Clemson-MUSC Bioengineering Program, Department of Bioengineering, Clemson University, Charleston, SC, USA; Department of Oral Health Science, Medical University of South Carolina, Charleston, SC, USA.
Clemson-MUSC Bioengineering Program, Department of Bioengineering, Clemson University, Charleston, SC, USA.
Exp Eye Res. 2021 Apr;205:108498. doi: 10.1016/j.exer.2021.108498. Epub 2021 Feb 15.
Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 μm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 μm/s in the SHAM group to 19.87 ± 10.10 μm/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 μm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 μm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.
扩散是营养物质和药物在无血管角膜基质中运输的重要机制。本研究的目的是研究紫外线 A（UVA）-核黄素诱导的角膜胶原交联（CXL）后基质转运特性的深度和方向依赖性变化及其与胶原结构变化的关系。在离体猪眼进行交联后，进行荧光恢复后光漂白（FRAP）以测量角膜深度为 100、200 和 300 μm 时鼻颞（NT）和前-后（AP）方向的荧光素扩散。还在这三个角膜深度进行二次谐波产生（SHG）成像以量化纤维排列。为了进一步确认，采用电导率法在 AP 方向量化角膜瓣中的离子通透性，并采用免疫组织化学（IHC）成像胶原结构。交联角膜与接受核黄素溶液但未接受 UVA 光（SHAM）照射的对照处理进行比较。FRAP 的结果表明，荧光素扩散系数从 SHAM 组的 23.39±11.60 μm/s 降低到 CXL 组的 19.87±10.10 μm/s。这种变化取决于深度和方向：与较深位置和 NT 方向相比，100 μm 深度（P=0.0005）和 AP 方向（P=0.001）的降低更为明显。电导率实验证实 AP 方向的溶质运输减少（P<0.0001）。FRAP 还检测到猪眼角膜的扩散各向异性：NT 方向的荧光素扩散系数高于 AP 方向的扩散系数。CXL 处理后这种各向异性增加。SHG 和 IHC 均显示 CXL 后胶原卷曲减少。与 SHAM 处理的角膜相比，CXL 处理的角膜前 200 μm 处的 SHG 图像分析显示相干性增加（P<0.01）。总之，CXL 导致基质溶质转运减少，这种减少集中在前部最前区域和 AP 方向。猪眼角膜中的溶质转运具有各向异性，CXL 后各向异性增加可能是由于胶原卷曲减少所致。
