,.
Invest Ophthalmol Vis Sci. 2020 Mar 9;61(3):28. doi: 10.1167/iovs.61.3.28.
Photoactivated cornea collagen cross-linking (CXL) increases corneal stiffness by initiating formation of covalent bonds between stromal proteins. Because CXL depends on diffusion to distribute the photoinitiator, a gradient of CXL efficiency with depth is expected that may affect the degree of stromal collagen organization. We used second harmonic generation (SHG) microscopy to investigate the differences in stromal collagen organization in rabbit eyes after corneal CXL in vivo as a function of depth and time after surgery.
Rabbit corneas were treated in vivo with either riboflavin/UV radiation (UVX) or Rose Bengal/green light (RGX) and evaluated 1 and 2 months after CXL. Collagen fibers were imaged with a custom-built SHG scanning microscope through the central cornea (350 µm depth, 225 × 225 µm en face images). The order coefficient (OC), a metric for collagen organization, and total SHG signal were computed for each depth and compared between treatments.
OC values of CXL-treated corneas were larger than untreated corneas by 27% and 20% after 1 month and 38% and 33% after 2 months for the RGX and UVX, respectively. RGX OC values were larger than UVX OC values by 3% and 5% at 1 and 2 months. The SHG signal was higher in CXL corneas than untreated corneas, both at 1 and 2 months after surgery, by 18% and 26% and 1% and 10% for RGX and UVX, respectively.
Increased OC corresponded with increased collagen fiber organization in CXL corneas. Changes in collagen organization parallel reported temporal changes in cornea stiffness after CXL and also, surprisingly, are detected deeper in the stroma than the regions stiffened by collagen cross-links.
光激活角膜胶原交联（CXL）通过在基质蛋白之间形成共价键来增加角膜硬度。由于 CXL 依赖于扩散来分布光引发剂，因此预计会出现随深度变化的 CXL 效率梯度，这可能会影响基质胶原组织的程度。我们使用二次谐波产生（SHG）显微镜研究了兔眼活体角膜 CXL 后基质胶原组织的差异，这些差异是作为手术深度和时间的函数。
兔角膜分别用核黄素/紫外线（UVX）或孟加拉玫瑰红/绿光（RGX）进行体内处理，并在 CXL 后 1 个月和 2 个月进行评估。使用定制的 SHG 扫描显微镜通过中央角膜（350 µm 深度，225×225 µm 面内图像）对胶原纤维进行成像。为每个深度计算了胶原组织的度量指标——有序系数（OC）和总 SHG 信号，并比较了两种处理方法之间的差异。
CXL 处理后的角膜的 OC 值在 1 个月后比未经处理的角膜分别增加了 27%和 20%，在 2 个月后增加了 38%和 33%，分别用于 RGX 和 UVX。在 1 个月和 2 个月时，RGX 的 OC 值比 UVX 的 OC 值分别大 3%和 5%。CXL 角膜的 SHG 信号比未经处理的角膜高，在手术后 1 个月和 2 个月时，分别增加了 18%和 26%，RGX 和 UVX 分别增加了 1%和 10%。
OC 的增加与 CXL 角膜中胶原纤维组织的增加相对应。胶原组织的变化与 CXL 后角膜硬度的时间变化平行，而且令人惊讶的是，这种变化比胶原交联增强的区域更深地出现在基质中。
