Human Corneolimbal Epithelium ex vivo: Bcl-2 and Bax in organ culture and in epithelium expanded on Human Amniotic Membranes

Abstract

Purpose: : We investigate damage and repair in donor corneolimbal epithelium, which has been obtained in Eye Bank Organ Culture (EBOC) and also expanded on Human Amniotic Membranes (HAMs) in two different media. Expression of apoptotic markers can thus be compared when in repair phase (early EBOC) and in an expanding mode (on HAMs), whereas the cultivating medium can be investigated for influencing cell survival driven pathways.

Methods: : Corneoscleral rims were obtained from donor corneas and either incubated in EBOC medium for 7 days, or cultivated on HAMs for 3 weeks. Samples were retrieved and examined for expression of genotypic and phenotypic apoptotic markers Bcl-2 and Bax by immunohistochemistry and qRT-PCR.

Results: : Corneal epithelium incubated in EBOC for 7 days shows a ~2-fold increase in expression of Bax compared to day 1, while there was virtually no change in the expression of Bcl-2. This gives a Bcl-2/Bax ratio of ~1:2. A significant increase in expression of Bcl-2 (~9-fold) and Bax (~4-fold) was found in epithelium cultured in the medium with HS on HAM, giving a Bcl-2/Bax ratio of ~8:1. No significant changes in expression of either of the apoptotic markers were seen in the cultures maintained in COM.

Conclusions: : Expression levels of anti-apoptotic marker Bcl-2 and the pro-apoptotic Bax in EBOC system for one week indicates a shift towards an apoptotic pathway, consistent with an ongoing degeneration and repair previously described to occur in the epithelium at this stage in culture. The Bcl-2/Bax ratio in epithelium on HAMs in HS indicates cells in a proliferate and expanding state promoted by a Bcl-2 driven cell survival pathway. This finding contrasts with the lack of Bcl-2 up regulation in COM and could possibly indicate an increased susceptibility to apoptosis in this medium.

Keywords: cornea: epithelium • cornea: storage • cell survival