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Background and Aim The role of the mucosal barrier on the esophagus has not been fully clarified. Tight junction (TJ) and Claudin are considered as one of the mucosal barrier of the esophagus. We have studied the protein expression of Claudin which composed of TJ using experimental model of the rat reflux esophagitis(RE). We suggested that Claudin-3 was one of the key TJ barrier proteins and alterations in Claudin-3 proteins might change TJ structures in the rat RE model (Asaoka D et al. J Gastroenterol 2005, Oguro M et al. J Gastroenterol 2011). However, the detailed structural change of TJ has not been investigated. The aim of this study was to explore the relationship between the morphological TJ structure and Claudin-3 protein in the rat esophageal epithelium in the control and RE model using electron microscopy. Materials and Methods Chronic acid RE model was prepared in male Wister rats by the method (Asaoka D et al. J Gastroenterol 2005), and Sham operation was implemented to the controls. Two weeks after the operation, the rats were sacrificed. After their esophagus was removed and fixed, the following studies were performed; 1) electron microscopy observation 2) freeze fracture electron microscopy observation 3) immunoelectron microscopy observation with anti-Claudin 3 antibody. Especially, we observed the samples in detail using the modified grid-mapped freeze fracture technique instead of the conventional freeze fracture method to prevent the occurrence of the fragmentation and specify the location of the fracture plane. Briefly, a gold finder grid was bonded to the replicated tissue by using 2% polycarbonate dissolved in dichloroethane. The polycarbonatestabilized samples were immersed in PBS and removed from the gold specimen disk. The samples were then stained with DAPI and mapped by using the confocal microscope. Results We observed TJ-related structures in the granular layer and spinous layer in the esophageal epithelia of both groups of the rats using transmission electron microscopy. However, no typical strands were observed by freeze fracture electron microscopy. The specimen wasn't stained with Claudin-3 in both the Sham and RE model rats by immunoelectron microscopy of ultrathin cryosections. Conclusion Claudin-3 protein wasn't involved in the composition of TJ structure. This result indicates that claudin-3 protein may play the role in the maintenance of the mucosal barrier in the different area from where TJ complex exists.

Background: Tight junction (TJ) is considered to be an important contributor to the epithelial defense system. However, TJ in the esophagus has not been well elucidated. Claudins are a family of TJ transmembrane proteins. They are both structurally and functionally essential components of TJ strands, and play a critical role in the TJ barrier In a previous study, we reported altered location pattern of epithelial claudin-1, -3, -4 proteins in a rat chronic acid esophagitis model using immunofluorescence analysis. The aim of this study is to characterize pathological lesions of the esophageal epithelial tight junction complex in a rat reflux esophagitis model in a search for a reliable diagnostic indicator. Methods: Rats underwent the operation to induce reflux esophagitis with or without Rabeprazole treatment (1.0mg/ kg/day and 10.0mg/kg/day). Sham-operated rats served as a control. Each group comprising more than 3 animals in each experiment. Fourteen days after the operation, esophagi were isolated from the rats and subjected to double-label confocal immunofluorescent microscopy, and biochemical analyses. Results: Immunofluorescent microscopy revealed claudins-1, -3, and -4 were located on the surface of epithelial cells in normal esophagus, although there were differences in the distribution patterns between claudin-3 and claudins-1 or -4 in the epithelial layer. However, in reflux esophagitis, the immunoreactivity of claudin-3 on the cell surface was significantly decreased, and it appeared instead as a faint granular pattern within the epithelial cytoplasm. Claudin-3 expression in the entire esophageal epithelium was also decreased. Expression and location of claudin-1 and -4 in epithelial cells were basically unaffected in reflux esophagitis. Gastric acid-induced dissociation of claudin-3 elicited instability of the epithelial tight junction complex, which was confirmed by sedimentation analysis using centrifugation in a sucrose density gradient. Rabeprazole (10.0mg/ kg/day) significantly attenuated these alterations. Conclusions: Our data indicate that dispersion of claudin-3 from esophageal epithelial plasmamembranes to cytoplasm and the resulting instability of the tight junction complex is a most specific and sensitive indicator for monitoring inflammatory and recovery processes in reflux esophagitis.

Gastroesophageal reflux (GER) and aspiration of gastric components has been implicated in the pathophysiology of ENT and chronic respiratory diseases. We previously demonstrated that patients with chronic cough, LTx and cystic fibrosis may have both, acid and non-acid reflux. Furthermore, non acid gastric juice from patients “on” PPI is able to trigger increased inflammatory reaction from human bronchial epithelial cells in culture. Animal experiments showed that only acid and bile acids in acidic environment were able to provoke damage in laryngeal mucosa. Patients “on” PPI treatment have persistent reflux of gastric contents with high pH, bile acids and increased bacterial subproducts (LPS). The role of this type of refluxate in extraesophageal GERD is still controversial. The aim of this study was to evaluate the effect of non acidic solutions with or without gastro-duodenal contents and LPS on functional tracheal mucosal integrity.Methods:New Zealand rabbits were euthanized and tracheal segments were mounted in Ussing Chambers for assessment of transepithelial mucosal resistance (TER). Substance P release (a neurotransmitter and marker of mucosal injury) was measured at the mucosal side. Tracheal mucosa was exposed for 20 min to a control solution (Krebs pH 7.4) or to test solutions containing deoxycholic acid (DC, 0.52mM), pepsin (0.4-1 mg/ml) or endotoxin (LPS) (1 g/ml). Results: Exposure of the tracheal mucosa to acidic solutions (pH 1.2, 1.5 and 2), without gastric components, induced a significant drop in TER of 82, 73 and 55% respectively (N=4). Less acidic solutions (pH 34) did not have an effect on the integrity of the tissue. However incubating the tissue with DC (0.5-2mM) at weakly acidic pH (pH 3-4) significantly reduced TER (by 34-30 %), whereas pepsin (0.4-1 mg/ml) was not able to provoke changes in TER at pH > 3 (N=4). Neutral solutions (pH 7.4) with DC (1.5mM) or pepsin (1 mg/ml) did not increase SP. However, exposure to LPS (1 μg/ml, pH 7.4) significantly increased SP release compared to control. Conclusions: Not only acidic solutions but also weakly acidic solutions (pH 34) containing bile acids can harm the functional integrity of the tracheal mucosa In Vitro. Local SP release, induced by endotoxins present in gastro-duodenal refluxate of patients “on” PPI, may further promote an inflammatory reaction.

tomy had more frequently erosive esophagitis than patients with cholecystectomy (p