Abstract
Purpose:
The corneal epithelium is the outermost layer of the cornea that is in direct contact with the outside environment. Exposure to environmental stressors, can results in stress-induced senescence whereby the cell no longer have the ability to divide. Previously (ARVO 2013) we demonstrated that rapamycin, an inhibitor of the mTOR pathway, prolongs the lifespan of corneal epithelial cells in culture. In this study we examined the specific effects of rapamycin on senescence and apoptosis of corneal epithelial cells. By reducing senescence and apoptosis, we hope to improve corneal epithelial stability and function in pathologic states.
Methods:
Primary human corneal epithelial cells were grown in keratinocyte serum free media and treated with a range of concentrations of rapamycin. Apoptosis and senescence were assessed using TUNEL assay, X-gal staining and Western blotting for the known markers. Toxicity and proliferation were evaluated using trypan blue exclusion assay and Ki67 immunostaining.
Results:
Rapamycin appeared to have toxic effects at higher concentrations but at low concentrations it appeared to reduce the proliferation of corneal epithelial cells without evidence of toxicity.Cells that were treated with rapamycin demonstrated significantly lower rates of apoptosis and senescence.
Conclusions:
These results suggest that rapamycin increased the lifespan of corneal epithelial cell in-vitro in part by inhibiting senescence. This is consistent with previous studies demonstrating that rapamycin inhibits the aging process. Further studies are needed to determine the mechanisms by which mTOR affects senescence of corneal epithelial cells.
Keywords: 482 cornea: epithelium •
480 cornea: basic science