Wertheimer Christian M, Mendes Bryan, Pei Qing, Brandt Katharina, Kochevar Irene E
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.
Harvard Medical School, Boston, MA, USA.
Transl Vis Sci Technol. 2020 Jul 14;9(8):24. doi: 10.1167/tvst.9.8.24. eCollection 2020 Jul.
Oxygen-independent cornea crosslinking (CXL) using rose bengal (RB) and green light may have unique clinical applications. These studies were designed to gain insight into the arginine (arg)-enhanced anaerobic crosslinking process, to maximize crosslinking efficiency, and to test a clinically feasible method for oxygen-free CXL.
Rabbit corneas were treated ex vivo using 1 mM RB and 532 nm light. RB photodecomposition, monitored by absorption spectrophotometry, was used to optimize arg concentration and to develop an irradiation and re-dying protocol. The minimal effective green light fluence was identified by linear tensile strength measurements. RB penetration into the stroma was determined by fluorescence microscopy. To favor the anaerobic pathway, a contact lens was used to minimize stromal oxygen level during irradiation. Stromal cell toxicity was evaluated by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay.
RB photodecomposition reached 75% of its maximal effect at 200 mM arg and the optimal fluence increment was 32.7 J/cm. The minimal effective fluence for cornea stiffening was 65.4 J/cm. Placement of a contact lens promoted oxygen-independent cornea stiffening, similar to that obtained on isolated, oxygen-deprived cornea. RB penetration into the stroma with arg present was limited to ∼120 µm, about 25% deeper than without arg. Stromal cell toxicity was limited to the depth of RB and arg penetration.
An oxygen-independent pathway in cornea for RB-CXL was characterized and optimized, including a possible clinical protocol for its use.
Oxygen-independent RB-CXL is an efficient and effective process that can be developed further for unique clinical applications.
使用孟加拉玫瑰红（RB）和绿光进行的非氧依赖性角膜交联（CXL）可能具有独特的临床应用。这些研究旨在深入了解精氨酸（arg）增强的厌氧交联过程，以最大化交联效率，并测试一种临床上可行的无氧CXL方法。
使用1 mM RB和532 nm光对兔角膜进行离体处理。通过吸收分光光度法监测RB光分解，以优化精氨酸浓度并制定照射和再染色方案。通过线性拉伸强度测量确定最小有效绿光通量。通过荧光显微镜确定RB在基质中的渗透情况。为了促进厌氧途径，在照射期间使用隐形眼镜将基质氧水平降至最低。通过末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）试验评估基质细胞毒性。
在200 mM精氨酸时，RB光分解达到其最大效应的75%，最佳通量增量为32.7 J/cm²。角膜硬化的最小有效通量为65.4 J/cm²。放置隐形眼镜可促进非氧依赖性角膜硬化，类似于在分离的缺氧角膜上获得的效果。存在精氨酸时RB在基质中的渗透限于约120 µm，比不存在精氨酸时深约25%。基质细胞毒性限于RB和精氨酸渗透的深度。
对角膜中RB-CXL的非氧依赖性途径进行了表征和优化，包括其使用的可能临床方案。
非氧依赖性RB-CXL是一种高效的过程，可进一步开发用于独特的临床应用。
