Corneal graft endothelial viability assessment using the triple labeling Hoechst/Ethidium homodimer/Calcein‐AM: technical improvements usind 3D microscopy

Abstract

Abstract Purpose: A reliable viability test for corneal endothelium is necessary for the assessment of new processes likely to affect it (storage media, lamellar cutting methods...). Aim: to improve the classical calcein‐AM (C)/Ethidium homodimer (E) double staining (live/dead assay) by adding Hoechst and by using a 3D microscope and image analysis that allow a quantitative assessment on the whole endothelium. As an example of application, the assessment of the actual endothelial viability immediately before graft is given. Methods: The endothelial side was incubated 45 min at 31°C with 200 μL of C (2μM), E (5μM), H (10μM). After flat mount, images of the whole endothelial area (81mm²) were taken using a microscope equipped with a motorized stage (IX81, Olympus) and a x4 objective. Z stacks were taken and an extended focal imaging algorithm allowed taking account of corneal folds. Endothelial cell density (ECD) and mortality were determined by automatic counting of H+ and E+ nuclei. Area of C+ cells was measured. Results: This upgraded staining on the whole endothelium allowed the exhaustive assessment of ECD, cell mortality and metabolic activity. On corneas immediately before graft, it highlighted a dramatic lower number of viable cells than expected with the routine assessment of ECD only, done 48H before graft in the eye bank. Conclusions: The triple HEC staining takes account of the heterogeneous pattern of lesions within the whole endothelial layer and is therefore far less biased than assessment done on small cell samples. Its use improves the assessment of all new processes likely to affect the endothelium ex vivo.