S M Osailan

Protein and mucin retention on oral mucosal surfaces in dry mouth patients

Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole-mouth saliva. High-molecular-weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane-bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.

Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative states

BackgroundReal-time PCR is a reliable tool with which to measure mRNA transcripts, and provides valuable information on gene expression profiles. Endogenous controls such as housekeeping genes are used to normalise mRNA levels between samples for sensitive comparisons of mRNA transcription. Selection of the most stable control gene(s) is therefore critical for the reliable interpretation of gene expression data. For the purpose of this study, 7 commonly used housekeeping genes were investigated in salivary submandibular glands under normal, inflamed, atrophic and regenerative states.ResultsThe program NormFinder identified the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative states, and GAPDH in the atrophic state. For normalisation to multiple housekeeping genes, for each individual state, the optimal number of housekeeping genes as given by geNorm was: ACTB/UBC in the normal, ACTB/YWHAZ in the inflamed, ACTB/HPRT in the atrophic and ACTB/GAPDH in the regenerative state. The most stable housekeeping gene identified between states (compared to normal) was UBC. However, ACTB, identified as one of the most stably expressed genes within states, was found to be one of the most variable between states. Furthermore we demonstrated that normalising between states to ACTB, rather than UBC, introduced an approximately 3 fold magnitude of error.ConclusionUsing NormFinder, our studies demonstrated the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative groups and GAPDH in the atrophic group. However, if normalising to multiple housekeeping genes, we recommend normalising to those identified by geNorm. For normalisation across the physiological states, we recommend the use of UBC.

Clinical assessment of oral dryness: development of a scoring system related to salivary flow and mucosal wetness

OBJECTIVE The aim of this study was to develop a clinical oral dryness score (CODS) for routine use in assessment of xerostomia patients and determine its relationship with salivary flow rates and mucosal wetness. STUDY DESIGN CODS was determined from 10 features of oral dryness, each scoring as 1 point for a total score of 0-10. CODS, salivary flow rates, and mucosal wetness were measured in 100 patients and 50 healthy control subjects. The reproducibility of CODS was 0.89-0.96 (intraclass correlation coefficient). RESULTS The mean ± SD CODS in patients was 6.0 ± 1.6 compared with 1.0 ± 0.9 for control subjects (P < .001), and the highest mean value was in the primary Sjögren syndrome group. There was a general inverse relationship in patients between mean CODS and salivary flow rate (P < .01) and mean CODS and mucosal wetness (P < .01). CONCLUSIONS The CODS was found to be useful, easy to use, and reliable for routine assessment of the severity of dry mouth.

Investigating the relationship between hyposalivation and mucosal wetness

BACKGROUND Mucosal wetness (MW) reflects the layer of residual saliva that covers the oral mucosal surfaces. OBJECTIVES The aim of this study was to determine MW at different oral mucosa sites and to investigate the relationship between MW, unstimulated whole salivary flow rates (UWS) and Clinical Oral Dryness Score (CODS). METHOD A total of 100 dry mouth patients and 50 healthy subjects participated in the study. MW was sampled with filter paper strips at four sites inside the mouth; anterior hard palate (AHP), buccal mucosa (BUC), anterior tongue (AT), lower lip (LL) and measured with a micro-moisture meter. Reproducibility was assessed by repeated sampling and diurnal variation was examined. RESULTS Mucosal wetness in healthy subjects differed according to site and means±SD were; AHP (11± 11.7μm), BUC (32±14.8μm), AT (65±17.2μm), and LL (25 ±13.5μm). Dry mouth patients with reduced UWS showed increased CODS. MW at all four sites was significantly reduced (P<0.05) in dry mouth patients compared with the healthy subjects. Reproducibility of MW measurement using the intra-class correlation coefficient showed agreement at different visits within subject. MW of the AT showed a positive correlation with UWS (P<0.05). CONCLUSION Mucosal wetness is a reliable measure of oral dryness and had a positive correlation with UWS.

