Abstract
PurposeTo establish a method for assessing graft viability, in-vivo, following corneal transplantation.MethodsOptimization 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.ResultsCalcein 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.ConclusionsIn-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.
Highlights
Corneal disease ranks second only to cataract as the leading cause of preventable blindness[1], and corneal transplantation is the commonest tissue transplant procedure performed worldwide
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
Summary
Corneal disease ranks second only to cataract as the leading cause of preventable blindness[1], and corneal transplantation is the commonest tissue transplant procedure performed worldwide. Ongoing endothelial cell loss occurs in the transplanted tissue, and proceeds at an accelerated rate compared to that observed in the native corneal endothelium of healthy adults (4.2% for penetrating keratoplasty vs 0.3% in the native corneal endothelium) [6,7]. This means that late graft failure is a common occurrence[8] requiring regrafting, which in itself is a common indication for keratoplasty [2]. There is concern that these more technically demanding procedures, may result in a lower number of endothelial cells being transplanted and may, have reduced survival times[9]
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