Characterisation of adherens and tight junctional molecules in normal animal larynx; determining a suitable model for studying molecular abnormalities in human laryngopharyngeal reflux

Abstract

Background: The disruption of intercellular junctions in the larynx is a pathological feature of laryngopharyngeal reflux (LPR). Good experimental models are necessary to gain greater insight into the molecular mechanisms and alterations that result from abnormal exposure of the laryngeal epithelium to acid refluxate.

Aims: To characterise laryngeal tissues from different species to determine the most suitable for use in experimental studies of LPR.

Methods: Human and non-human laryngeal tissues (mouse, rat, guinea pig, porcine, and rabbit) were studied. Histological characterisation was performed by light microscopy. The expression and subcellular localisation of adherens junctional molecules (E-cadherin and beta catenin) was evaluated by immunohistochemistry, and tight junction molecules (occludin and zonula occludens 1 (ZO-1)) by western blotting. The ultrastructural features of porcine and human tissue were assessed by electron microscopy.

Results: Porcine tissue revealed both respiratory-type and stratified squamous epithelium, as seen in the human larynx. The expression and subcellular localisation of the E-cadherin-catenin complex was detected in all species except mouse and rat. The pattern of ZO-1 and occludin expression was preserved in all species.

Conclusion: The expression of intercellular junctional complexes in porcine epithelium is similar to that seen in humans. These results confirm the suitability of these species to study molecular mechanisms of LPR in an experimental system.

Figure 1

Morphological characterisation of human, rat, and mouse laryngeal mucosal tissue by haematoxylin and eosin staining. (A) In the human larynx, the surface epithelial cell layer consists of ciliated columnar epithelial cells. Vascular channels and scattered chronic inflammatory cells are seen in the subepithelial fibroconnective tissue. The squamous epithelium of the laryngeal mucosa is largely non-keratinising and differs from rat sections (B). Respiratory-type epithelium was not seen in (B) rat or (C) mouse laryngeal tissue.

Figure 2

E-cadherin immunostaining in laryngeal epithelium from (A) human, (B) mouse, and (C) rat.

Figure 3

Immunostaining for β catenin in laryngeal epithelium from (A) human, (B) mouse, and (C) rat.

Figure 4

Western blot analysis of E-cadherin and β catenin. Normal laryngeal tissue from (A) human, (B) pig, (C) guinea pig, (D) rat, (E) mouse, and (F) rabbit; α tubulin was used as a loading control. Note the presence of low intensity bands for mouse and rat tissue compared with the other species.

Figure 5

Western blotting analysis of zonula occludens 1 (ZO-1) and occludin. Normal laryngeal tissue from (A) human, (B) pig, (C) guinea pig, (D) rat, (E) mouse, and (F) rabbit; α tubulin was used as a loading control.

Figure 6

(A) Transmission electron micrograph of the apical region of ciliated columnar and goblet cells from the vocal fold of human larynx tissue (original magnification, ×5200). (B) Cell membranes of adjacent ciliated cells closely apposed at the junctional complex (arrow) (original magnification, ×11 600). (C) Transmission electron micrograph of the apical region of columnar epithelial and goblet cells from the porcine larynx (original magnification, ×5200). (D) Cell membranes of adjacent cells closely apposed at the tight junctional complex (original magnification, ×11 600).