Non-linear Optical Collagen Cross-linking (NLO CXL) for Treatment of Keratoconus

项目摘要/摘要 胶原蛋白交联(CXL)利用单光子，380 nm，UVA光激活核黄素(RF)是 已知会导致胶原蛋白硬度增加两倍，角膜变平的屈光度为1-3。而这些 结果表明，治疗圆锥角膜和矫正圆锥角膜都有显著的疗效。 轻微的屈光不正，这种手术有几个主要的缺点，包括术后疼痛和 视力恢复延迟，细菌感染和角膜创伤的风险增加。在我们最近 完成了NEI资助的研究项目，我们开发了一种双光子、非线性光学(NLO)CXL方法 使用红外飞秒激光(FS)光，将RF光激活限制为双光子焦点体积 物镜。该装置使用放大的FS激光产生760 nm、0.3μJ的脉冲，脉冲频率小于46.1. MW总功率(低于ANSI激光眼睛曝光的热极限)，并可交叉连接直径4 mm的区域 在不到4分钟的时间内，基质硬度显著增加1.6倍，并诱导角膜屈光度1~2度 在活的兔子身上压平。作为这个项目的一部分，我们还开发了一种基于FS激光的新型角膜 上皮微机械加工方法通过上皮创造微通道(Microch)来极大地 增强跨上皮(TE)射频扩散。这个项目的目标是检验这样一种假设，即术后疼痛 视力恢复延迟是由于UVA CXL和TE-NLO造成的细胞过度损伤 CXL显示出更小的细胞损伤和更快的视觉恢复。这一假设将通过 以下具体目标：目标1.通过确定以下影响来优化微通道射频基质穿透 脉冲能量、密度和深度对射频间质浓度和上皮/间质完整性的影响 其他赋形剂TE方法使用体外兔眼。目的2.确定TE射频穿透的效果 比较MicroCH和最佳赋形剂RF方法对角膜上皮和基质完整性的影响 SA1利用体外兔眼模型测量上皮/基质细胞死亡和诱导的炎症基因 在有无UVA或NLO CXL的情况下，在3小时和24小时表达。目的3.测定微循环的影响 射频给药和UVA与NLO CXL对活体兔角膜上皮完整性、角膜的影响 敏感性、间质炎症、伤口愈合和视力恢复。

Project Summary/Abstract Collagen crosslinking (CXL) using single photon, 380 nm, UVA light to photoactivate riboflavin (Rf) is known to induce a two-fold increase in collagen stiffness and 1-3 diopters of corneal flattening. While these effects have been shown to have significant therapeutic benefits for both treating Keratoconus and correcting minor refractive errors, there are several major drawbacks to this procedure, including post-operative pain and and delayed visual recovery with increased risk of bacterial infection and corneal scaring. In our recently completed, NEI funded research project we developed a two-photon, nonlinear optical (NLO) CXL approach using infrared femtosecond laser (FS) light that limits Rf photoactivation to the two-photon focal volume of an objective lens. This device uses an amplified FS laser to generate 760 nm, 0.3 μJ pulses at less than 46.1 mW total power (under the ANSI thermal limit for laser eye exposure), and can crosslink a 4 mm diameter area in less than 4 min, significantly enhance stromal stiffness 1.6 fold, and induce 1 to 2 diopters of corneal flattening in live rabbits. As part of this project, we have also developed a novel, FS laser based, corneal epithelial micromachining approach to create Microchannels (MicroCh) through the epithelium to greatly enhance transepithelial (TE) Rf diffusion. Goal of this project is to test the hypothesis that post-operative pain and delayed visual recovery is due to the excessive cellular damage caused by UVA CXL and that TE-NLO CXL shows significantly less cell damage with faster visual recovery. This hypothesis will be tested through the following specific aims: Aim 1. Optimize MicroCh TE Rf stromal penetration by determining the effects of pulse energy, density and depth on Rf stromal concentration and epithelial/stromal integrity as compared to other excipient TE approaches using ex vivo rabbit eyes. Aim 2. Determine the effects of TE Rf penetration on corneal epithelial and stromal integrity comparing MicroCh to optimal excipient Rf approaches identified in SA1 using an ex vivo rabbit eye model to measure epithelial/stromal cell death and induced inflammatory gene expression at 3 hours and 24 hours with and without UVA or NLO CXL. Aim 3. Determine the effects MicroCh Rf delivery and UVA versus NLO CXL in live rabbits by assessing the effects on epithelial integrity, corneal sensitivity, stromal inflammation, wound healing and visual recovery.