Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK

• Published in: PloS one 2017-10, Vol.12 (10), p.e0184824
• Main Authors: Bhogal, Maninder, Lwin, Chan N, Seah, Xin-Yi, Murugan, Elavazhagan, Adnan, Khadijah, Lin, Shu-Jun, Peh, Gary, Mehta, Jodhbir S
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Aged; Alizarin; Animals; Apoptosis; Biocompatibility; Biology and Life Sciences; Calcein; Cataracts; Cells, Cultured; Confocal; Cornea; Corneal Endothelial Cell Loss - etiology; Corneal transplantation; Damage assessment; Damage detection; Descemet Stripping Endothelial Keratoplasty - adverse effects; Descemet Stripping Endothelial Keratoplasty - methods; Electron microscopy; Endothelial cells; Endothelium; Eye; Eye surgery; Female; Fluoresceins; Fluorescence; Grafting; Grafts; Human performance; Humans; Insertion; Male; Medicine and Health Sciences; Methods; Microscopy, Fluorescence; Middle Aged; Models, Animal; Neurodegeneration; Optimization; Postoperative period; Quantum dots; Research and Analysis Methods; Scanning electron microscopy; Sensitivity; Sensitivity analysis; Studies; Surgery; Swine; Tissue Donors; Toxicity; Transplantation; Transplants & implants; Wounds; Young Adult
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0184824

To establish a method for assessing graft viability, in-vivo, following corneal transplantation. Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques. Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1%) and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7-35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage. In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo.