依达拉奉对大鼠视神经钳夹伤后视网膜细胞的保护作用

Protective effects of edaravone on retinal cells after optical nerve crash in rats

目的探讨大鼠视神经钳夹伤致视网膜细胞凋亡过程中活性氧(reactive oxygen species,ROS)的含量和依达拉奉对视网膜细胞的保护作用。方法选择健康清洁级SD大鼠192只(384眼),雌雄各半,随机分为正常组、假手术组、对照组、治疗组,每组各48只。对照组、治疗组用视神经钳夹法制作大鼠视神经夹挫伤模型;治疗组造模后腹腔注射依达氉拉奉30mg·kg-1,用生理盐水稀释后每隔12h腹腔注射给药。给药时间为30min,共14d。分别于造模后3d、6d、10d、14d处死大鼠。光镜下观察各组不同时间点视网膜结构,通过激光共聚焦显微镜观察视网膜切片TUNEL荧光标记,2’,7’-二氯双氢荧光素双乙酸酯为标记探针,通过流式细胞术定量检测各组不同时间点视网膜细胞内2’,7’-二氯双氢荧光素的荧光强度。以AnnexinV/PI双染色法通过流式细胞术定量检测各组不同时间点视网膜细胞凋亡率。结果造模后3～14d,正常组及假手术组视网膜细胞偶见TUNEL阳性标记;对照组见大量TUNEL阳性标记细胞,但治疗组明显少于对照组。视网膜细胞中ROS的表达量在造模后3d、6d、10d、14d时正常组分别为32.530±2.203、43.600±3.635、32.800±1.952、45.330±2.902,治疗组分别为84.507±3.754、65.130±5.308、63.400±5.226、66.990±6.320,对照组分别为92.830±2.875、99.900±0.100、99.300±1.212、92.470±2.159,每个时间点治疗组和对照组比较、正常组和对照组比较、正常组和治疗组比较,差异均有统计学意义(均为P<0.05);假手术组与正常组间相比差异均无统计学意义(均为P>0.05)。细胞总凋亡率也明显升高,造模后3d、6d、10d、14d时正常组分别为(9.550±0.325)%、(10.380±0.642)%、(11.540±1.224)%、(10.520±0.839)%,治疗组分别为(22.960±2.574)%、(27.590±1.369)%、(33.950±4.580)%、(25.810±2.170)%,对照组分别为(35.510±3.086)%、(37.550±2.357)%、(40.220±5.011)%、(34.320±3.351)%,每个时间点治疗组和对照组、正常组和对照组、正常组和治疗组之间比较,差异均有统计学意义(均为P<0.05),假手术组与正常组间相比差异均无统计学意义(均为P>0.05)。结论依达拉奉通过清除ROS,抑制了视网膜细胞凋亡,具有一定的视网膜细胞保护作用。

Objective To investigate the content of reactive oxygen species （ROS） in the process of retinal ceils apoptosis after optical nerve crash in rats,and dis- cuss the protective effects of edaravone on retinal cells. Methods A total of 192 SD rots with half males and half females were randomly divided into normal group, sham- operation group, control group and treatment group ,48 rats in each group. In the control and treatment group, the optic nerve crush model was made. In the treatment group, di- luted edaravone（30 mg· kg^-1 ） with normal saline was intraperitoneally injected every 12 hours after modeling. The delivery time was 30 minutes with a total of 14 days. Rats were killed at 3 days,6 days, 10 days and 14 days during the experiment. The TUNEL la- beled apoptotic was done, apoptotic ceils in each group were observed by laser scanning confocal microscope,retinal structure was checked by light microscopic,2＇, 7 ＇-dichloro fluorescein pair of dihydroartemisinin acetate ester（DCFH-DA） as the labeled probe, fuorescence intensity of 2＇ ,7＇-dichloro fluorescein dihydrotestosterone in retinal cells was checked by flow cytometry at different time points of four groups. Retinal cell ap- optosis rate was checked by double staining of AnnexinV/PI via flow cytometry at dif- ferent time points of four groups, Rtesults From 3 days to 14 days after modeling, TUNEL-positive labeling could be observed occasionally in retinal cells of normal group and shana-operation group, a large number of TUNEL-positive labeled cells were ob- served in the control group ,but the treatment group was significantly less than the con- trol group. At 3 days,6 days,10 days and 14 days,the expression of ROS in retinal cells in normal group were 32.530 ± 2.203,43. 600 ± 3.635,32. 800 ± 1. 952,45.330 ± 2.902, respectively,treatment group were 84. 507 ± 3. 754,65. 130 ± 5. 308,63. 400 ± 5. 225, 66.990±6. 320, respectively, control group were 92. 830 ± 2. 875,99. 900 ± 0. 100, 99.300 ± 1. 212,92. 470 ± 2. 159, respectively, there were statistical differences among three groups at each time point （ all P 〈 0. 05 ）, but no difference between sham-opera- tion group and normal group（ all P 〉0.05 ）. At 3 days,6 days,10 days and 14 days,the apoptosis rate of cells in normal group was （ 9.550 ± 0. 325 ）% , （ 10. 380 ± 0. 642 ）%, （11.540 ± 1. 224）% and （10. 520 ± 0. 839）% , respectively, treatment group were （22. 960 ± 2.574） %, （27.590 ± 1. 369） %, （33. 950 ± 4.580） %, （25.810 ± 2. 170） %, respectively,control group were （35. 510 ± 3. 086）% , （37. 550 ± 2. 357）% , （40. 220 ± 5. 011）% , （34. 320 ± 3. 351 ）% , respectively, there were statistical differences among three groups at each time point （ all P 〈 0. 05 ）, but no difference between sham group and normal group （ all P 〉 0.05 ）. Conelusion Edaravone inhibits the apoptosis of ret- inal cells by eliminating ROS,and has certain protective effects on retinal cells.