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

描述(由申请人提供)：这项建议的主要目标是测试以下假设：利用飞秒激光(FS)的非线性光学(NLO)、双光子过程(TP)对核黄素进行光活化可以诱导空间分辨(X-Y-Z)胶原交联(CXL)和完整的活体兔眼的机械硬化。最近的研究证实，370 nm光(UVA)对核黄素的光激活会导致自由基诱导的胶原CXL的产生和角膜硬化，这对圆锥角膜患者显示出显著的治疗效果。虽然UVA和核黄素本身无毒，但UVA准直光路中自由基的产生可能会导致细胞损伤，影响角膜内皮和晶体蛋白晶状体，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，UVA CXL的治疗性给药已被滴定，以限制自由基的产生到角膜前部，从而降低了CXL的疗效，并将治疗范围限制在受影响的角膜厚度超过400微米的患者。我们建议用近红外FS激光取代准直UVA光，在聚焦透镜确定的空间分辨焦体积内诱导NLO CXL，将极大地提高CXL的安全性和有效性。与传统UVA CXL相比，NLO CXL的主要好处包括：1)扩大角膜CXL的应用范围，用于治疗较薄的扩张性角膜(&lt；400 um)，包括LASIK术后扩张的患者以及目前建议用于传统UVA CXL的其他角膜疾病，包括大疱性角膜病变、角膜溃疡和微生物感染；2)除前基质外，还提供额外的治疗区域，包括中、后和全厚度角膜基质；以及3)扩大使用由低紫外线(UVB和UVC)和可见光(400~780 nm)激活的替代光敏剂，这将使用单光子方法对视网膜产生光毒性。为了验证这一假设，我们提出了以下具体目标：1)适应当前的FS投放系统用于活体兔角膜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NLO CXL与UVA CXL相比对角膜CAF、3D角膜弹性模量和细胞损伤的短期影响(1天和3天)，4)确定NLO CXL与UVA CXL相比对角膜CAF、3D角膜弹性模数和细胞修复的长期影响(1月和6个月)。

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.