly decreased in STZ-treated mice as compared to sham-treated mice. CONCLUSIONS: In STZ-treated mice, significant reduction in salivary flow rate was observed without any concomitant modification in AQP5 expression and localization.

Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation.

Rat submandibular gland can regenerate following ligation-induced atrophy, eventually recovering its normal morphology and function. Previous studies have suggested that the regeneration process implies both self-proliferation of existing acini and formation of new acinar cells. One hypothesis is that new acinar cells may differentiate from the ductal cells in a similar fashion to the process of cytodifferentiation occurring during submandibular glandular development. In this study atrophy was induced, under recovery anaesthesia, by applying a metal clip on the main duct of the submandibular gland without including the chorda lingual nerve. After 2 weeks the duct was deligated for 3, 5 or 7 days or 8 weeks and the glands collected. Tissue was prepared for immunohistochemistry, biochemical analysis and RNA extraction. The histology of the regenerated glands shows several normal-looking acini, which have regained their glycoprotein content (AB/PAS positive), data also confirmed by biochemical analysis (SDS-PAGE/PAS). Regenerating tissue was characterized by the presence of embryonic-like branched structures ending with AB/PAS positive acinar cells. The proteins SMG-B and PSP are normally expressed in acinar cell precursors during development but only by intercalated ductal cells in the adult stage. In the adult regenerating gland mRNA levels of both SMG-B and PSP were found to be up-regulated compared to ligated glands and SMG-B expression localized to acinar cells whilst the ductal cells were negative. This study of rat submandibular gland regeneration suggests new acinar cells have differentiated from ducts and express markers of acinar cell precursors in a similar manner to the cytodifferentiation process occurring during glandular development.

Evidence of interlobular repulsion during branching morphogenesis in mouse salivary glands.

We developed a culture method for detecting repulsion among epithelial lobules during branching morphogenesis in mouse submandibular glands. Three epithelia were placed at each vertex of an imaginary triangle apart but near enough to meet with one another if each of them expands radially during the culture period. No repulsion was observed following cultivation with neuregulin 1 and lysophosphatidic acid; the epithelia actively branched and nearly contacted one another in the triangle's center. In contrast, strong repulsion was observed among the epithelia cultured with fibroblast growth factor 1 (FGF1), which exhibited less branching and moved away from the center. The localization of DiI- (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate) and BrdU- (5-bromodeoxyuridine) labeled cells in the cultures exposed to FGF1 indicated that the cells were unable to move and proliferate in the center. SB431542, an inhibitor of transforming growth factor-beta (TGFbeta) signaling, was unable to abolish this repulsion, suggesting that TGFbetas will not probably act as repellants in this case.

[Importance of submandibular salivary glands for organism].

Based upon analysis of postoperative morbidity the authors concluded that salivary glands extirpation led to considerably often development of different gastrointestinal tract diseases in comparison with other operative interventions. The most often (62%) these diseases appeared in patients with submandibular salivary gland removal in anamnesis and rarer (21%) - with parotid salivary gland removal. So it could be claimed that salivary glands were an important constituent part of digestive system. Salivary glands removal was only proper in case of impossibility of carrying organ preserving operative intervention.

Smad7 attenuates TGF-ß-mediated aging-related hypofunction of submandibular glands.

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients' quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-ß/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-ß/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-ß-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.