二氧化碳激光致小鼠角膜损伤模型的构建及损伤修复观察

Construction of a carbon dioxide laser-induced mouse corneal injury model and observation of corneal wound healing

目的 构建二氧化碳(CO_(2))激光致小鼠角膜损伤模型,观察照射后小鼠角膜损伤修复过程。方法 将10只C57BL/6J小鼠的20眼分为4组,用波长10.6μm、光斑直径2 mm、功率0.94 W的CO_(2)激光照射角膜中央,各组照射剂量分别为3.0 J·cm^(-2)、4.5 J·cm^(-2)、7.5 J·cm^(-2)和10.5 J·cm^(-2),对应照射时间分别为0.10 s、0.15 s、0.25 s和0.35 s。于照射后1 d通过裂隙灯显微镜、OCT和组织病理学评估角膜损伤程度,以确定构建角膜损伤模型的最佳照射剂量。随后,构建小鼠角膜损伤模型,采用相同的方法分别于照射前及照射后0 h至6个月进行角膜损伤修复观察。结果 照射剂量为3.0 J·cm^(-2)时,角膜未观察到明显损伤。照射剂量为4.5 J·cm^(-2)时,角膜中央可见灰白色损伤区,受损区角膜上皮和内皮缺失。照射剂量为7.5 J·cm^(-2)或10.5 J·cm^(-2)时,角膜损伤区为瓷白色,角膜厚度增加,角膜边缘与虹膜粘连。故选用4.5 J·cm^(-2)照射剂量的CO_(2)激光构建角膜损伤模型。该照射剂量的激光损伤后角膜迅速肿胀增厚,损伤后1 d厚度达到峰值,之后逐渐恢复,14 d时基本恢复正常。损伤早期(0 h~3 d)角膜发生了受损上皮和内皮脱落、新生上皮和内皮迁移、炎症细胞浸润与消退等过程;损伤后期(7 d~6个月)角膜逐渐恢复并趋于正常,但部分小鼠角膜存在基质增生现象。结论 照射剂量为4.5 J·cm^(-2)的CO_(2)激光可用于构建小鼠角膜损伤模型。角膜损伤后的急性反应期主要发生在损伤后3 d内。损伤后期角膜的生理结构趋于正常,但透明度未能完全恢复。

Objective To construct a carbon dioxide(CO_(2))laser-induced corneal injury model in mice and observe the process of corneal wound healing.Methods Twenty eyes of ten C57BL/6J mice were divided into 4 groups.The central corneas in each group were irradiated by CO_(2)laser with a wavelength of 10.6μm,spot diameter of 2 mm and power of 0.94 W.The exposure doses were 3.0 J·cm^(-2),4.5 J·cm^(-2),7.5 J·cm^(-2)and 10.5 J·cm^(-2),with corresponding exposure durations of 0.10 s,0.15 s,0.25 s and 0.35 s.Corneal injury severity was assessed using a slit lamp microscope,optical coherence tomography and histopathological evaluation at 1 day after the exposure to determine the proper exposure dose for constructing a corneal injury model.Subsequently,the corneal injury model was constructed and the same methods were used to monitor wound healing before,and 0 hours to 6 months after the exposure.Results No obvious corneal injury was observed at an exposure dose of 3.0 J·cm^(-2).At an exposure dose of 4.5 J·cm^(-2),an off-white injury area appeared on the central cornea with loss of epithelium and endothelium.At an exposure dose of 7.5 J·cm^(-2)or 10.5 J·cm^(-2),the injury area became porcelain white,the cornea was thickened,and the iris was seen adhering to the margin of the cornea.Therefore,4.5 J·cm^(-2)CO_(2)laser was selected to construct a corneal injury model.At this exposure dose,the cornea swelled and thickened rapidly after injury,reached the maximum thickness 1 day after the exposure,and then gradually recovered,returning to normal by 14 days after the exposure.In the early stage(0 hours to 3 days after the exposure),the cornea showed shedding of injured epithelium and endothelium,migration of new epithelium and endothelium,and infiltration and regression of inflammatory cells.At the late stage(7 days to 6 months after the exposure),the cornea gradually returned to a normal physiological state,but some of the injured cornea exhibited stromal hyperplasia.Conclusion A CO_(2)laser with an exposure dose of 4.5 J·cm^(-2)can be used to construct a corneal injury model in mice.The acute phase of corneal injury primarily occurs within 3 days after the exposure.The cornea tends to restore its original physiological structure,but the corneal transparency cannot return to a normal state.