*Advanced Optical Imaging Group, School of Physics, University College Dublin, Dublin, Ireland; †Wellington Eye Clinic, Dublin, Ireland; and ‡IROC Science to Innovation AG, Zurich, Switzerland.
Cornea. 2014 Feb;33(2):125-30. doi: 10.1097/ICO.0000000000000015.
The aim of this study was to investigate morphological changes, using second-harmonic (SH) optical imaging, in the corneal stroma after normal- and high-intensity collagen crosslinking in postmortem enucleated porcine eyes at controlled intraocular pressures (IOPs).
Reflection-mode SH optical imaging of the stroma was realized after standard collagen cross-linking (CXL) and accelerated crosslinking (AXL) of porcine corneas, and the results were compared with the results for untreated controls. Ultraviolet-X lamps (365 nm) were used during riboflavin crosslinking with intensities of 3, 10, 30, and 100 mW/cm2 for constant 5.4-J/cm2 exposure doses. The IOP was varied using saline solution injected through the optic nerve and monitored using a Schiotz tonometer. SH optical imaging was realized with an in-house build multiphoton microscope using an ultrafast dispersion-compensated Ti-sapphire laser.
SH reflection images of CXL and AXL porcine corneas obtained at IOPs of 8, 11, 16, and 26 mm Hg showed a similar fibrillar structure of collagen lamellae. All crosslinked corneas showed an increased fibrillar contrast in comparison with untreated baseline images. At low IOPs, strong variations in the scattering were observed that reduced with an increase in the IOP, when fibrils tended to straighten out. At low and normal IOPs, no significant difference between CXL and AXL results could be observed. At very elevated IOPs, however, the impact of AXL was found to alter the fibrillar structure of the collagen becoming less apparent in SH images when compared with that of CXL.
We found a strong influence of the IOP on SH reflection imaging of postmortem porcine corneal stroma. CXL and AXL led to similar SH images indicative of a similar tensile strength. Only at very elevated IOPs (26 mm Hg) did the results for AXL deviate from those of CXL, suggesting an IOP-related threshold for reliable applications of AXL.
本研究旨在通过在受控眼内压（IOP）下对死后猪眼进行正常强度和高强度胶原交联后的角膜基质的二次谐波（SH）光学成像，研究形态学变化。
在猪角膜标准胶原交联（CXL）和加速交联（AXL）后，实现基质的反射模式 SH 光学成像，并将结果与未经处理的对照组进行比较。在 365nm 紫外线-X 灯下，使用 3、10、30 和 100mW/cm2 的强度进行核黄素交联，以实现恒定的 5.4J/cm2 暴露剂量。通过视神经注入生理盐水来改变 IOP，并使用 Schiotz 眼压计进行监测。SH 光学成像使用内部构建的多光子显微镜，使用超快色散补偿钛宝石激光来实现。
在 IOP 为 8、11、16 和 26mmHg 时获得的 CXL 和 AXL 猪角膜的 SH 反射图像显示出类似的胶原板层纤维状结构。与未经处理的基线图像相比，所有交联的角膜均显示出纤维状对比度增加。在低 IOP 下，观察到强烈的散射变化，随着 IOP 的增加而减少，当纤维趋于变直时。在低和正常 IOP 下，CXL 和 AXL 的结果之间没有观察到显著差异。然而，在非常高的 IOP 下，发现 AXL 的影响改变了胶原的纤维状结构，与 CXL 相比，SH 图像中的纤维状结构变得不那么明显。
我们发现 IOP 对死后猪角膜基质的 SH 反射成像有很强的影响。CXL 和 AXL 导致相似的 SH 图像，表明具有相似的拉伸强度。只有在非常高的 IOP（26mmHg）下，AXL 的结果才与 CXL 的结果不同，这表明 AXL 的应用存在与 IOP 相关的阈值。
