The existence of dead cells in donor corneal endothelium preserved with storage media
The existence of dead cells in donor corneal endothelium preserved with storage media
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DOI：
10.1136/bjophthalmol-2017-310913
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发表时间：
2017-12-01
影响因子：
4.1
通讯作者：
Kinoshita, Shigeru
中科院分区：
文献类型：
--
作者：
Kitazawa, Koji;Inatomi, Tsutomu;Kinoshita, Shigeru
目的 通过组织学检查探讨保存在储存介质中的供体角膜内皮细胞（CEC）的活力。方法从SightLife眼库（西雅图，华盛顿）获取28个供体角膜，取出中心角膜移植物进行移植后，使用这些角膜多余的周边部分进行组织学检查。为了评估角膜内皮细胞的活力，使用碘化丙啶、钙黄绿素-AM、Hoechst 33 342、膜联蛋白V、抗波形蛋白抗体和甲苯胺蓝进行生物染色实验。结果内皮组织学分析显示，死细胞的细胞质具有低强度荧光，细胞核被染成红色，而几乎所有活细胞的细胞质都呈绿色和蓝色。原子核。 28 个供体角膜的平均死细胞率为 4.9%+/-3.3%（平均值 +/- SD）（范围：0.6%-10.5%）。碘化丙啶阳性细胞膜联蛋白 V 染色呈阳性，波形蛋白呈阴性，甲苯胺蓝呈浅色。标本在培养基中孵育后，红核死细胞从蓝核活细胞水平脱落。结论我们的研究结果表明，在保存介质保存的供体角膜内皮中存在死细胞，并且它们在培养后脱落，因此表明角膜移植术后CEC的减少可能与死细胞的存在有关。
Aim To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination.Methods Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue.Results Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%+/-3.3% (mean +/- SD) (range: 0.6%-10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells.Conclusion Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.