Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy
Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy
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DOI：
10.1097/ico.0000000000000743
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发表时间：
2016-04-01
期刊：
影响因子：
2.8
通讯作者：
Paik, David C.
中科院分区：
文献类型：
--
作者：
Kim, Su-Young;Babar, Natasha;Paik, David C.
目的：利用甲醛释放剂羟甲基甘氨酸钠（SMG）和核黄素- uva光化学角膜交联（CXL）建立内皮细胞毒性与组织固定（毒性/固定）之间的关系，用于角膜的治疗性组织交联。方法：采用11个新鲜尸体兔头进行离体角膜交联模拟。上皮清创后，将组织暴露于pH 8.5下1/4 max （9.8 mM）或1/3 max (13 mM) SMG或核黄素- uva （CXL）中30分钟。对侧角膜作为配对对照。暴露后，通过热变性温度（Tm）确定交联效果，并使用钙黄蛋白AM和乙啶同型二聚体染色（The Live/Dead Kit）评估内皮损伤。使用共聚焦激光扫描荧光显微镜使用标准化算法生成活/死细胞计数。结果：CXL、1/3 SMG、1/4 SMG术后Delta Tm分别为2.2 +/- 0.9℃、1.3 +/- 0.5℃、1.1 +/- 0.5℃。内皮细胞损伤表示为每个高倍视场计数的死细胞/活细胞+死细胞的百分比。值分别为3 +/- 1.7%（对照组）和8.9 +/- 11.1%（对照组）（P = 0.390）；1 + / - 0.2%(控制)和19.5 + / - 32.2%(1/3马克斯SMG) (P = 0.426);和2.7 + / - 2.4%(控制)和2.8(1/4马克斯SMG) + / - 2.2% (P = 0.938)。然后将内皮毒性值与Tm的变化进行索引，以产生毒性/固定指数。数值如下：CXL为2.7,1/3 max为14,1/4 max为0.1。结论：四分之一max (1/4 max = 9.8 mM) SMG有效交联组织，对内皮细胞无毒。因此，SMG是一种潜在的化合物，可以实现这两种预期的效果。
Purpose:To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea.Methods:Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm.Results:The Delta Tm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 +/- 0.9 degrees C, 1.3 +/- 0.5 degrees C, and 1.1 +/- 0.5 degrees C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 +/- 1.7% (control) and 8.9 +/- 11.1% (CXL) (P = 0.390); 1 +/- 0.2% (control) and 19.5 +/- 32.2% (1/3 max SMG) (P = 0.426); and 2.7 +/- 2.4% (control) and 2.8 +/- 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max.Conclusions:Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.