Tu1133 Ultrastructural Localization of Claudin-3 in the Rat Esophageal Stratified Epithelium Under Normal Conditions and Chronic Reflux Esophagitis

Abstract

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.