Corneal Endothelial Cell Fate After Organismal Death And The Post-mortem Survival Mechanism

Purpose:To investigate the structural and functional changes of post-mortem corneal endothelial cells(CECs)under 4? or 25? and explore the post-mortem CECs survival mechanism.Methods:C57BL/6 mice were sacrificed and dead bodies were preserved at 4?or 25? for up to 72h.At different post-mortem interval(PMI),the anterior segment were evaluated by slit-lamp microscopy,and bacterial contamination of aqueous humor were detected by agar plate test.Hematoxylin and eosin,N-cadherin and Na+/K+-ATPase staining and scanning electron microscope(SEM)were used to investigate CECs structure and function.Cell apoptosis and necroptosis was detected with the TUNEL assay and phosphate RIP3 staining.Altered gene expression pattern of rabbit CECs at 12h post-mortem were detected by RN A sequencing(RNA-seq)and quantitative Real-time RT-PCR.Etanercept,a tumor necrosis factor(TNF)inhibitor,was injected to protect mouse CECs at 25?.Results:At 48h post-mortem,intact endothelium with strong cell-cell junctions were retained only in 4?.Hypothermia maintained a normal anterior chamber depth,along with sterile aqueous humor,but little necrosis.RNA-seq analysis and quantitative Real-time RT-PCR(q-RT-PCR)showed that eye development genes and negative regulation of apoptotic signaling pathway genes were upregulated at 4?.Furthermore,the TNF-a signaling pathway has a key role in post-mortem CECs death and dysfunction at 25?,which was blocked by Etanercept injection.Conclusion:Post-mortem corneal endothelium preserved under hypothermia maintain the intact structure,normal function,high viability,and healthy cell pool.Our study indicates that the TNF-a signaling pathway is involved in the post-mortem CECs survival process,which may be used in eye bank tissue processing and corneal endothelial tissue engineering.