The Roles Of Autophagy In Rat Corneal Endothelial Cells Preserved In Optisol

Purpose:To investigate the roles of autophagy in corneal endothelial cells(CEC)during mid-term corneal preservation.Methods:To establish the mid-term corneal preservation model,rat cornea was stored in Optisol.We applied alizarin red staining to count the number and observed the morphology of CECs.The expression of ZO-1,F-actin,N-cadherin and Na+-K+-ATPase,were detected by immunofluorescence staining(IF).We examined the ultrastructural changes of endothelial cells by scanning electron microscope.The expressions of autophagy-related proteins,such as LC 3,Beclin 1 and P62 were measured by Western blotting and IF.TUNEL staining and IF of activated-caspase3 were conducted to investigate the apoptosis of CECs.We also performed corneal transplantation in rats and examined the effects of agonist or inhibitor of autophagy:Rapamycin or Chloroquine.Results:The number of CECs was gradually decreased during corneal storage,.It showed that there were nuclear shrinkage,alterations of cell junction ZO-1,damage of cytoskeleton F-actin and cell adhesion molecules N-cadherin,as well as the abnormal expression of Na+-K+-ATPase in CECs.The images by scanning electron microscopy demonstrated cell edema,and fractured Y-type connection between CECs,and disappearance of surface microvilli.In addition,the expression of LC3 ? and Beclin 1 was increased at beginning and decreased later,reaching the peak value on the 15th day.The trend of P62 was opposite.Meanwhile,TUNEL-positive cells and the level of activated-caspase3 were gradually increased.Furthermore,administration of Chloroquine,an inhibitor of autophagy,significantly increased the ratio of TUNEL-positive cells and the expression activated-caspase3,resulting in increased endothelial cell death and destruction,and it could be found that the cornea remained cloudy after one week of corneal transplantation.On the other hand,it was shown that treatment of Rapamycin,an autophagy agonist,decreased apoptosis rate,increased endothelial cell viability,ameliorated structure and function related indicators,and induced better corneal transparence after one week of corneal transplantation.Conclusion:The activity of corneal endothelial cells was gradually decreased and autophagy was induced during corneal storage.We provided novel evidence that autophagy can reduce apoptosis,promote endothelial cell survival,improve endothelium quality and prolong the preservation time of cornea,suggesting the beneficial role of autophagy in the corneal preservation.