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

描述（由申请方提供）：本提案的主要目的是检验以下假设：使用飞秒激光（FS）光通过非线性光学（NLO）、双光子过程（TP）对核黄素进行光活化，诱导完整活体兔眼的空间分辨（X-Y-Z）胶原交联（CXL）和机械硬化。 最近的研究已经确定，370 nm光（UVA）对核黄素的光活化导致自由基诱导的胶原蛋白CXL和角膜硬化的产生，这对患有圆锥角膜的患者显示出显著的治疗益处。 虽然UVA和核黄素本身是无毒的，但在UVA准直光路内产生的自由基可导致影响角膜内皮和晶状体蛋白透镜的细胞损伤，从而导致大泡性角膜病变和潜在的白内障。 为了避免这种并发症，已经滴定UVA CXL的治疗性施用以限制前角膜的自由基产生，从而降低CXL的功效并将治疗范围限制在受影响角膜厚度大于400 μ π ι的患者。 我们建议用近红外FS激光替代准直UVA光，以在由聚焦透镜确定的空间分辨焦点体积内诱导NLO CXL，这将大大提高CXL的安全性和有效性。 与传统UVA CXL相比，NLO CXL的主要益处包括：1）角膜CXL的应用扩展到治疗较薄的扩张角膜（<400 μ m），包括LASIK术后扩张患者和目前建议用于传统UVA CXL的其他角膜疾病，包括大泡性角膜病变、角膜溃疡和微生物感染; 2）前基质以外的额外治疗区域，包括中、后和全层角膜基质;和3）扩大使用由低紫外线激活的替代光敏剂使用单光子方法，可以检测对视网膜具有光毒性的紫外线（UVB和UVC）和可见光（400至780 nm）。 为了验证这一假设，我们提出以下具体目标：1）使当前FS输送系统适用于活体兔角膜NLO CXL，2）确定不同TP焦点体积、FS激光功率和扫描速度对NLO CXL角膜硬化的影响，3）确定短期影响与UVA CXL相比，NLO CXL（1天和3天）对角膜CAF、3D角膜弹性模量和细胞损伤的影响，4）确定长期影响与UVA CXL相比，NLO CXL（1个月和6个月）对角膜CAF、3D角膜弹性模量和细胞修复的影响。

DESCRIPTION (provided by applicant): The major objective of this proposal is to test the hypothesis that photoactivation of riboflavin by non- linear optical (NLO), two photon processes (TP) using femtosecond laser (FS) light induces spatially resolved (X-Y-Z) collagen cross-linking (CXL) and mechanical stiffening of intact, live rabbit eyes. Recent studies have established that photoactivation of riboflavin by 370 nm light (UVA) leads to the generation of free radical-induced collagen CXL and corneal stiffening that shows significant therapeutic benefits for patients suffering from Keratoconus. While UVA and riboflavin are themselves non-toxic, the generation of free radicals within the UVA collimated light path can lead to cellular damage affecting the corneal endothelium and crystallin lens, resulting in bullous keratopathy and potentially cataract. To avoid this complication, therapeutic administration of UVA CXL has been titrated to limit free radical generation to the anterior cornea, thus reducing the efficacy o CXL and limiting the therapeutic range to patients with affected corneas thicker than 400 um. We propose that replacement of collimated UVA light with near-infrared FS laser light to induce NLO CXL within a spatially resolved focal volume determined by the focusing lenses would greatly improve the safety and efficacy of CXL. Major benefits of NLO CXL compared to conventional UVA CXL would included: 1) expanded application of corneal CXL to the treatment of thinner ectatic corneas (<400 um) including post LASIK ectasia patients and other corneal disorders currently proposed for conventional UVA CXL including, bullous keratopathy, corneal ulceration and microbial infection; 2) additional treatment areas beyond that of the anterior stroma, including middle, posterior and full thickness corneal stroma; and 3) expanded use of alternative photosensitizers activated by low ultraviolet (UVB and UVC) and visible light (400 to 780 nm) that would be phototoxic to the retina using a single photon approach. To test this hypothesis we propose the following Specific Aims: 1) Adapt the current FS delivery system for live rabbit corneal NLO CXL, 2) Determine the effect of varying TP focal volume, FS laser power and scanning speed on NLO CXL corneal stiffening, 3) Determine the short-term affect (1 and 3 days) of NLO CXL on corneal CAF, 3D corneal elastic modulus, and cell damage compared to UVA CXL, 4) Determine the long-term affects (1 and 6 months) of NLO CXL on corneal CAF, 3D corneal elastic modulus, and cell repair compared to UVA CXL.