Department of Ophthalmology, St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, UK.
Instituto Universitario Fernandez-Vega, Universidad de Oviedo and Fundacion de Investigacion on Oftalmologica, Oviedo, Spain.
Br J Ophthalmol. 2018 Apr;102(4):549-555. doi: 10.1136/bjophthalmol-2017-310906. Epub 2017 Nov 13.
BACKGROUND/AIMS: Descemet membrane endothelial keratoplasty (DMEK) preparation is technically demanding and is a limiting factor for uptake of this kind of surgery. Supply methods that simplify the procedure for surgeons are key to increasing uptake. This study compares two different shipping protocols for DMEK.
An 8.5 mm DMEK graft was punched, marked and loaded for transportation in two different conditions: (A) endothelium trifolded inwards in organ culture conditions (n=7) and (B) endothelium rolled outwards in hypothermic conditions (n=7). Tissues were shipped from Italy to the UK, then analysed for orientation, endothelial cell density, denuded areas, cell mortality, triple viability staining (Hoechst/ethidium homodimer/calcein AM (HEC)), immunolocalisation of ZO-1 and Na/K-ATPase proteins, visualisation of actin filaments using phalloidin and histological analysis using H&E on paraffin-embedded sections.
All tissues clearly showed the mark used for graft orientation. After shipping in condition A, there was an increase in cell mortality of 8.1% and in denuded areas of 22.4%, whereas for condition B there was an increase in cell mortality of 14.2% and in denuded areas of 34.3% after shipping. HEC staining revealed areas of viable cells and apoptotic cells, with large denuded areas found in the periphery for condition B and within folds for condition A.
Prestripped preloaded DMEK grafts retained sufficient viable cells for transplantation, with condition A (endothelium-in) offering the advantage of greater flexibility of use due to a longer shelf-life. HEC analysis provides further detailed information as to the status of DMEK grafts and should be used in future similar studies.
背景/目的：Descemet 膜内皮角膜移植术（DMEK）的准备工作技术要求较高，是限制该手术应用的一个因素。为外科医生简化手术流程的供应方法是增加手术接受度的关键。本研究比较了两种不同的 DMEK 运输方案。
将 8.5mm 的 DMEK 移植物打孔、标记并装载，在两种不同条件下进行运输：（A）在器官培养条件下将内皮向内对折（n=7）和（B）在低温条件下将内皮向外卷起（n=7）。组织从意大利运往英国，然后分析其取向、内皮细胞密度、裸露区、细胞死亡率、三重活力染色（Hoechst/ethidium homodimer/calcein AM（HEC））、ZO-1 和 Na/K-ATPase 蛋白的免疫定位、使用鬼笔环肽对肌动蛋白丝的可视化以及使用 H&E 对石蜡包埋切片的组织学分析。
所有组织均清晰显示了用于移植取向的标记。在条件 A 下运输后，细胞死亡率增加了 8.1%，裸露区增加了 22.4%，而在条件 B 下运输后，细胞死亡率增加了 14.2%，裸露区增加了 34.3%。HEC 染色显示了有活力细胞和凋亡细胞的区域，在条件 B 下发现较大的裸露区位于周边，而在条件 A 下发现较大的裸露区位于折叠处。
预折叠预装的 DMEK 移植物保留了足够的有活力细胞用于移植，条件 A（内皮向内）具有更长的货架寿命，因此使用更灵活。HEC 分析为 DMEK 移植物的状态提供了进一步的详细信息，应在未来的类似研究中使用。
