Effects Of Low-intensity He-Ne Laser Irradiation On Proliferation And Ultrastructural Changes Of Cultured Rabbit Corneal Endothelial Cells In Vitro

Objective(1) To investigate the effects of different low-intensity He-Ne laser irradiation on proliferation of cultured rabbit corneal endothelial cells(CECs) in vitro.(2) To observe the ultrastructural changes of rabbit corneal endothelium in vitro after lowintensity laser irradiation.Methods The Subculturing rabbit corneal endothelial cells(CECs) were isolated and suspended in DMEM supplemented with relatively low concentrations of newborn calf serum(NCS).The CECs were maintained at 37℃in a humidified atmosphere(95% air and 5% CO₂) at a density of 4×10³ cells/well in 96-well microplates. The interval wells and the gaps between them were filled with sterile black ink in order to minimize the light reflections. Those wells were treated by He-Ne laser(632.8 nm, spot diameter 0.64cm; HN-500C, Guangzhou, China) irradiation with the power of 2,5or10mW, for 2, 5, 10 or 15 minutes daily, respectively, for 7 days. The irradiation was performed on monolayer cells. After irradiation, the 96-well microplates were returned to the incubator for a further culture at 37℃, 5% CO₂. In all cases, the control non-irradiated cells were kept in the same conditions as the treated cells. After treatment the CECs were incubated till the ninth day, and the proliferation and activity were assessed by a MTT tests. At the indicated times, MTT was added to the cells and incubated for 4h. OD₄₉₀, the absorbance value at 490 nm, was read with a 96-well plate reader(BIO-RID 450, USA). The changes of cell cycle before and after laser irradiated were examined by flow cytometry. The morphological changes of CECs before and after laser irradiation were observed by biological microscope and histochemical staining. The ultrastructural changes of CECs after laser irradiation were surveyed by scanning electron microscope(SEM) and transmission electron microscope(TEM).Results The results of MTT assay indicated that the proliferative activity of cultured rabbit CECs in vitro was increased after laser irradiation and culturing, the CECs grew rapidly and increased quantitatively. The optimal laser irradiation energy intensity and time in this experiment was 5mW·cm^-2,600s. The results of flow cytometry showed that the proliferative index in laser irradiated group was much higher than that in the control group, but there was no difference compared with the basic fibroblast growth factor group(bFGF). Under SEM, there were more microvilli on the CECs surface of laser irradiation group than non-laser irradiation group. Under TEM, observation indicated that there were more nucleoli in CECs nucleus of the former group compared with the latter.Conclusion:(1) low-intensity laser can promote the proliferation of cultured rabbit CECs in vitro in certain extent.(2) The ultrastructural changes demonstrated that low-intensity laser had some proliferative effects towards the cultured rabbit corneal endothelial cells in vitro.