Denton Michael L, Foltz Michael S, Estlack Larry E, Stolarski David J, Noojin Gary D, Thomas Robert J, Eikum Debbie, Rockwell Benjamin A
Northrop Grumman, Life Sciences Research and Applications, San Antonio, Texas 78228-1330, USA.
Invest Ophthalmol Vis Sci. 2006 Jul;47(7):3065-73. doi: 10.1167/iovs.05-1066.
Until reliable nonanimal systems of analysis are available, animal models will be necessary for ocular laser hazard analysis and for evaluating clinical applications. The purpose of this work was to demonstrate the utility of an in vitro system for laser bioeffects by identifying photothermal and photochemical cytotoxicity thresholds for continuous-wave (cw) and mode-locked (ml) laser exposures.
Exogenous melanosomes were added to hTERT-RPE1 cells in exposure wells 1 day before laser exposure. Thermal or photochemical laser exposures were delivered to artificially pigmented retinal pigment epithelial (RPE) cultures, with subsequent assay for viability 1 hour after exposure. Beam delivery for the 1-hour photochemical exposures was via a modified culture incubator. The cytoprotective effect of pretreatment with two antioxidants was investigated.
Phagocytosis of melanosomes by the RPE cells was efficient, yielding cultures of uniform pigmentation. The damage threshold for the thermal exposure was consistent with published in vivo results. Thresholds for both blue exposures (cw and ml) were identical. Overnight treatment of cells with ascorbic acid (AA) minimized cell death from both cw and ml blue laser exposure, whereas similar treatment with N-acetyl-L-cysteine (NAC) was less effective.
The in vitro system described is suitable for measuring meaningful thermal and photochemical laser damage thresholds. The system is also useful in comparative laser bioeffects studies, such as comparisons between cw and ml laser exposures, cells with various degrees of pigmentation, and studies determining the efficacy and mechanisms of treatments altering the response of cells to lasers.
在获得可靠的非动物分析系统之前，动物模型对于眼部激光危害分析和评估临床应用将是必要的。这项工作的目的是通过确定连续波（cw）和锁模（ml）激光照射的光热和光化学细胞毒性阈值，来证明体外系统在激光生物效应方面的实用性。
在激光照射前1天，将外源性黑素体添加到暴露孔中的hTERT-RPE1细胞中。将热或光化学激光照射施加到人工色素沉着的视网膜色素上皮（RPE）培养物上，照射后1小时随后进行活力测定。1小时光化学照射的光束传输通过改良的培养箱进行。研究了两种抗氧化剂预处理的细胞保护作用。
RPE细胞对黑素体的吞噬作用有效，产生色素沉着均匀的培养物。热暴露的损伤阈值与已发表的体内结果一致。两种蓝光照射（cw和ml）的阈值相同。用抗坏血酸（AA）过夜处理细胞可使cw和ml蓝光激光照射引起的细胞死亡最小化，而用N-乙酰-L-半胱氨酸（NAC）进行类似处理效果较差。
所描述的体外系统适用于测量有意义的热和光化学激光损伤阈值。该系统在比较激光生物效应研究中也很有用，例如cw和ml激光照射之间的比较、不同色素沉着程度的细胞之间的比较，以及确定改变细胞对激光反应的治疗方法的疗效和机制的研究。
