Thy1-YFP转基因小鼠视网膜神经节细胞的存活率

Survival rate of retinal ganglion cells in transgenic Thy1-YFP mice

目的:研究转基因小鼠Thy1-YFP在视神经钳夹(ONC)模型和谷氨酸兴奋性毒性(NMDA)模型中YFP阳性视网膜神经节细胞(YFP^(+)RGCs)的存活率与总体RGCs(RBPMS^(+)RGCs)的存活率差异,以期为Thy1-YFP小鼠在视神经退行性病变中的进一步研究提供实验数据支持。方法:8周龄Thy1-YFP小鼠(雌雄不限)共20只,采用随机数字表法将小鼠随机分为正常组、ONC模型组和NMDA模型组。正常组小鼠进行左右眼对照;模型组小鼠均采用单眼造模,对侧眼为对照眼。其中,ONC小鼠于钳夹后7 d取材,NMDA小鼠于NMDA玻璃体腔注射后24 h取材。取材后,通过全视网膜铺片荧光染色和激光共聚焦拍照的方式获取RGCs信息,利用Image J计数YFP^(+)RGCs和总体RBPMS^(+)RGCs。统计分析ONC模型和NMDA模型中YFP^(+)RGCs的存活率与总体RBPMS^(+)RGCs的存活率差异,存活率差异以实验眼与对照眼的YFP^(+)RGCs(或RBPMS^(+)RGCs)的密度比值计算。结果:Thy1-YFP转基因小鼠视网膜上YFP^(+)RGCs和RBPMS^(+)RGCs的平均密度左右眼一致,具有可比性,其中YFP^(+)RGCs约占总RGCs的0.19%。在ONC模型中,与对照眼相比,ONC眼的YFP^(+)RGCs与RBPMS^(+)RGCs的密度都出现了大幅度下降。YFP^(+)RGCs的平均存活率为(59.4±8.2)%,而RBPMS^(+)RGCs为(33.0±1.7)%,差异达26.4%(P<0.001)。在NMDA模型中,与对照眼相比,NMDA注射眼的YFP^(+)RGCs与RBPMS^(+)RGCs的密度亦都出现了大幅度下降。YFP^(+)RGCs的存活率为(46.1±6.0)%,而RBPMS^(+)RGCs为(32.4±3.5)%,差异达13.7%(P<0.01)。结论:转基因小鼠ONC模型与NMDA模型表明YFP^(+)RGCs存活率并不能良好地反映总RGCs的存活率,说明Thy1-YFP小鼠的RGCs并不能代表总体RGCs,存在一定的RGCs亚类特异性。本研究结果为Thy1-YFP小鼠在视神经退行性病变中的研究提供了数据支持,进一步加深了研究者对于Thy1-YFP转基因小鼠的认识,为将来的研究提供了方向和借鉴。

Objective:To study the difference in survival rate between Thy1-YFP positive retinal ganglion cells(YFP^(+)RGCs)and total RGCs(RBPMS^(+)RGCs)in optic nerve crush(ONC)model and gluta-mate excitotoxicity(NMDA)model,to provide a further experimental data support to the study of transgenic Thy1-YFP mice in the neuronal degenerative disease.Methods:Eight-week-old transgenic Thy1-YFP mice were randomly divided into the ONC group and the NMDA group.One eye received ONC or NMDA injection,while the contralateral eye was set as sham-operated control.The ONC group mice were sacrificed after a week of ONC,and the NMDA group mice were sacrificed 24 hours after NMDA injection.After sampling,RGCs information was obtained by whole-retina patch fluo-rescence staining and laser confocal photography.Image J was used to count YFP^(+)RGCs and total RBPMS^(+)RGCs.The difference between the survival rate of YFP^(+)RGCs in the ONC model and NMDA model and the overall survival rate of RBPMS^(+)RGCs was statistically analyzed.The survival rate difference was calculated by the density ratio of YFP^(+)RGCs(or RBPMS^(+)RGCs)between the ex-perimental eyes and control eyes.Results:The mean densities of YFP^(+)RGCs and RBPMS^(+)RGCs in the retinas of Thy1-YFP transgenic mice were consistent and comparable between the two eyes,with YFP^(+)RGCs accounting for 0.19%of the total RGCs.In the ONC model,compared with the control eye,the density of YFP^(+)RGCs and RBPMS^(+)RGCs in the ONC eye showed a significant decrease.The average survival rate of YFP^(+)RGCs was(59.4±8.2)%,and that of RBPMS^(+)RGCs was(33.0±1.7)%;the difference reached 26.4%(P<0.001).In the NMDA model,compared with the control eye,the density of YFP^(+)RGCs and RBPMS^(+)RGCs in the NMDA injection eye also de-creased significantly.The survival rate of YFP^(+)RGCs was(46.1±6.0)%,while that of RB-PMS^(+)RGCs was(32.4±3.5)%,and the difference reached 13.7%(P<0.01).Conclusion:The ONC and NMDA models of transgenic mice indicate that the survival rate of YFP^(+)RGCs does not re-flect the survival rate of total RGCs well,suggesting that RGCs of Thy1-YFP mice are not represen-tative of total RGCs and there is a certain subclass specificity of RGCs.This work provided the data for the study of optic degenerative diseases and enhanced the knowledge of Thy1-YFP mice.