治疗性650 nm低功率半导体激光照射鸡视网膜的安全剂量研究

Safe dose of 650 nm low-power semiconductor laser irradiation in chicken retina

目的:观察650 nm半导体激光(功率2 mW)照射以视锥细胞为主的鸡视网膜后是否存在慢性光损伤,探讨该波段激光对视网膜的安全性。方法:采用随机数字表法将60只自然光线下饲养1个月的小鸡分为正常对照组、照射3-min组、照射6-min组和照射30-min组,每组15只。采用650 nm激光并按照分组不同各组小鸡双眼每日接受不同时间的激光照射。分别于激光照射后1个月(2月龄鸡)、3个月(4月龄鸡)和6个月(7月龄鸡)各组任选5只进行活体光相干断层扫描(OCT)检查,测定鸡的相对视网膜面积;采用过量麻醉法处死并制备视网膜切片,行苏木精-伊红染色和TUNEL染色,分别观察各组鸡视网膜面积变化和视细胞凋亡情况;实验眼制备视网膜匀浆,分别采用TBA法和NBT法检测鸡视网膜中丙二醛质量摩尔浓度和超氧化物歧化酶(SOD)比活性;采用Western blot法检测鸡视网膜中L/M视蛋白和视紫红质蛋白的表达。结果:2月龄时鸡照射30-min组视网膜中丙二醛质量摩尔浓度高于正常对照组,差异有统计学意义(P<0.05),4月龄时激光照射6-min组和照射30-min组丙二醛质量摩尔浓度均高于正常对照组,差异均有统计学意义(P=0.026、0.003),7月龄时所有激光照射组鸡视网膜中丙二醛质量摩尔浓度均高于正常对照组,差异均有统计学意义(P=0.038、0.032、<0.01)。7月龄时激光照射30-min组鸡视网膜中SOD比活性和视紫红质蛋白相对表达量均低于正常对照组[SOD:(140.20±5.99)(nmol/s·mg)与(160.57±3.13)(nmol/s·mg);视紫红质蛋白:0.392±0.065与0.566±0.072],差异均有统计学意义(均P<0.05)。OCT检查显示,照射3-min组、照射6-min组和照射30-min组6个月内鸡视网膜相对面积及形态与正常对照组相比无明显差别;组织病理学检查显示各照射组鸡视网膜厚度均接近正常对照组,TUNEL染色显示各组鸡视网膜细胞排列整齐,未发现TUNEL阳性染色细胞。结论:实验鸡眼接受650 nm半导体激光照射时,用2 mW的功率每日照射6 min持续6个月不引起明显光损伤;每日照射30 min持续6个月引起视网膜中自由基含量增高和视紫红质减少,提示存在光损伤。

Objective To observe whether there was a chronic light damage after the irradiation of 650 nm semiconductor laser(power 2 mW)in chicken cone-rich-retina and discuss the safety of this laser for retina.Methods Sixty 1-month-old chicken reared under natural light were divided into a normal control group,an irradiation 3-min group,an irradiation 6-min group and an irradiation 30-min group by using a random number table and 15 for each group.The chicken eyes were irradiated with 650 nm laser for different duration according to a grouping.Relative retina area was measured with optical coherence tomography(OCT)at 1 month(2-month-old chicken),3 months(4-month-old chicken)and 6 months(7-month-old chicken)after laser irradiation.Chickens were sacrificed by overdose anesthesia and the histopathology of chiken retina was examined by hematoxylin-eosin staining.The apoptosis of the retinal cells was evaluated by TUNEL staining.Chicken retinal homogenate was prepared,and the content of malondialdehyde and activity of superoxide dismutase(SOD)in the retina were detected by TBA method and NBT method,respectively.Western blot was employed to detect the expression of L/M opsin and rhodopsin in the retina.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results In 2-month-old chicken,the molar concentration of malondialdehyde in retina was significantly higher in the irradiation 30-min group compared with the normal control group(P<0.05).In 4-month-old chicken,the molar concentration of malondialdehyde was statistically higher in the irradiation 6-min group and the irradiation 30-min group in comparison with the normal control group(P=0.026,0.003).In 7-month-old chicken,the concentrations of retinal malondialdehyde in the irradiation 3-min group,irradiation 6-min group and irradiation 30-min group were statistically higher than those of the normal control group(P=0.038,0.032,<0.01).In 7-month-old chicken,the SOD activity and the relative expression of rhodopsin in the retina of the irradiation 30-min group were statistically reduced incomparison with the normal control group(SOD:[140.20±5.99][nmol/s·mg]vs.[160.57±3.13][nmol/s·mg]);Rhodopsin:(0.392±0.065 vs.0.566±0.072)(both at P<0.05).OCT showed that there was no significant difference in relative retinal area within 6 months among the four groups.Histopathological examination showed that the thickness of the retina in each irradiation group was close to the normal control group.TUNEL staining showed that the retinal cells were regularly arranged,and no TUNEL positive staining cells were found in all of the groups.Conclusions Irradiation of a 650 nm semiconductor laser(2 mW)in chicken's eyes for 6 minutes is safe for retina within 6 months.The lasser irradiation 30 minutes for 6 months results in an increase of free radical content in the retina and a decrease in rhodopsin,suggesting the presence of photo damage.