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.

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation.

OBJECTIVES: Salivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration. MATERIALS AND METHODS: We used a duct ligation/deligation-induced submandibular gland regeneration model of Sprague-Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin-eosin staining, real-time PCR (RT-PCR), immunohistochemistry and Western blotting. We counted the number of Ki67-positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases. RESULTS: Intact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67-positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases-1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration. CONCLUSIONS: Our findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.

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.

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.

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.

Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

Morphological differences between regenerating salivary glands after salivary gland duct ligation and embryonic salivary glands.

BACKGROUND: Most animals and organs have regenerative capabilities. Whether regeneration is a developmental process or a distinct phenomenon that is independent of development is debatable. METHOD: We examined the differences between developing and regenerating salivary glands using duct-ligation models. We performed morphological analyses comparing submandibular gland regeneration and development. To reveal the proliferation processes that occur during salivary gland regeneration and development, we counted the number of Ki67-positive cells over time. In addition, we examined the expression of the following markers: aquaporin 5, smooth muscle actin, cytokeratin 7, and tubulin beta 3. RESULT: The proliferation patterns seen during regeneration differed from those observed during development. Different salivary gland marker expression patterns were seen during development and regeneration. CONCLUSION: This study showed that regenerating salivary glands do not follow the same growth process as developing salivary glands.

Specific complexes derived from extracellular matrix facilitate generation of structural and drug-responsive human salivary gland microtissues through maintenance stem cell homeostasis.

Three-dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s-Ecx) and analyzed the components of the s-Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s-Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s-Ecx. s-Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s-Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.

5-fluorouracil and cyclophosphamide modify functional activity in submandibular gland of rats / El 5-fluorouracilo y la ciclofosfamida modifican la actividad funcional en la glándula submandibular de las ratas

Objective: Chemotherapy treatment against cancer produce systemic toxicities, among which are those related to important structures of the stomatognathic system and its functional activity. 5 Fluorouracil (5-FU) and cyclophosphamide (Cf) are drugs widely used in solid tumors and in bone marrow transplantation, respectively. The objective of this work was to evaluate the toxicity of these drugs regarding functional activity of the submandibular glands, by measuring the percentage of glycogen consumption in two experimental models. Material and Methods: 84 male Wistar rats aged three months were used, housed in individual cages, with controlled temperature and lighting and ad libitum diet. They were divided into four experimental groups: 1) Control (C); 2) Treated with 5-FU+leucovorin (LV) at 20 and 10mg/Kg of body weight respectively for five consecutive days; 3) treated with Cf i.p. at 50mg/Kg of body weight for two consecutive days; and 4) rats with paired feeding (PF): for five and two days respectively, the amount administered resulted from the average of the ingested food of groups 2 and 3. Both submandibular glands were excised. The submandibular glycogen concentration was analyzed at initial time (t0) and after 60 minutes of mechanical stimulation (t60). Results: the average variation changed significantly between time 0 and 60 in the groups C and PF. (p-value=0.0001), the 5-FU + LV treatment group had an average concentration higher at t0 than groups C and PF, without significant consumption at T60. While group Cf showed a lower average concentration at time 0 with respect to groups C and PF, without significant consumption at T60. Conclusion: 5-FU+LV and Cf affect the metabolism of carbohydrates, decreasing the use of glycogen as a metabolic substrate. In the present experimental model, the toxicity of these drugs affected the functional activity of the submandibular gland.

Objetivo: el tratamiento de quimioterapia contra el cáncer produce toxicidades sistémicas, entre las que se encuentran las relacionadas con estructuras importantes del sistema estomatognático y su actividad funcional. El 5-fluorouracilo (5-FU) y la ciclofosfamida (Cf ) son fármacos ampliamente utilizados en tumores sólidos y en trasplantes de médula ósea, respectivamente. El objetivo de este trabajo fue evaluar la toxicidad de estos fármacos con respecto a la actividad funcional de las glándulas submandibulares, midiendo el porcentaje de consumo de glucógeno en dos modelos experimentales. Material y Métodos: se utilizaron 84 ratas Wistar machos de tres meses de edad, alojadas en jaulas individuales, con temperatura e iluminación controladas y dieta ad libitum. Se dividieron en cuatro grupos experimentales: 1) Control (C); 2) Tratados con 5-FU+leucovorina (LV) a 20 y 10mg/Kg de peso corporal, respectivamente, durante cinco días consecutivos; 3) tratados con Cf i.p. a 50mg/Kg de peso corporal durante dos días consecutivos; y 4) ratas con alimentación por parejas (PF): durante cinco y dos días respectivamente, la cantidad administrada resultó del promedio de los alimentos ingeridos de los grupos 2 y 3. Ambas glándulas submandibulares fueron extirpadas. La concentración de glucógeno submandibular se analizó en el momento inicial (t0) y después de 60 minutos de estimulación mecánica (t60). Resultados: la variación promedio cambió significativamente entre el tiempo 0 y 60 en los grupos C y PF. (p=0,0001), el grupo de tratamiento 5-FU+LV tuvo una concentración promedio más alta en t0 que los grupos C y PF, sin un consumo significativo en T60. Mientras que el grupo Cf mostró una concentración promedio más baja en el tiempo 0 con respecto a los grupos C y PF, sin un consumo significativo en T60. Conclusión: 5-FU + LV y Cf afectan el metabolismo de los carbohidratos, disminuyendo el uso de glucógeno como sustrato metabólico. En el presente modelo experimental, la toxicidad de estos medicamentos afectó la actividad funcional de la glándula submandibular.