Intraoral duct ligation without inclusion of the parasympathetic nerve supply induces rat submandibular gland atrophy

The atrophic effect of ligating the main duct of the right submandibular gland was examined in rat using a novel intraoral approach that did not include the chorda lingual (CL) nerve. Comparison was made with the effect of duct ligation including the attached CL nerve as carried out in previous studies. In all animals, the contralateral, unligated left submandibular gland was used as a control. At different times (1, 2, 7, 14 and 21 days) after ligation, glands were removed and weighed. Tissue was fixed for morphological analysis and homogenized for biochemical assay of secretory proteins. After 21 days, ligated glands showed a significant decrease in wet weight compared with unligated glands. Weight loss was the greatest (P < 0.05) in glands ligated with the CL nerve included. Light microscopy revealed that following ligation, an initial inflammatory reaction was followed by severe atrophy of acini and granular ducts. The atrophy was less severe when the CL nerve was not ligated. Secretory proteins were decreased from day 1 onwards following duct ligation in both groups. It can be concluded that most of the atrophy induced by duct ligation is independent of damage caused to the parasympathetic nerve supply, although the latter causes a greater atrophy presumably due to denervation.

Recovery of rat submandibular salivary gland function following removal of obstruction: a sialometrical and sialochemical study

Functional recovery of the rat submandibular gland following ligation of the main excretory duct was examined. Rat submandibular glands were ligated for 1, 4 and 8 weeks using a micro‐clip with a plastic tube. Micro‐clips were removed and glands were allowed to recover for periods of 8, 16 and 24 weeks. Submandibular glands were stimulated with autonomimetic drugs (methacholine and isoprenaline) and salivas were collected from atrophic or de‐ligated and contralateral control glands. Glands recovered almost full size (92% of control gland) following 24 weeks of de‐ligation. Saliva volume secreted by ligated/de‐ligated (RSM) and control (LSM) glands were similar with different doses of agonists. Protein output expressed per gram of tissue wet weight was similar from both ligated/de‐ligated and control glands with all doses of agonist. Sodium and chloride levels were higher from de‐ligated glands than contralateral control glands. Protein electrophoresis showed similar profiles of salivary proteins in all samples with some minor differences. Acinar cells in de‐ligated glands showed a normal morphology, as indicated by light microscopy, whilst granular ductal cells were fewer and contained fewer secretory granules. Sodium potassium ATPase staining of striated ducts in de‐ligated glands was similar to that of control glands. It can be concluded that rat submandibular glands can regenerate following severe atrophy and secrete normal amounts of saliva containing broadly a full profile of secretory proteins. In contrast to acinar cells, ductal cells appear not to recover full function.

Rat salivary gland ligation causes reversible secretory hypofunction

Aim:  To determine the influence of inflammation on salivary secretion. Secretion by salivary glands involves interactions between nerves, blood vessels and salivary cells. The present study investigated the effects of inflammation on rat submandibular gland function following acute ductal obstruction.

Altered plasticity of the parasympathetic innervation in the recovering rat submandibular gland following extensive atrophy.

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro‐effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra‐oral approach to avoid damaging the chorda‐lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda‐lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole‐body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland.

Acute salivary gland hypofunction in the duct ligation model in the absence of inflammation

Objective The commonly associated aetiology of salivary gland inflammation and salivary hypofunction has led to the widely held belief that inflammation causes salivary gland hypofunction. Indeed, our own recent study seemed to support this contention. Here, we tested the hypothesis that, in an acute duct ligation model, eliminating inflammation the submandibular gland would recover normal function. Materials and methods Ligation of the rat submandibular gland excretory duct for 24 h was used to induce inflammation and salivary gland hypofunction. A group of duct ligated rats was compared with a second group given dexamethasone, on the day of duct ligation. Twenty-four hours later salivary gland function was assessed and salivary glands were collected. Results Histology and myeloperoxidase activity assay revealed a profound decrease in inflammatory cell infiltration of ligated glands from rats given dexamethasone, compared with ligated glands in the absence of dexamethasone. Salivary flow rate evoked by methacholine was decreased (P < 0.01) by approximately 56% (ligated vs control, 79 ± 9 μl min−1 g−1vs 177 ± 11 μl min−1 g−1) and salivary flow from ligated dexamethasone-treated and ligated glands was similar. Conclusion Despite eliminating the inflammatory reaction in the ligated gland, salivary hypofunction was not reversed, suggesting that other mechanisms must be at work in the ligation-induced salivary hypofunction.