International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation, Venice, Italy.
Department of Ophthalmology, Riga Stradins University, Riga, Latvia.
Acta Ophthalmol. 2018 Sep;96(6):e718-e726. doi: 10.1111/aos.13746. Epub 2018 Mar 9.
To compare different Descemet membrane endothelial keratoplasty (DMEK) graft preparation methods.
Stripping from the trabecular meshwork (M1) using epithelial spatula; stripping by scoring the peripheral endothelium (M2) using Sinskey hook; stripping by punch method (M3) using donor trephine; Submerged hydro-separation (M4); and pneumatic dissection method (M5) were evaluated. Preparation time, costs, endothelial cell loss (ECL) postpreparation, cell death and morphology were compared. Hoechst/Ethidium/Calcien AM (HEC) staining and Zonula Occludens-1 (ZO-1) expression were analysed. Statistical analysis was performed using one-way anova and; Tukey as post hoc test.
A total of 35 corneas (seven per group) were used. Endothelial cell loss (ECL) represented as Mean (SD), in M1, M2, M3, M4 and M5 was 2.7 (5.0), 3.0 (7.4), 1.2 (7.4), 3.3 (7.3) and 4.1 (7.1)%, respectively not showing any difference between the groups (p = 0.96). A significantly higher cell death (p < 0.05) was observed in M4 and M5 compared with M1, M2 and M3. Graft preparation time was significantly shorter in M4 and M5 and longest in M3 (p < 0.05). M3 was the most expensive preparation technique. Minimum pleomorphic cells were observed in M1, M2 and M3, whereas moderate pleomorphism was seen in M4 and M5. Hoechst, Ethidium homodimer and Calcein AM (HEC) staining showed high Ethidium positivity (dead cells) in M4 and M5 with minimum positivity in M1, M2 and M3. Zonula Occludens-1 (ZO-1) was expressed in all the conditions except the denuded areas.
Graft preparation using Sinskey hook (M2) and donor punch (M3) are reliable methods in terms of efficiency and quality with acceptable range of ECL. The preparation time and associated costs could be a limitation for M3.
比较不同的撕囊膜内皮角膜移植术（DMEK）供体准备方法。
使用上皮刮刀从小梁网（M1）上剥离；使用 Sinskey 钩在周边角膜内皮上划痕（M2）；使用供体环钻打孔（M3）；浸没式水分离（M4）；以及气动分离法（M5）。比较准备时间、成本、准备后内皮细胞丢失（ECL）、细胞死亡和形态。分析 Hoechst/Ethidium/Calcien AM（HEC）染色和 Zonula Occludens-1（ZO-1）表达。采用单因素方差分析和 Tukey 作为事后检验进行统计分析。
共使用 35 个角膜（每组 7 个）。M1、M2、M3、M4 和 M5 中的内皮细胞丢失（ECL）分别表示为平均值（标准差）：2.7（5.0）、3.0（7.4）、1.2（7.4）、3.3（7.3）和 4.1（7.1）%，各组之间无差异（p=0.96）。与 M1、M2 和 M3 相比，M4 和 M5 中的细胞死亡明显更高（p<0.05）。M4 和 M5 的移植片准备时间明显短于 M1、M2 和 M3，而 M3 的准备时间最长（p<0.05）。M3 是最昂贵的准备技术。M1、M2 和 M3 中观察到的细胞形态最小，而 M4 和 M5 中观察到的细胞形态中度不规则。Hoechst、Ethidium homodimer 和 Calcein AM（HEC）染色显示 M4 和 M5 中 Ethidium 阳性（死亡细胞）较高，而 M1、M2 和 M3 中阳性最低。除了裸露区域外，所有条件下均表达 Zonula Occludens-1（ZO-1）。
就效率和质量而言，使用 Sinskey 钩（M2）和供体环钻（M3）进行供体准备是可靠的方法，ECL 可接受。准备时间和相关成本可能是 M3 的限制因素。
