Bottós Katia Mantovani, Schor Paulo, Dreyfuss Juliana L, Nader Helena Bonciani, Chamon Wallace
Department of Ophthalmology, Universidade Federal de São Paulo, Brazil.
Arq Bras Oftalmol. 2011 Sep-Oct;74(5):348-51. doi: 10.1590/s0004-27492011000500008.
To determine if the corneal epithelium prevents the collagen cross-linking effect. Using immunofluorescence microscopy after CXL, we indirectly analyzed the role of the epithelium as ultraviolet-A (UVA) shield as well as a barrier to riboflavin penetration.
Fifteen freshly enucleated porcine eyes were divided into 3 groups. The corneal epithelium was kept intact in all groups. Five eyes served as control (Group 1). On group 2, eyes received tetracaine anesthetic drops and topical 0.1% riboflavin solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500). On Group 3, riboflavin was injected into the anterior chamber to allow penetration of the drug through the endothelium. Groups 2 and 3 were exposed to UVA (365 nm, 3 mW/cm(2)) for 30 minutes. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen type I and DAPI (4,6-diamidino-2-fenilindole dihydrocloride) and analyzed with fluorescence microscopy.
Corneas treated with UVA irradiation and intracameral injection of riboflavin (Group 3) showed greater pattern of collagen organization compared to groups 1 (Control) and 2 (riboflavin and tetracaine eye drops). A yellow stromal staining, which represents the riboflavin diffusion into the stroma, was only observed in eyes injected with riboflavin into the anterior chamber.
Using immunofluorescence microscopy in porcine corneas, we demonstrated that the corneal epithelium reduces the effectiveness of CXL by preventing the penetration of the drug and not by limiting the UVA transmittance. An inadequate intrastromal concentration of riboflavin may impair CXL effect.
确定角膜上皮是否会阻止胶原交联效应。通过在角膜交联（CXL）后使用免疫荧光显微镜，我们间接分析了上皮作为紫外线A（UVA）屏障以及核黄素渗透屏障的作用。
将15只新鲜摘除的猪眼分为3组。所有组的角膜上皮均保持完整。5只眼作为对照组（第1组）。在第2组中，给眼滴用丁卡因麻醉滴眼液并局部应用0.1%核黄素溶液（10mg核黄素-5-磷酸溶于10mL 20%右旋糖酐-T-500中）。在第3组中，将核黄素注入前房以使药物通过内皮渗透。第2组和第3组暴露于UVA（365nm，3mW/cm²）30分钟。将角膜的超薄切片（8μm）用抗I型胶原和DAPI（4,6-二脒基-2-苯基吲哚二盐酸盐）染色，并通过荧光显微镜进行分析。
与第1组（对照组）和第2组（核黄素和丁卡因滴眼液）相比，经UVA照射和前房内注射核黄素处理的角膜（第3组）显示出更大的胶原组织模式。仅在前房内注射核黄素的眼中观察到代表核黄素扩散到基质中的黄色基质染色。
通过在猪角膜中使用免疫荧光显微镜，我们证明角膜上皮通过阻止药物渗透而非限制UVA透射率来降低CXL的有效性。基质内核黄素浓度不足可能会损害CXL效果。
