Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.
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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5627903?pdf=render |
| _version_ | 1819122833457938432 |
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| author | Maninder Bhogal Chan N Lwin Xin-Yi Seah Elavazhagan Murugan Khadijah Adnan Shu-Jun Lin Gary Peh Jodhbir S Mehta |
| author_facet | Maninder Bhogal Chan N Lwin Xin-Yi Seah Elavazhagan Murugan Khadijah Adnan Shu-Jun Lin Gary Peh Jodhbir S Mehta |
| author_sort | Maninder Bhogal |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T06:58:44Z |
| format | Article |
| id | doaj.art-2dbf91cc5f8b4ba78e9cc7dc6290a7ac |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T06:58:44Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-2dbf91cc5f8b4ba78e9cc7dc6290a7ac2022-12-21T18:34:51ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011210e018482410.1371/journal.pone.0184824Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.Maninder BhogalChan N LwinXin-Yi SeahElavazhagan MuruganKhadijah AdnanShu-Jun LinGary PehJodhbir S MehtaTo 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.http://europepmc.org/articles/PMC5627903?pdf=render |
| spellingShingle | Maninder Bhogal Chan N Lwin Xin-Yi Seah Elavazhagan Murugan Khadijah Adnan Shu-Jun Lin Gary Peh Jodhbir S Mehta Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. PLoS ONE |
| title | Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. |
| title_full | Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. |
| title_fullStr | Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. |
| title_full_unstemmed | Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. |
| title_short | Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK. |
| title_sort | real time assessment of corneal endothelial cell damage following graft preparation and donor insertion for dmek |
| url | http://europepmc.org/articles/PMC5627903?pdf=render |
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