Perrot Sébastien, Dutertre-Catella Hélène, Martin Chantal, Warnet Jean-Michel, Rat Patrice
Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, Paris, France.
Cytometry A. 2003 Sep;55(1):7-14. doi: 10.1002/cyto.a.10067.
Three-dimensional culture or human corneal equivalents for safety testing are difficult to investigate with classic cytometric or biochemical methods. So a fluorometric method is proposed using resazurin probe.
Absorbance and fluorescence spectra of the oxidized and reduced forms of resazurin were performed to determine optimal measurement conditions. More than 100 enucleated porcine eyes were used for this experiment. Twelve corneas were immersed in resazurin solution and fluorescence was measured hourly from 1 to 10 h. Ninety benzalkonium chloride-treated corneas and control corneas were used as toxicity controls, and corneal viability was compared with in vivo rabbit eye irritation.
After analysis of spectra, the optimal measurement condition of resazurin metabolism proved to be a fluorescence measurement using 570 nm excitation wavelength and 590 nm emission wavelength. The reduction of resazurin was optimal after 6 h of incubation. Resazurin metabolism by isolated pig corneas varied proportionately with the benzalkonium chloride concentration employed, clearly showing significant differences (P < 0.001) in agreement with the in vivo data.
The resazurin metabolism test can be used to evaluate corneal viability and can thus be a potential alternative for toxicological assessments. This assay allows rapid assessment of non destructed samples, with simple equipment and at a reduced cost for continuous monitoring of corneal viability and possibly other three-dimensional cellular models.
用于安全性测试的三维培养物或人角膜等效物难以用经典的细胞计数法或生化方法进行研究。因此，提出了一种使用刃天青探针的荧光法。
对刃天青氧化型和还原型的吸光度和荧光光谱进行检测，以确定最佳测量条件。本实验使用了100多只摘除的猪眼。将12只角膜浸入刃天青溶液中，在1至10小时内每小时测量一次荧光。90只经苯扎氯铵处理的角膜和对照角膜用作毒性对照，并将角膜活力与兔眼体内刺激实验进行比较。
光谱分析后，刃天青代谢的最佳测量条件被证明是使用570nm激发波长和590nm发射波长进行荧光测量。孵育6小时后，刃天青的还原效果最佳。分离的猪角膜的刃天青代谢与所用苯扎氯铵浓度成比例变化，与体内数据一致，明显显示出显著差异（P < 0.001）。
刃天青代谢试验可用于评估角膜活力，因此可能成为毒理学评估的潜在替代方法。该试验允许使用简单设备对未破坏的样品进行快速评估，并以较低成本持续监测角膜活力以及可能的其他三维细胞模型。
