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A. W. Taylor, J. A. Dixon, F. Carley, I. Zambrano, M. C. Hillarby; Endothelial Cell Loss in Corneal Organ Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2699.
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Prior to transplantation corneas are stored in organ culture for up 4 weeks. However, up to 30% of corneas are discarded, mainly due to a poor endothelial cell count. An understanding of the mechanisms that are involved in endothelial cell loss could lead to improved culture conditions and a reduced discard rate. We have previously shown that apoptosis plays a role in endothelial cell loss. In this study we have further investigated the genes expressed during the process. In this study we investigated the gene expression controlling this cell loss.
RNA was extracted from corneal endothelium with either a low or high cell count after 10 days in organ culture. The RNA was used to probe micro arrays. Genes shown to be either up or down regulated were selected for study by immunohistochemistry and real time PCR.
The micro array showed several genes to be up or down regulated in corneas with low endothelial counts. Of particular interest were genes involved in signalling pathways, cell cycling and apoptosis. We have confirmed the up regulation for several genes including TNFRI, Fas, Caspase 3, Caspase 8 and MADD using both immunohistochemistry and real-time PCR.
The presence of caspase 3 in the endothelium confirms apoptosis occurs during cell loss. The presence of Caspase 8 confirms apoptosis is induced by cell surface receptor binding. Both Fas and TNFR1 are present on the corneal endothelial cells and the presence of MADD confirms the TNF induced apoptotic pathway has been activated. However, we have not confirmed or disproved the activation of the Fas induced apoptotic pathway. These results are helping to elucidate the apoptotic pathways involved in endothelial cell loss and we are hoping to block these pathways to preserve the endothelial cells during organ culture. Corneal transplantation is limited by the number of corneas. A better understanding of endothelial cell loss in culture could potentially increase the number of corneas available for transplantation.
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