LncRNA Tsix在NMDA诱导小鼠视网膜兴奋性毒性模型中的表达及其意义

Expression of lncRNA Tsix in a mouse model of N-methyl-D-aspartic acid-induced retinal excitotoxicity and its significance

目的研究不同剂量N-甲基-D-天冬氨酸(NMDA)溶液对小鼠视网膜神经节细胞(RGCs)的损伤作用,并检测新型长链非编码RNA(lncRNA)Tsix在小鼠视网膜兴奋性毒性模型中的表达水平及其对视网膜和RGCs的保护作用。方法选取7~8周龄C57B6/J小鼠105只,采用随机数表法将小鼠随机分为正常对照组、2 mmol/L NMDA组、10 mmol/L NMDA组、20 mmol/L NMDA组和40 mmol/L NMDA组,每组21只。正常对照组小鼠右眼玻璃体腔内注入1μl氯化钠溶液,不同剂量NMDA组分别注射相应剂量的NMDA溶液各1μl。1周后,分别采用光相干断层扫描(OCT)、苏木精-伊红染色、视网膜铺片和免疫荧光染色法分析不同剂量NMDA组视网膜各层厚度、神经节细胞层(GCL)细胞数量和RGCs数量。采用RNAscope原位杂交技术检测不同剂量NMDA组GCL中lncRNA Tsix的表达。采用实时荧光定量PCR技术检测不同剂量NMDA组Tsix基因的转录本水平。结果OCT结果显示,2、10、20和40 mmol/L NMDA组视网膜全层厚度分别为(255.00±6.63)、(252.40±6.41)、(248.67±6.20)和(229.11±10.37)μm,较正常对照组的(269.60±20.01)μm明显变薄,差异均有统计学意义(均P<0.05)。苏木精-伊红染色结果显示,正常对照组小鼠GCL细胞排列均匀、紧密,呈单层,细胞核大而圆,NMDA注射后细胞出现体积不均和空泡,有核固缩现象。各剂量NMDA组GCL细胞数量随NMDA剂量的增加而显著降低,与正常对照组相比差异均有统计学意义(均P<0.05)。当NMDA浓度增至20 mmol/L时,GCL的细胞数量减少至正常对照组的一半。视网膜铺片结果提示,β3-微管蛋白阳性RGCs细胞数量随NMDA剂量的增加显著降低,与正常对照组比较差异均有统计学意义(均P<0.05);10 mmol/L NMDA组RGCs数量减少至正常对照组的一半。RNAscope结果显示,lncRNA Tsix主要在GCL细胞的细胞质中表达,且随着NMDA剂量的增加,表达lncRNA Tsix的阳性细胞率显著降低,各组总体比较差异有统计学意义(F=13.670,P<0.01)。实时荧光定量PCR结果验证Tsix随NMDA剂量的增加表达趋势与RNAscope结果一致。结论NMDA对视网膜厚度和RGCs的损伤呈剂量依赖性,小鼠视网膜lncRNA Tsix的表达主要集中在GCL细胞的细胞质,且转录本水平随着NMDA剂量的增加而降低,对RGCs发挥保护作用。

Objective To investigate the damage effect of different concentrations of N-methyl-D-aspartic acid(NMDA)to retinal ganglion cells(RGCs)in mice and explore the expression of long noncoding RNA(lncRNA)Tsix in the retina of mice with excitotoxicity as well as the protective effect of lncRNA Tsix on retina and RGCs.Methods A total of 105 C57B6/J mice at 7-8 weeks of age were selected and randomly divided into the normal control group,2 mmol/L NMDA group,10 mmol/L NMDA group,20 mmol/L NMDA group and 40 mmol/L NMDA group using a random number table method,with 21 mice in each group.In the normal control group,the mice were intravitreally injected with 1μl of sodium chloride solution in the right eye,and mice were given intravitreal injection of 1μl of different doses of NMDA according to grouping.At one week after the injection,the thickness of each retinal layer,the number of ganglion cell layer(GCL)cells and the number of RGCs were analysed and compared among different groups through optical coherence tomography(OCT),hematoxylin-eosin staining,retinal whole mount staining and immunofluorescence staining.RNAscope in situ hybridization was used to verify the expression of lncRNA Tsix in the GCL of different groups.The quantitative real-time PCR was used to detect the transcript levels of Tsix in different groups.This study was approved by an Ethics Committee of Tianjin Medical University(No.SYXK2018-0004),and the use of experimental animals was in accordance with the regulations of Tianjin Medical University and ARVO statement.Results The OCT results showed that the total retinal thickness of mice in the 2,10,20 and 40 mmol/L NMDA groups were(255.00±6.63),(252.40±6.41),(248.67±6.20)and(229.11±10.37)μm,respectively,which were thinner than(269.60±20.01)μm in the normal control group,and the differences were statistically significant(all at P<0.05).Hematoxylin-eosin staining showed that the cells in the GCL of the normal control group were uniform and compact,and arranged in a single layer with large and round nuclei.In the NMDA groups,the cells were uneven in volume with vacuoles and nuclear pyknosis.The cell density in the GCL was decreased significantly with the increasing NMDA doses in NMDA groups in comparison with the normal control group,and the differences were statistically significant(all at P<0.05).In the 20 mmol/L NMDA group,the cell density in the GCL was reduced to half of the normal control group.The results of retinal whole mount staining showed that the density ofβ3-tubulin-positive RGCs was decreased significantly as the dose of NMDA increased in NMDA groups,and the differences were statistically significant compared with the normal control group(all at P<0.05).The number of RGCs in the 10 mmol/L NMDA group was reduced to half of that in the normal control group.RNAscope results showed that lncRNA Tsix was mainly expressed in the cytoplasm of the GCL cells.The proportion of lncRNA Tsix-positive cells was significantly reduced with the increase of the NMDA dose(F=13.670,P<0.01).The quantitative real-time PCR results verified that the trend of Tsix expression was consistent with the RNAscope result.Conclusions NMDA exerts a dose-dependent damage to the layer thickness of mouse retina and RGCs.The expression of lncRNA Tsix in mouse retina is mainly enriched in the cytoplasm of the cells in the GCL,and the transcript level of Tsix is reduced with the increase of NMDA concentration and have a protective effect on RGCs.