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H. Lin, W. Li, L. Quyang, Z. Geng, N. Dong, C. Li, Z. Liu; Air Exposure Induced Similar Characteristics to Dry Eye in Conjunctival Tissue Culture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3668.
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© ARVO (1962-2015); The Authors (2016-present)
To investigative an air exposure induced ex vivo conjunctival tissue culture model with similar pathological characteristics to dry eye.
Human conjunctival explants were cultured under airlift or submerged manner for different duration up to 2 weeks. Epithelial cell proliferation, differentiation, and mucin alteration were studied using certain markers such as K10, K12 and K16 keratins, p63, Pax6, MUC5AC, MUC19, MUC4 and MUC16. Apoptosis of epithelial and stroma cells in cultured conjunctiva was studied by Flurorescence TUNEL assay. MMP9, TNF- and IL-1β concentrations in the condition medium were detected by ELISA. Wnt signaling related molecules as β-catenin and phosphor-β-catenin were studied by immunofluoresece staining.
The ex-vivo dry eye model was successfully created by culturing human conjunctival explants at air-liquid interface (airlift) after 8 days. Increased stratification only happened in conjuctiva epithelium in airlift, but not submerged, cultures. Abnormal epidermal differentiation as shown by positive expression of K10 keratin in suprabasal cells and decreased expression of K19. Pax6 nuclear staining was markly attenuated as early as 2 days in airlift cultures, and showed the perinuclear and cytoplasm expression pattern gradually. MUC5AC expression decreased from day 4 and diminished on day 10 in each group. The gel-forming kind of MUC19 disappeared from day 2, while the membrane-bound expression gradually decreased with time in airlift manner. MUC4 and MUC16 lost the full thickness expression patern in the conjunctival epithelium after culture. Inflammatory mediators as MMP-9, TNF-and IL-1βin the condition medium significantly increased after 8 days airlifting culture. Apoptosis of conjunctival epithelial and stroma cells emerged from day 2 and obviously increased after airlift culture for 10 days. Wnt signaling pathway showed transient activation in airlift manner from day 2 to day 8.
Dryness due to air exposure activates Wnt signaling coupled with abnormal epidermal differentiation, squamous metaplasia, apoptosis and inflammation in ex vivo conjunctival tissue. Airlift cultured model might serve as a good manner to study the mechanism of dry eye as well as to screen for potential treatment.
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