Wollensak Gregor, Spoerl Eberhard, Wilsch Michaela, Seiler Theo
Department of Ophthalmology, Technical University of Dresden, Dresden, Germany.
Cornea. 2004 Jan;23(1):43-9. doi: 10.1097/00003226-200401000-00008.
Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo.
Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35- 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis.
In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 microm of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5-0.7 mW/cm2 could be deduced.
Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 microm using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.
已证实联合核黄素/紫外线A（UVA）治疗可诱导角膜中的胶原蛋白交联，从而增加角膜的生物力学硬度，并已成功用于治疗进行性圆锥角膜。本研究旨在探讨联合核黄素/UVA治疗对体内角膜基质细胞可能产生的细胞毒性作用。
34只新西兰白兔接受0.1%核黄素溶液和表面UVA辐照度为0.75至4 mW/cm²（1.35 - 7.2 J/cm²）的照射，持续30分钟。术后4小时（n = 6）或24小时（n = 28）对动物实施安乐死。另外4只对照眼仅进行上皮清创。对摘除眼球的角膜进行常规组织学切片评估。此外，采用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）技术和透射电子显微镜检测基质细胞凋亡。
仅进行角膜上皮清创的对照眼中，术后4小时在角膜基质前50微米处发现凋亡的基质细胞。然而，核黄素/UVA诱导的凋亡仅在术后24小时摘除的兔眼中可见。在这些眼中，我们发现基质细胞凋亡至不同的基质深度，这取决于所施加的UVA辐照度。可以推断出对基质细胞具有细胞毒性的UVA辐照度范围为0.5 - 0.7 mW/cm²。
核黄素/UVA治疗会导致剂量依赖性的基质细胞损伤，使用表面UVA剂量5.4 J/cm²时，在人角膜中预期可损伤至300微米深度。未来应开展研究以检查基质细胞的再生情况，并排除基质细胞丢失可能产生的不良后遗症，如基质瘢痕形成或变薄。
