Department of Ophthalmology and Neuroscience Center of Excellence, LSU Health Sciences Center, School of Medicine, Louisiana State University, New Orleans, LA 70112, USA.
Cornea. 2010 Apr;29(4):412-7. doi: 10.1097/ICO.0b013e3181bdf1cc.
To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A exposure using immunofluorescence microscopy.
Twenty adult New Zealand albino rabbits weighing 2.0–3.0 kg were used in this study. Two rabbits served as controls.The animals had their epithelia removed and were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL of 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to ultraviolet A (360 nm, 3 mW/cm2) for 30 minutes. Four rabbits were humanely euthanized at each time point of 1, 3, and 11 days and at 3 and 5 weeks after the procedure. Immunohistochemistry studies of thin sections of each cornea were performed using terminal deoxynucleotyl transferase–mediated uridine triphosphate biotin nick-end labeling staining, alpha smooth muscle actin (a-SMA),CD-3, myeloperoxidase antibodies, and 4',6-diamidino-2-phenylindole(DAPI) counterstaining. In another experiment, 6 additional rabbits were treated as above, and after 10 days of cross-linking, 5 mL of lipopolysaccharide endotoxin (1 mg/mL) was injected in the midstroma.
Cross-linked corneas showed early stromal edema. By 5 weeks, complete resolution of the edema and a pronounced highly-organized anterior 200-mm fluorescent zone was observed. Terminal deoxynucleotyl transferase mediated uridine triphosphate biotin nick-end labeling staining showed keratocyte death by both necrosis and apoptosis between days 1 and 3 after cross-linking. At day 1,the limbal area close to the cross-linking zone showed some inflammatory cells and a-SMA–positive cells, indicative of the presence of myofibroblasts. By day 3, some myofibroblasts had migrated to the area beneath the cross-linked stroma. Between days 3 and 5 weeks, there was an increase in a-SMA staining in the area surrounding the cross-linked stroma. The area of cross-linking remained acellular up to 5 weeks.
Collagen cross-linking results in early edema,keratocyte apoptosis, and necrosis, appearance of inflammatory cells in the surrounding area of treatment and transformation of surrounding keratocytes into myofibroblasts. Compaction of anterior stroma fibers, keratocyte loss, and displacement of cell nuclei including inflammatory cells may have clinical implications in the long-term risk of further corneal thinning in keratoconus and in the cross-linked corneal immune response.
使用免疫荧光显微镜评估核黄素和长波紫外线照射后兔眼角膜成纤维细胞的超微结构改变和炎症细胞反应。
本研究使用 20 只成年新西兰白兔（体重 2.0-3.0kg）。其中 2 只兔子作为对照。去除上皮后，用核黄素 0.1%溶液（10mg 核黄素-5-磷酸在 10ml 20%葡聚糖 T-500 中）交联，每 3 分钟应用一次，共 30 分钟，然后用长波紫外线（360nm，3mW/cm2）照射 30 分钟。程序后第 1、3、11 天和 3、5 周，每一时间点处死 4 只兔子。用末端脱氧核苷酸转移酶介导的尿嘧啶三磷酸生物素缺口末端标记染色、α平滑肌肌动蛋白（α-SMA）、CD-3、髓过氧化物酶抗体和 4'，6-二脒基-2-苯基吲哚（DAPI）复染对每个角膜的薄切片进行免疫组织化学研究。在另一个实验中，另外 6 只兔子如上所述处理，交联 10 天后，在中基质注射 5ml 脂多糖内毒素（1mg/ml）。
交联的角膜表现出早期基质水肿。5 周后，观察到水肿完全消退，并出现明显的、高度有序的前 200μm 荧光带。交联后第 1 天至第 3 天，末端脱氧核苷酸转移酶介导的尿嘧啶三磷酸生物素缺口末端标记染色显示成纤维细胞通过坏死和凋亡死亡。第 1 天，靠近交联区的角膜缘区域有一些炎症细胞和 α-SMA 阳性细胞，表明存在肌成纤维细胞。第 3 天，一些肌成纤维细胞已迁移到交联基质下方的区域。第 3 天至第 5 周，交联基质周围区域的 α-SMA 染色增加。交联区域直到第 5 周仍然没有细胞。
胶原交联导致早期水肿、成纤维细胞凋亡和坏死、治疗区域周围炎症细胞的出现以及周围角膜细胞向肌成纤维细胞的转化。前基质纤维的紧密化、角膜细胞的丢失和包括炎症细胞在内的细胞核的移位，可能对视锥角膜长期进一步角膜变薄的风险和交联角膜免疫反应具有临床意义。
