红外激光致角膜损伤的修复机制研究

Corneal wound healing mechanism after infrared laser irradiation

目的探讨3.74μm红外激光致角膜损伤的修复机制。方法取27只6~8周龄C57BL/6J小鼠作为研究对象,将小鼠随机分为正常组(3只)和实验组(24只)。正常组不做任何处理,实验组小鼠用3.74μm红外激光照射双眼,光斑直径2 mm,曝光时间0.8 s,辐照量23.2 J·cm^(-2)。激光照射损伤后3 h、6 h、12 h、1 d、3 d、7 d、14 d、21 d各取3只实验组小鼠,连同正常组小鼠角膜组织进行病理切片,采用免疫组织化学染色检测中性粒细胞弹性蛋白酶、CD68、CD163以及血栓调节蛋白阳性细胞情况,鉴定新生血管发生情况。结果正常组小鼠角膜基质中均未见中性粒细胞弹性蛋白酶、CD68、CD163以及血栓调节蛋白阳性细胞。实验组小鼠中性粒细胞弹性蛋白酶阳性细胞在激光照射后3 h出现于角膜组织损伤边缘,12 h迁移至损伤区,1 d时数量最多,之后逐渐减少,21 d仍有少量阳性细胞;CD68阳性细胞在激光照射后12 h出现于角膜组织损伤边缘,1 d时出现在损伤区,21 d时仍有少量阳性细胞;CD163阳性细胞在激光照射后7 d出现于角膜组织损伤边缘,14 d时出现在损伤区,21 d仍有少量阳性细胞;血栓调节蛋白阳性细胞在激光照射后14~21 d出现于角膜基质损伤区。结论3.74μm红外激光可致角膜全层损伤,大量炎症细胞自损伤边界或角膜缘向损伤区迁移并参与修复过程。早期以中性粒细胞和M1型巨噬细胞浸润为主,后期以M2型巨噬细胞参与为主,并伴有新生血管生成。

Objective To explore the corneal wound healing mechanism after 3.74μm infrared laser irradiation.Methods Twenty-seven C57BL/6J mice(six to eight weeks old)were randomly divided into a normal group(3 mice)and an experimental group(24 mice).Mice in the normal group were not subjected to any treatment.The corneas of mice in the experimental group were damaged by infrared laser at a wavelength of 3.74μm.The spot diameter was 2 mm,the exposure duration 0.8 s,and the radiant exposure 23.2 J·cm^(-2).Pathological sectioning of corneas was performed at 3 h,6 h,12 h,1 d,3 d,7 d,14 d,and 21 d after laser irradiation in the experimental group,with 3 mice at each time point.It was the same for mice in the normal group.Immunohistochemical staining was conducted to examine neutrophil elastase,CD68,CD163 and thrombomodulin-positive cells and identify neovascularization.Results Neutrophil elastase,CD68,CD163,and thrombomodulin-positive cells were not detected in the corneal stroma of mice in the normal group.Neutrophil elastase-positive cells were detected in the damaged corneal periphery of mice in the experimental group at 3 h,migrated to the damaged area at 12 h,peaked at 1 d,and then decreased gradually with time,but still existed slightly at 21 d after laser irradiation.CD68-positive cells were detected in the damaged corneal periphery of mice in the experimental group at 12 h and in the damaged area at 1 d through 21 d after laser irradiation.CD163-positive cells were found in the damaged corneal periphery of mice in the experimental group at 7 d and in the damaged area at 14 d and 21 d after laser irradiation.Thrombomodulin-positive cells were found in the damaged area of the corneal stroma at 14 d and 21 d after laser irradiation.Conclusion During the wound healing process after 3.74μm infrared laser-induced full-thickness corneal injury in mice,a large number of inflammatory cells migrate from the damaged corneal periphery or limbus to the damaged area.Neutrophils and M1 macrophages infiltrate in the early stage,while M2 macrophages are involved in the later stage,accompanied by neovascularization.