Abstract
Purpose:
The number of corneal endothelial cells (CECs) decreases with age because corneal endothelium does not divide in vivo. After corneal transplantation, the endothelial cell density on donor corneas rapidly declines by acute intraoperative trauma. Therefore it is important to protect CECs during surgery for the survival of the allograft. The corneal endothelium are mitochondria-rich cells, indicate that CECs are metabolically active. It is also reported that cell death is associated with reduced functioning of mitochondria. In glia cell and cardiomyocyte, Cyclosporin A (CsA) suppress cell death by inhibition of the permeability transition pore (PTP) which lead to stabilization of mitochondrial membrane potential as well as immunosuppressive effect. The purpose of this study was to investigate whether CsA inhibit cell death in human cultured corneal endothelial cells (hCECs) and mouse corneas.
Methods:
HCECs were stimulated with H2O2(0~1000nM) for 1 hour. And whole mount cornea, endothelial cell side up from C57BL/6 were stored DMEM serum free at 4C and then subjected to various concentrations of H2O2 (0-1000M) for 1 hour with/without various concentration CsA. Apoptosis were detected using Caspase 3/7 (Invitrogen). Mitochondria in corneal endothelial cells were labeled by the addition of MitoTracker Red CMXRos to the corneal cups (125 nM final concentration; Invitrogen) and maintaining for 30 minutes in a 37C in a 5% CO2 incubater.
Results:
1) Oxidative stress induced cell death was observed in a concentration-dependent manner in hCECs. 2) CsA decreased cell death by about 10%, and suppressed the reduction of mitochondrial membrane potential at 100nM in hCECs. Only the highest concentration of CsA (1000nM) showed cytotoxicity in hCECs. 3) CsA suppressed the reduction of mitochondrial membrane potential at 100nM in whole mount mouse corneas.
Conclusions:
We conclude that CsA suppress the reduction of mitochondrial membrane potential from oxidative stress, and inhibit cell death in corneal endothelial cells.
Keywords: 481 cornea: endothelium •
426 apoptosis/cell death •
489 cyclosporine