摘　要：为观察3.74 μm远红外激光致角膜损伤的特点和损伤修复过程，利用该激光在光斑直径为2 mm、照射时间为0.8 s、辐照量为23.2 J/cm2的条件下照射C57BL/6J小鼠角膜，采用大体观察、裂隙灯显微镜、光学相干断层扫描（OCT）以及组织病理方法，在角膜损伤后3 h、6 h、12 h、1 d、3 d、7 d、14 d和21 d进行观察。大体观察角膜损伤即刻可见灰白色损伤斑，表面凹凸不平，角膜混浊随时间逐渐加重，1 d达到顶峰，3～7 d混浊减轻，14～21 d再次加重。裂隙灯下角膜损伤累及全层，角膜厚度随时间先增大后逐渐恢复。OCT观察角膜损伤后明显外凸，反射光带全层性增强，3 h角膜显著增厚，12 h达到最厚，后逐渐恢复至正常。经组织切片观察：上皮层损伤后3 h核固缩深染，6～12 h核染色变淡消失，1 d 时1～2层新生上皮完全覆盖损伤区，3～7 d上皮细胞增至3～4 层，14～21 d恢复正常；基质层损伤后3～6 h核染色质大量脱失，12 h出现浸润细胞，后浸润细胞增多，由基质深层向浅层迁移，14～21 d浸润细胞减少，纤维排列仍不规则；内皮层损伤后3 h细胞脱落，1 d出现少量新生细胞，其后逐渐增多并趋向恢复。结果表明，3.74 μm激光在23.2 J/cm2照射剂量下可致角膜全层损伤，角膜损伤反应随时间先加重后逐渐恢复，21 d时上皮和内皮层基本恢复正常，但基质层并未恢复透明。本研究为红外激光角膜损伤危害评价和损伤治疗研究提供了试验依据。
关键词：3.74 μm激光；照射剂量；小鼠；角膜；损伤修复
中图分类号：R779.12       　    　  文献标志码： A                DOI：10.3969/j.issn.1007-7146.2022.03.010

Academy of Military Medical Science, Academy of Military Science, Beijing 100039, China)
Abstract: In order to observe corneal wound healing process after injury induced by infrared laser at the wavelength of 3.74 μm. The cornea of C57BL/6J mice was damaged by infrared laser at the wavelength of 3.74 µm for exposure duration of 0.8 s and the beam diameter of 2 mm with corneal radiant exposures of 23.2 J/cm2. Gross observation through digital camera, slit lamp microscopy, optical coherence tomography (OCT) and histological evaluation of the cornea were preformed pre-exposure and 3 h, 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, 21 d after injury. It was shown that the corneal lesion was grey-white and could be seen immediately after laser exposure. The anterior surface became unsmooth. The corneal opacity increased gradually and peaked at 1 d and then subsided at 3～7 d, but worsened again at 14～21 d. By slit illumination it could be seen that the corneal lesion involved the whole layer. The corneal thickness increased gradually, then subsided with time, whereas the light scattering increased again at 21 d. OCT showed that the high-reflectance band and the corneal thickness from the damaged region increased, and the cornea protruded significantly. At 12 h, the corneal thickness reached maximum, then decreased gradually with time. Histological section showed that at 3 h, the epithelial nuclei were hyperchromatic with pyknosis, the chromatin of epithelial nuclei staining faded and then disappeared at 6～12 h. At 1 d, 1～2 layers of newborn epithelial cells covered the injury area completely. 3～4 layers of epithelial cells could be observed at 3～7 d, and the epithelium returned to normal from 14 d to 21 d. Most chromatin in the stroma cells lost after injury at 3 h, the chromatin of stromal nuclei disappeared and infiltrating cells appeared in the stroma layer at 6～12 h. At 1 d, infiltrating cells increased in the stroma, and the infiltrating cells of the stroma migrated from the deep layer to the shallow layer at 3～7 d. From 14 d to 21 d, the infiltrating cells in the stroma decreased, whereas the stromal fibers were still irregularly arranged. The endothelial layer disappeared completely at 3 h, and a few endothelial cells appeared in the endothelium at 1 d. At 3～7 d, the endothelial cells increased and flattened, and the endothelium returned to normal from 14 d to 21 d. Thus, the cornea lesion induced by 3.74 μm laser at the irradiation dose of 23.2 J/cm2 in mice model involved the whole cornea layer. After injury, the corneal response was enhanced at first, and then recovered gradually. The epithelium and endothelium recovered to normal, while the structure of the stroma layer did not return to normal during the observation period. Providing the experimental basis for hazard evaluation of corneal injuryed by laser and treatment.
Key words: 3.74 μm laser; radiation dose; mice; cornea; wound healing
（Acta Laser Biology Sinica, 2022, 31(3): 261-267）