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Dongfang Yu, Richard M Davis, Megumi Aita, Phillip W Clapp, Wanda K O, Scott Randell, Richard C Boucher; Characterization of Rat Meibomian Gland Ion Transport. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1303.
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© ARVO (1962-2015); The Authors (2016-present)
The meibomian gland plays an essential role to maintain ocular surface health and integrity by providing the tear film outer lipid layer. This study was designed to explore the ion and fluid transport activities of rat meibomian gland and to establish a primary rat meibomian gland cell culture system.
Messenger RNA expression of selected sodium and chloride channels in rat meibomian gland tissues were determined by RT-PCR. Localization of mRNA for the α, β, and γ subunits of the epithelial sodium channel (ENaC) was determined by in situ hybridization. Protein expression and localization of β-ENaC were evaluated by western blot and immunohistochemistry, respectively. Freshly excised rat meibomian gland tissues were enzymatically digested and co-cultured with mitomycin C-treated 3T3 cells. Analyses for ion channels, other biomarkers of cell proliferation and differentiation, and lipid production of rat meibomian gland tissues and primary cell culture were performed with molecular biological and histochemical techniques. Meibomian gland cells were seeded onto permeable supports for air-liquid interface culture and further Ussing chamber electrophysiological studies. Fluid transport by meibomian gland cells on inserts was studied using labeled-dextran and confocal microscopy.
ENaC, sodium/glucose co-transporter (Slc5a1), cystic fibrosis transmembrane conductance regulator (Cftr), and transmembrane protein family 16 (Tmem16a and f) gene expression was detected in rat meibomian gland tissues. ENaC (α, β, and γ subunits) mRNA was localized mostly to rat meibomian gland peripheral acini. Consistent with its mRNA distribution, β-ENaC protein had a similar distribution pattern. Primary rat meibomian gland cells expanded well in culture, expressed similar profiles of selected molecules as fresh meibomian gland tissues, and produced neutral lipids. Electrophysiological studies of primary rat meibomian gland cells identified functional ENaC, SLC5A1, CFTR, and TMEM16. Active fluid transport across rat meibomian gland cells was detected by labeled-dextran confocal studies.
This study illustrates the presence of molecules involved in active ion and fluid transport in the rat meibomian gland. We established an in vitro primary rat meibomian gland cell culture model that will serve as a platform for investigating the roles of specific ion channels likely to be essential for normal meibomain gland function.
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