Purchase this article with an account.
Gary S L Peh, Maninder S Bhogal, Chan Nyein Lwin, Xin-Yi Seah, Khadijah Binte Adnan, Elavazhagan Murugan, Jodhbir Mehta; Real-Time Assessment of Corneal Endothelial Cell Damage Following DMEK Graft Preparation and Insertion. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1478.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
This study describes a method that enable accurate evaluation of graft viability with a single incubation of a fluorescent viability dye that can be tracked following in-situ graft preparation through to implantation and its subsequent assessment in-vivo.
Optimization of viability dye was performed on cultured corneal endothelial cells (CEnCs) expanded for at least 2 passages, isolated from donor corneas unsuitable for transplantation. Cells were incubated with varying concentrations of calcein AM (CAM), and fluorescence intensity was measured. Cellular toxicity of CAM to cultured CEnCs was also assessed by flow cytometry. For assessment of the cell viability of DMEK graft in-situ, transplant-grade corneas were procured and prepared as per for DMEK surgery. Imaging was performed at 3 time points: prior to graft preparation, following preparation and imaging of the unfolded graft within the anterior chamber of a porcine globe. All images were exported to ImageJ for processing and quantification, where viability based on CAM/eithidium homodimer/Hoechst labelling was assessed. Trypan blue/Alizarin red viability staining was also performed. All statistical analyses were performed using GraphPad Prism, and a p-value of <0.05 was deemed to be significant.
Of the concentrations evaluated, 2.67 µM were found to be sufficient for imaging with the Spectralis HRA. At 2.67 µM, viability of labelled CEnCs was not significantly affected compared to unlabeled cells (2.72% vs 3.96%, p = 0.25), and morphometric analysis showed no significant differences between labelled and control corneas. Following implantation of pre-labelled graft, high quality images can be obtained when imaging through fresh or 24hr-old edematous porcine cornea. Areas of non-viability detected using in-situ imaging showed good agreement with the in-vivo global viability method. Estimates of cell density based on CAM fluorescence showed a strong positive correlation with Hoechst positive cells from the same graft region ex-vivo (R=0.74, p<0.0001)
We have developed and validated a method for sequential graft viability assessment that can be applied at every stage of transplantation. The methodology utilizes current clinical imaging device, where with minor adaptation enabled in vivo assessments of overall graft viability and cell density of the whole graft.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only