A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix electrophoresis.

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galß1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.

Quantitative Assessment of Thyroid, Submandibular, and Parotid Glands Elasticity With Shear-Wave Elastography in Children.

Tissue elasticity is becoming a more commonly used parameter in evaluation of parenchyma in inflammatory diseases. Considering the changes in the thyroid and salivary glands with adolescence, determination of mean elasticity ranges with a function of age is necessary to apply ultrasound elastography more widely in the pediatric population.The thyroid, submandibular, and parotid glands of 127 healthy volunteers (66 males, 61 females; mean age = 10.3 ± 3.9 years; range = 3-17 years) were evaluated with shear-wave elastography.The mean elasticity values for the thyroid, submandibular, and parotid glands were 14.6 ± 3.3, 11.8 ± 2.2, and 11.8 ± 2.6 kPa, respectively. There was a significant positive correlation between age and elasticity of the thyroid, submandibular, and parotid glands. There was a significant correlation between age and elasticity value of the thyroid gland adjusted for weight and height.This study provided the baseline quantitative elasticity measures of thyroid, submandibular, and parotid glands, which would be a reference for upcoming studies. In addition, an increase in elasticity value in thyroid gland as a function of age independent of change in weight and height was demonstrated.

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.

Regenerative capacity of allogenic gingival margin- derived stem cells with fibrin glue on albino rats' partially dissected submandibular salivary glands.

OBJECTIVE: To evaluate the possible regenerative effect of allogenic gingival margin-derived stem cells (GMSCs) with or without autologous fibrin glue on partially dissected submandibular salivary glands of albino rats. METHODS: Forty rats were randomly divided into four equal groups. Group I, where no operation was performed, was considered the negative control. Group II rats were considered the positive control and were subjected to a rectangular cut on the outer surface of the center right of the submandibular salivary gland and received no other treatment. Groups III and IV rats were handled as those in group II, but the cut areas of group III were filled with fibrin glue and the cut borders of group IV were injected with 1×105cell/ml GMSCs and then glued with fibrin glue. Five animals from each group were euthanized at the end of the first postoperative week, while the remaining animals were euthanized at the end of the second postoperative week, i.e., end of the experiment. RESULTS: Regeneration of ductal, acinar, and myoepithelial cells was better in group IV. A two-way ANOVA for proliferating cell nuclear antigen and α-smooth muscle actin revealed an overall significant difference between the different groups (P<0.05). In addition, an LSD post hoc test for multiple comparisons revealed a significant difference between each two groups. An independent sample t-test revealed significant differences between time periods for groups II, III, and IV, but there were no significant differences between the time periods for group I. CONCLUSION: Injecting GMSCs at the cut borders and gluing the cut area with autologous fibrin glue ameliorates the regeneration of partially dissected submandibular salivary gland better than using fibrin glue alone.

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.

[Paying attention to the protection of the function organ of submandibular gland].

Submandibular gland is an important human function organ. With the wide application of sialoendoscope and new understanding of IgG4 related sialadenitis, a lot of submandibular glands which were previously considered to be removed are preserved. However, some submandibular glands which might be preserved are still unfortunately sacrificed. Therefore, we advocate the popularization and promotion of new knowledge and techniques and should pay more attention to the protection of the function organ of submandibular gland.

L1 Peptide-Conjugated Fibrin Hydrogels Promote Salivary Gland Regeneration.

Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.

Similar synapse elimination motifs at successive relays in the same efferent pathway during development in mice.

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting.

Endothelial Tight Junctions Are Opened in Cholinergic-Evoked Salivation In Vivo.

Blood vessels provide the original supplies for the formation of primary saliva, which is regulated by the tight junctions (TJs) between endothelial cells. Previous studies have shown that blood flow increases with vasodilatation during cholinergic-evoked salivation. However, changes in vascular paracellular permeability and the role of endothelial TJs in salivation are unknown. Here, we established an in vivo paracellular permeability detection system and observed that the endothelial TJs were permeable to 4-kDa fluorescein isothiocyanate (FITC)-dextran while impermeable to 40- and 70-kDa FITC-dextran under an unstimulated condition in mouse submandibular glands (SMGs). Pilocarpine increased the flux of 4- and 40-kDa FITC-dextran out of blood vessels but did not affect 70-kDa FITC-dextran. Claudin 5, a TJ protein specifically localized in salivary endothelial cells, was redistributed from the apicolateral membranes to the lateral and basolateral membranes and cytoplasm in cholinergic-stimulated mouse SMGs and freshly cultured human SMG tissues. In the transplanted SMGs from epiphora patients, we found that claudin 5 was present in the basolateral membranes and cytoplasm, instead of the apical region in control SMGs. Moreover, the level of phospho-myosin light chain 2 increased within the blood vessels of the pilocarpine-stimulated mouse SMGs and transplanted human SMGs, while the downstream molecule F-actin was reorganized in the endothelial cells of the transplanted human SMGs. Taken together, our findings provide direct visual evidence that the opening of endothelial TJs and the redistribution of claudin 5 are essential events contributing to cholinergic-evoked salivation, thus enriching our understanding of the secretory mechanisms that link blood flow to primary saliva formation by regulating the endothelial paracellular permeability.

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.