Szurman Peter, Sierra Ana, Kaczmarek Radoslaw, Jaissle Gesine B, Wallenfels-Thilo Barbara, Grisanti Salvatore, Lüke Matthias, Bartz-Schmidt Karl U, Spitzer Martin S
Department of Ophthalmology I, Eberhard-Karls University of Tuebingen, Schleichstrasse 12, 72076 Tuebingen, Germany.
Exp Eye Res. 2007 Jul;85(1):44-53. doi: 10.1016/j.exer.2007.03.003. Epub 2007 Mar 19.
Epiretinal deposits of triamcinolone acetonide (TA) can be detrimental to retinal cells in vitro as several laboratory studies have shown. This contrasts with the good clinical experience of intravitreal TA use. We investigated the effect of TA crystals on retinal cells concerning the critical dose range, a potential cell recovery, the drug-tissue interaction and what protective biological factors could explain the discrepancy between in vivo and in vitro results. A human retinal pigment epithelium cell line (ARPE19) and transformed rat retinal ganglion cells (RGC5) were used. Purified TA crystals were either added directly on top of the cell cultures or on top of membrane filter inserts, basement membrane sheets or porcine vitreous with the cells growing underneath. To determine the number of live versus dead cells fluorescent stains were used. Proliferation and viability were measured using the MTT assay and the mean inhibitory dose (ID(50)) calculated with or without a filter. Cell recovery was measured after transient TA exposure (0.01-1 mg/ml) compared to continuous exposure after 7 days. To exclude a mere mechanical effect of epicellular deposits the TA crystals were replaced by glass pearls in a serum-free medium and the MTT toxicity assay was performed after 24 h. Without direct contact of TA crystals with the cells only a moderate decrease of mitochondrial activity was observed that fully recovered after transient exposure and showed a clinically safe ID(50) of 7.7 mg/ml. In contrast, direct exposure to even minute crystalline deposits for 7 days caused a rapid progressive and irreversible cell death being significant far below clinically used concentrations (ID(50) 0.058 mg/ml). Direct exposure to glass pearls did not show any loss of viability. Both basement membrane sheets and vitreous reliably prevented direct cytotoxicity to underlying retinal ganglion cells. Our findings suggest that irreversible TA cytotoxicity in a cell culture setting occurs earlier than previously assumed in the presence of even minute epicellular deposits. But in most clinical situations epiretinal TA deposits seem not to be harmful to ocular cells as protective biological factors may prevent close apposition of TA crystals to susceptible retinal cells. However, in eyes that have undergone vitrectomy with ILM peeling epimacular deposits could be critical.
正如多项实验室研究所示，曲安奈德（TA）的视网膜前沉积物在体外可能对视网膜细胞有害。这与玻璃体内使用TA的良好临床经验形成对比。我们研究了TA晶体对视网膜细胞的影响，涉及关键剂量范围、潜在的细胞恢复情况、药物与组织的相互作用，以及哪些保护性生物因素可以解释体内和体外结果之间的差异。使用了人视网膜色素上皮细胞系（ARPE19）和转化的大鼠视网膜神经节细胞（RGC5）。纯化的TA晶体要么直接添加到细胞培养物顶部，要么添加到膜滤器插入物、基底膜片或猪玻璃体顶部，细胞在其下方生长。为了确定活细胞与死细胞的数量，使用了荧光染色。使用MTT法测量增殖和活力，并计算有无滤器时的平均抑制剂量（ID(50)）。与7天后的持续暴露相比，在短暂暴露TA（0.01 - 1 mg/ml）后测量细胞恢复情况。为了排除细胞外沉积物的单纯机械作用，在无血清培养基中用玻璃珠代替TA晶体，并在24小时后进行MTT毒性试验。在TA晶体与细胞没有直接接触的情况下，仅观察到线粒体活性有适度下降，短暂暴露后完全恢复，临床安全ID(50)为7.7 mg/ml。相比之下，即使直接暴露于微小的晶体沉积物7天也会导致快速进行性和不可逆的细胞死亡，远远低于临床使用浓度时就具有显著影响（ID(50) 0.058 mg/ml）。直接暴露于玻璃珠未显示任何活力丧失。基底膜片和玻璃体都可靠地防止了对下方视网膜神经节细胞的直接细胞毒性。我们的研究结果表明，在细胞培养环境中，即使存在微小的细胞外沉积物，TA的不可逆细胞毒性也比之前假设的发生得更早。但在大多数临床情况下，视网膜前TA沉积物似乎对眼细胞无害，因为保护性生物因素可能会阻止TA晶体与易感视网膜细胞紧密贴附。然而，在接受了玻璃体切除术并剥除内界膜的眼中，黄斑前沉积物可能至关重要。
