目的:探讨来自宁夏的枸杞提取物(Lycium Barbarum extracts,LBE)对视神经挤压损伤(optic nerve crush,ONC)后小鼠视网膜结构和功能的保护作用.方法:首先比较健康组、2 d模型组和7 d模型组中闪光视网膜电图(flash electroretiongram,fERG)和视网膜结构的改变;然后比较腹腔注射磷酸盐缓冲液(phosphate buffered saline,PBS)组及不同剂量的LBE对造模7 d的ONC小鼠视网膜神经节细胞(retinal ganglion cells,RGCs)存活的影响;最后利用LBE最佳保护作用的剂量,与PBS对照组比较,进一步研究LBE对ONC手术7 d小鼠视网膜fERG和结构的影响.RGCs计数利用视网膜平铺片进行免疫荧光染色(immunofluorescence,IF)标记,视网膜冰冻切片用于IF染色标记双极细胞、视锥细胞、突触小泡蛋白(synaptophysin,SYN)和突触后致密蛋白95(postsynaptic density protein 95,PSD95),二氢乙锭(dihydroethidium,DHE)标记视网膜产生的活性氧化产物(reactive oxygen species,ROS).用ImageJ软件计数RGCs、双极细胞和视锥细胞的数目,测量双极细胞突起在内丛层(inner plexiform layer,IPL)所占荧光阳性面积百分比(percentage of the fluorescence-positive area,PP)、SYN 在 IPL 和外丛层(outer plexiform layer,OPL)的免疫反应荧光强度(fluorescence intensity,FI)和PSD95在OPL的FI及PP,计算ROS反应后的红色FI.结果:与正常组和ONC-2 d组相比,ONC-7 d后存活的RGCs数目、双极细胞突起在IPL所占PP、双极细胞数目、视锥细胞数目、SYN在IPL和OPL的FI强度减少(P＜0.05);PSD95在OPL层PP减少(P＜0.05);fERG显示a波和b波的波幅在暗适应(P＜0.01)和明适应下(P＜0.05)减少,二者的潜伏期没有明显变化(P＞0.05);DHE染色显示ROS在术后明显增加(P＜0.01).与对照组相比,上述不同剂量的LBE经腹腔注射到小鼠体内,均可使RGCs存活增加(P＜0.01),其中1.0 mg/kg LBE组存活的RGCs均数最大(P＜0.01);此外,1.0 mg/kg LBE腹腔注射可以增加双极细胞突起所占IPL层PP和细胞数目(P＜0.05)、视锥细胞数目(P＜0.01)、SYN在IPL(P＜0.01)及在OPL(P＜0.05)的FI、PSD95在OPL表达的FI及PP(P＜0.05)、a波和b波暗适应(P＜0.05)和明适应时(P＜0.01)的幅值,降低ROS产生(P＜0.05).结论:LBE对小鼠ONC后视网膜的结构和功能具有保护作用,其保护作用可能与其抑制视网膜的氧化应激反应有关.
Extracts of Lycium barbarum protected the structure and function of the mouse retina after optic nerve crush
Objective:To investigate the protective effect of Lycium barbarum extracts(LBE,Ningxia,China)on the retinal structure and function in the mice administrated with optic nerve crush(ONC).Methods:The changes of flash electroretinogram(fERG)and retinal structure in the normal group,ONC-2 d group and ONC-7 d group were compared.Then,the effects of intraperitoneal injection of various dosages of LBE on the survival of retinal ganglion cells(RGCs)in the ONC-7 d group were compared.After that,the LBE dosage with the best protective effect was selected for the next experiment.Finally,we further studied the effects of 1.0 mg/kg LBE on the retinal ERG and structure preservation ONC-7 d group.In all the mentioned experiments,the full-field flash ERG was used to detect the changes in the a-wave and b-wave amplitude and latency under dark and light conditions in the living animals.The collected retinas were processed as the retinal flat whole-mounts and used for the immunofluorescent labeling of the RGCs,or as the frozen sections to be utilized for the fluorescent staining of the retinal bipolar cells,cones,synaptophysin(SYN),anti-postsynaptic density protein 95(PSD95)and the reactive oxygen species(ROS).ImageJ software was used to count the numbers of the surviving RGCs,bipolar cells and cones,to calculate the percentage of the fluorescence-positive area(PP)of the bipolar cell protrusions in the inner plexiform layer(IPL),to measure the fluorescence intensity(FI)of the SYN in the IPL and outer plexiform layer(OPL),to measure the FI and PP of the PSD95 in the OPL,and to detect the red FI after the DHE detection.Results:Compared with the N and ONC-2 d groups,the numbers of the survival RGCs,bipolar cells and cones,the PP of the bipolar cell protrusion in the INL,and the FI of the SYN in the IPL and OPL decreased(P＜0.05)in the ONC-7 d group.The FI of the PSD95 did not change(P＞0.05);but the PP decreased in the OPL(P＜0.050).The results of ERG examination showed that the amplitudes of a-and b-waves decreased under dark adaptation(P＜0.01)and the reduction in the amplitude of b wave during light adaptation was significant 7 days after surgery(P＜0.05).DHE staining showed that the ROS was increased 7 days after the operation(P＜0.01).Compared with the control group,treatment with LBE could increase the survival of the RGCs(P＜0.01)with the largest survival average density of RGCs in the 1.0 mg/kg LBE group(P＜0.01).In addition,compared with the control group,1.0 mg/kg LBE could increase the PP occupied by the bipolar cell protrusions in the IPL(P＜0.05),the number of surviving cone cells(P＜0.01),the FI of SYN in the IPL(P＜0.01)and OPL(P＜0.05),and the FI and the PP of PSD95 in the OPL(P＜0.05).The improved function of the retina was shown by the increasing amplitudes under dark adaptation(P＜0.05)and light adaptation(P＜0.01).LBE reduced the FI of ROS(P＜0.05).Conclusion:LBE has a protective effect on the retina after ONC injury in mice,and its protective effect may be related to its inhibition of oxidative stress in the retina.
万方数据
维普
