Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty.
Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty.
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DOI：
10.1136/bjophthalmol-2015-307534
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发表时间：
2016-04
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影响因子：
--
通讯作者：
Allan BD
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文献类型：
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作者：
Bhogal M;Balda MS;Matter K;Allan BD
描述一种用于后弹力层内皮角膜移植术 (DMEK) 的整体细胞活力评估的新方法，并比较两种当代供体组织制备方法。使用两种不同的方法制备DMEK移植物：液泡分离和手动剥离（每组n=8）。将样品与 Hoechst、钙黄绿素-AM 和乙锭同二聚体一起孵育，然后安装在弯曲成像室上。将 Z 堆叠荧光显微镜图像组合起来，生成能够分辨所有细胞核的聚焦全局图像。使用图像处理软件定义钙黄绿素阳性活细胞区域，对该区域内的所有细胞核进行计数，并减去乙锭阳性死细胞以获得活内皮细胞总数。校正后的整体细胞密度是通过将活细胞数量除以移植物面积来计算的，移植物面积已针对曲面成像进行了校正。两组校正后的整体细胞密度均低于中央内皮细胞密度：剥离组为制备前中央内皮细胞密度的85.5%，气泡组为75.8%。液体气泡分离组的校正整体细胞密度显着低于剥离组（p=0.04）。在 DMEK 中制备移植物后，眼库对中央内皮细胞密度的估计高估了真实细胞密度。剥离法比液泡法破坏性更小且更一致。细胞损失与液体气泡组中气泡分离之前的基质水合程度密切相关。
To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group.