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

项目概要/摘要 使用单光子、380 nm、UVA光来光活化核黄素（Rf）的胶原交联（CXL）是 已知其诱导胶原硬度增加两倍和角膜变平1-3屈光度。虽然这些 已经显示出对于治疗圆锥角膜和矫正圆锥角膜 虽然屈光不正较小，但该手术存在几个主要缺点，包括术后疼痛和 延迟视力恢复，增加细菌感染和角膜瘢痕形成的风险。在我们最近 完成，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，其将Rf光活化限制在 物镜透镜。该装置使用放大的FS激光器产生760 nm，0.3 μJ的脉冲，小于46.1 mW的总功率（低于ANSI对激光人眼照射的热限值），并且可以交联4 mm直径的区域 在小于4 min内，显著增强基质硬度1.6倍，并诱导1 - 2屈光度的角膜 在活兔子中变平。作为该项目的一部分，我们还开发了一种新型的基于FS激光的角膜 上皮微加工方法，以创建通过上皮的微通道（MicroCh）， 增强跨上皮（TE）Rf扩散。这个项目的目标是测试手术后疼痛的假设， 延迟的视力恢复是由于UVA CXL引起的过度细胞损伤， CXL显示出明显更少的细胞损伤，视力恢复更快。这一假设将通过 具体目标如下：目标1。通过确定以下因素的影响优化MicroCh TE Rf基质渗透 脉冲能量、密度和深度对Rf基质浓度和上皮/基质完整性的影响， 使用离体兔眼的其他赋形剂TE方法。目标2.确定TE Rf穿透的影响 对角膜上皮和基质完整性的影响，比较MicroCh与 SA 1使用离体兔眼模型测量上皮/基质细胞死亡和诱导的炎症基因 在有和没有UVA或NLO CXL的情况下，在3小时和24小时的表达。目标3。确定MicroCh的影响 通过评估对上皮完整性、角膜完整性和角膜完整性的影响，在活兔中比较Rf递送和UVA与NLO CXL 敏感性、基质炎症、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.