Abstract
Purpose :
Corneal endothelial cells, non-regenerative in nature, face irreversible damage due to dystrophy or intraocular surgery, with transplantation as the sole available remedy. While assessing endothelial cell density via a specular microscope is a common practice, evaluating cell function remains elusive. We describe fundamental experiments in this proof of concept study that leverage our innovative apoptosis visualization technology, Detection of Apoptosing Retinal Cells (DARC), to target endothelial cells. DARC employs a fluorescent Annexin A5 molecule to label phosphatidylserine (PS), a phospholipid that is externalized on the cell membrane's outer layer during early apoptosis and cell stress. As stress-related PS externalization is not confined to retinal cells and spans all cells, it was applied to corneal endothelial cells of rodents to investigate the application of DARC in assessing cell stress.
Methods :
Rodents' corneas were excised, and central endothelium wounds were induced using various sized needles (18G, 21G or 31G needles) or forceps. Following a 22-hour immersion in a stabilizing solution (L-15 medium), corneas were treated with DARC solution, featuring fluorescently labeled Annexin A5, for 2 hours and fixed with 4% PFA and stained with DAPI. Imaging with bright field microscopy (Olympus BX40, ImagePro Plus 7.0) verified the extent of corneal endothelial cell damage. DARC and DAPI fluorescence were imaged to quantify cell stress and cell density, respectively. Images taken were analyzed using ImageJ software.
Results :
Our results show DARC can visualise apoptosis in cases of mechanical induced corneal endothelial injury. Using ImageJ the areas of DARC staining intensity around the mechanical injury versus areas at least 100 pixels from the scratch area were assessed. Our results indicated significant higher DARC staining at the scratch (p=0.01) (mean 59.2 SD 23.3 versus mean 5.5, SD 1.6 in non-scratch areas) when assessing all images. The quantity of apoptosis-positive cells identified by DARC was associated with the extent of damage and areas of induced mechanical injury.
Conclusions :
We have shown initial positive results showing DARC's potential to quantify mechanically induced corneal endothelial damage. Further work in the field will enable corneal endothelial cell assessment with functional evaluation and is encouraging for potential of in vivo DARC assessment of the cornea.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.