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

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 1 μl of sodium chloride solution in the right eye, and mice were given intravitreal injection of 1 μl 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 of β3-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.

Retinal excitotoxicity; N-methyl-D-aspartic acid; Retinal ganglion cell; Tsix; Long noncoding RNA