ABSTRACT
@#【Objective】To test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel（ENaC） will increase ocular hydration and cure the rabbit′ s dry eye induced by scopolamine. 【Methods】In the experiment，24 New Zealand rabbits weighing about 2.0- 2.5 kg were divided into 3 groups：tested group（8 rabbits）， control group （8 rabbits），and treatment group （8 rabbits）. For the rabbits in the tested group，they were given subcutaneous injection of scopolamine 4 times a day for 12 consecutive days to induce the dry eye. For the rabbits in the control group，they accepted subcutaneous injection of saline solution. For the rabbits in the treatment group ，they were firstly given the same treatment as the rabbits in the tested group. Then，the local regions in their right eyes received the application of 100 mmol/L amiloride （a sodium channel inhibitor）. Meanwhile，the local regions in their left eyes received the application of equivalent saline solution. We detected ENAC α and ENAC γ subunits of epithelial sodium channel in conjunctival epithelium by fluorescence quantitative PCR and detected the opening of epithelial sodium channel by short- circuit current technology. Finally，we compared the ENaC α and ENaC γ gene expression，corneal fluorescein sodium staining and tear secretion among the 3 groups. Fluorescence quantitative PCR was used to detect ENaC α and ENaC γ subunits of epithelial sodium channel in conjunctival epithelium and short-circuit current was used to detect the opening of epithelial sodium channel. The ENaC α and ENaC γ gene expression，corneal fluorescein sodium staining and tear secretion （immerged length） were compared among 3 groups.【Results】 The results showed that the quantity of tear secretion was（17.00 ± 0.37）mm for the control group，（4.42 ± 1.34）mm for the tested group（P<0.001 vs Control，n=8） and（14.25 ± 0.54）mm for the treatment group（P>0.05 vs Control，n=8）. The results of short-circuit current detection showed that the sodium current was（5.72 ± 0.35）μA /cm2 in normal model and（12.24 ± 0.54）μA /cm2 in dry eye model（P<0.001）. After Amiloride treatment，the sodium current decreased to（4.00 ± 0.61）μA/cm2 （P>0.05） and there was no statistical difference compared with the normal group. According to the results of fluorescence quantitative PCR，the expression of ENaC α and ENaC γ subunits and IL- β in in dry eye model were up-regulated（P<0.01） compared with that in normal group. After topical amiloride application，there were no statistical differences in inflammatory cytokines IL- 1 β，ENaC α，and ENaC γ between the normal group and treatment group（P>0.05，n=8）. After treatment，tear secretion increased （P<0.001），and ocular surface staining improved significantly.【Conclusion】 Topical application of amiloride increase the quantity of preocular tears owing to inhibition of conjunctival sodium channels and could provide an effective new therapy for chronic dry eye.
ABSTRACT
Objective To study the culture method of conjunctival epithelium in vitro.Methods The rabbit conjunctival epithelium was cultured by tissue inoculation and was explanted in culture dish or on human amnionic membrane for fwo weeks.The cell morphology and growth features were observed.Results The rabbit conjunctival epithelium explanted in culture dish connected each other into membrane at eighth days around,which were monolayer cells was observed.The rabbit conjunctival epithelium explanted on human amnionic membrane connected each other into membrane at sixth day,and multi-layer cells were observed.All cultured cells showed positive in CK13 staining by indirect immunofluorescence staining.Conclusions Tissue inoculation is a good approach of conjunctival epithelium culture,and multi-layer cells can be gained if the tissue be exlpanted on human amnionic membrane.
ABSTRACT
PURPOSE: To reconstruct a cultured conjunctival equivalent that closely resembles normal conjunctival epithelium in three-dimensional culture systems. METHODS: Human conjunctival epithelial cells were cultured on dead de-epidermized dermis in the air-exposed state. After 2 weeks of culture, the sections were stained with hematoxylin and eosin. Immunohistochemical and electron microscopic studies were performed. The results were compared with those of normal conjunctiva and cultured eyelid skin equivalent. RESULTS: In the cultured conjunctival equivalent, nonkeratinizing stratified epithelium was formed similarly to normal conjunctival epithelium. Keratin 13 was expressed, but not keratin 10, in the cultured conjunctival equivalent, similarly to normal conjunctival epithelium. However, in the cultured eyelid skin equivalent, keratinizing stratified epithelium was formed. In addition, keratin 10 was expressed, but not keratin 13, contrary to those of the cultured conjunctival equivalent. In the cultured conjunctival equivalent, ultrastructurally, keratin intermediate filaments and desmosomes were found. In addition, microvilli were seen in the uppermost epithelial cells. CONCLUSIONS: These findings demonstrate that the cultured conjunctival equivalent resembles normal conjunctival epithelium morphologically, biochemically and ultrastructurally, thereby suggesting that the cultured conjunctival equivalent may have a great potential in the study of conjunctival epithelium.