Wollensak Gregor, Iomdina Elena, Dittert Dag-Daniel, Herbst Hermann
Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia.
Cornea. 2007 Jun;26(5):600-5. doi: 10.1097/ICO.0b013e318041f073.
This study was undertaken to investigate the wound healing process of the first 6 weeks after photodynamic cross-linking treatment in the rabbit cornea, using the photosensitizer riboflavin and UVA.
After removal of the central epithelium, the right corneas of 8 Chinchilla rabbits were cross-linked with a photosensitizing 0.1% riboflavin solution and UVA light (370 nm; irradiance, 3 mW/cm(2); dose, 5.4 J/cm(2)) for 30 minutes. Two animals were euthanized 3 days, 7 days, 4 weeks, and 6 weeks postoperatively. The corneas of the enucleated eyes were evaluated using 4-microm light microscopic sections with routine stains and avidin-biotin complex immunostaining with anti-alpha-smooth muscle actin.
By day 3 after treatment, complete apoptotic damage and loss of the endothelial cells and the stromal keratocytes were found in the irradiated area through the entire thickness of the stroma. There was marked stromal edema (850 +/- 66 vs. 332 +/- 43 microm in the untreated controls; P < 0.01). The epithelium was already closed again. At the margins of the lesion, there was a mild inflammatory reaction with scattered macrophages, lymphocytes, and neutrophils. By day 7, the endothelium was already intact again, and keratocyte repopulation of the posterior stroma was noted. By week 4, the keratocyte repopulation of the anterior stroma was observed with some acellular areas between. By week 6, the cytoarchitecture of the cornea seemed normal again. By weeks 4 and 6, alpha-actin-positive keratocytes were identified, especially in the periphery of the irradiated area.
After riboflavin/UVA cross-linking of rabbit cornea, a complete cell loss occurs in the irradiation area with an irradiance of 3 mW/cm(2). The cytotoxic damage is repaired by repopulation after approximately 4-6 weeks. A combination of cross-linking with other procedures such as the implantation of intracorneal rings should be performed only after a sufficient time interval of approximately 2 months, allowing cellular regeneration.
本研究旨在利用光敏剂核黄素和紫外线A（UVA），研究兔角膜光动力交联治疗后前6周的伤口愈合过程。
去除8只青紫蓝兔右眼中央上皮后，用0.1%核黄素光敏溶液和UVA光（370nm；辐照度，3mW/cm²；剂量，5.4J/cm²）交联30分钟。术后3天、7天、4周和6周分别对2只动物实施安乐死。对摘除眼球的角膜进行评估，制作4微米厚的光学显微镜切片，进行常规染色，并使用抗α平滑肌肌动蛋白的抗生物素蛋白-生物素复合物免疫染色。
治疗后第3天，在照射区域的整个基质厚度中发现内皮细胞和基质角膜细胞完全凋亡损伤并丧失。基质明显水肿（未治疗对照组为332±43微米，照射组为850±66微米；P<0.01）。上皮已经再次闭合。在病变边缘，有轻度炎症反应，伴有散在的巨噬细胞、淋巴细胞和中性粒细胞。到第7天，内皮已经再次完整，并且观察到后基质中有角膜细胞重新填充。到第4周，观察到前基质中有角膜细胞重新填充，其间有一些无细胞区域。到第6周，角膜的细胞结构似乎再次正常。到第4周和第6周，鉴定出α肌动蛋白阳性角膜细胞，尤其是在照射区域的周边。
兔角膜经核黄素/UVA交联后，辐照度为3mW/cm²时，照射区域会出现完全的细胞丧失。细胞毒性损伤在约4-6周后通过细胞重新填充得以修复。交联与其他手术（如角膜内环植入）联合进行时，应仅在约2个月的足够时间间隔后进行，以允许细胞再生。
