To investigate the site of barrier function to the passive diffusion of a small molecule (phalloidin) in the corneal epithelium in the mouse.Methods:
Penetration of phalloidin (molecular weight 1115 daltons) into the cornea was evaluated by studying fluorescent binding of phalloidin to actin in tissue sections, in whole mount preparations, and in the fixed intact globe by confocal microscopy. In addition, the location of tight junction proteins in the individual layers of the corneal epithelium was determined by immunohistochemistry.Results:
Phalloidin staining of corneal sections was positive in all corneal layers in tissue sections and in all layers of the corneal epithelium except the suprabasal layer in excised fixed whole mounts of the cornea. However, when phalloidin staining was attempted in intact fixed globes, before excision of the cornea for whole mount preparation, only the most superficial layer of cells was stained indicating that phalloidin could not penetrate the tissue beyond the suprabasal epithelial layer. Detergent (Triton X-100) treatment of the excised cornea and the intact fixed globe, allowed penetration of phalloidin into the suprabasal epithelial layer. Tight junction proteins occludin, ZO-1 and claudin were present in most layers of the cornea but while ZO-1 and occludin were distributed in a typical pericellular pattern, claudin seemed to be particularly prominent in the suprabasal layer and appeared only as a discontinuous punctate pericellular pattern in the superficial layer. Intraepithelial leukocytes were detected in the superficial epithelium and the basal epithelium but not in the suprabasal epithelium.Conclusion:
The suprabasal epithelium cell layer appears to represent the main barrier site to the passage of small molecules and cells in the mouse cornea and this property may be attribuatable to prominent claudin expression in this layer.