Gallego-Muñoz Patricia, Ibares-Frías Lucía, Lorenzo Elvira, Marcos Susana, Peréz-Merino Pablo, Bekesi Nandor, Kochevar Irene E, Martínez-García M Carmen
Departamento de Biología Celular, Histología y Farmacología, GIR de Técnicas Ópticas para el Diagnóstico, Universidad de Valladolid, Valladolid, Spain.
Instituto de Óptica, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3471-3480. doi: 10.1167/iovs.16-21365.
To evaluate corneal wound healing after treatment with a new collagen crosslinking protocol using rose bengal dye and green light (RGX).
One cornea of 20 New Zealand rabbits was de-epithelialized (DE) in an 8-mm diameter circle and, in another group (n = 25), the DE corneas were then stained with 0.1% rose bengal for 2 minutes and exposed to green light (532 nm) for 7 minutes (RGX). The contralateral eyes without treatment acted as controls. The animals were clinically followed including fluorescein staining and pachymetry. Healing events were analyzed after euthanasia at 2, 30, and 60 days. Cell death (TUNEL assay), cell proliferation (5-bromo-2'-deoxyuridine incorporation), and cell differentiation to myofibroblasts (α-SMA labeling) were carried out. In addition, loss of keratocytes and subsequent repopulation of the corneal stroma were quantified on hematoxylin-eosin-stained sections.
Wound closure was slower after RGX (4.4 days) then after DE (3.3 days). Cell death was restricted to the anterior central stroma, and the cellular decrease did not differ significantly between RGX and DE corneas. Cell proliferation in the epithelium and stroma appeared at 2 days. In both DE and RGX corneas, recovery of the epithelium was complete at day 30, although cell repopulation of the stroma was not complete at 60 days.
The healing response in corneas after RGX is very similar to that observed after DE alone, suggesting that, along with its short treatment time and limited effect on keratocytes, RGX displays good potential for clinical cornea stiffening.
评估使用孟加拉玫瑰红染料和绿光（RGX）的新型胶原交联方案治疗后角膜伤口的愈合情况。
20只新西兰兔的一只眼睛的角膜在直径8毫米的圆圈内进行上皮去除（DE），在另一组（n = 25）中，然后将DE角膜用0.1%的孟加拉玫瑰红染色2分钟，并暴露于绿光（532纳米）下7分钟（RGX）。未治疗的对侧眼睛作为对照。对动物进行临床随访，包括荧光素染色和角膜厚度测量。在2天、30天和60天安乐死后分析愈合情况。进行细胞死亡（TUNEL检测）、细胞增殖（5-溴-2'-脱氧尿苷掺入）以及细胞向肌成纤维细胞的分化（α-SMA标记）。此外，在苏木精-伊红染色切片上对角膜细胞的损失和随后角膜基质的重新填充进行定量。
RGX治疗后伤口闭合（4.4天）比DE治疗后（3.3天）更慢。细胞死亡局限于中央前部基质，RGX和DE角膜之间细胞减少没有显著差异。上皮和基质中的细胞增殖在2天时出现。在DE和RGX角膜中，上皮在30天时恢复完全，尽管基质中的细胞重新填充在60天时未完成。
RGX治疗后角膜的愈合反应与单独DE治疗后观察到的非常相似，这表明，连同其短的治疗时间和对角膜细胞的有限影响，RGX在临床角膜强化方面具有良好的潜力。
