Excitatory toxicity of glutamate on meningothelial cells of optic nerve sheaths and its oxidative stress mechanism

Background 

Meningothelial cells (MECs) occupy the predominant cell component of barrier between optic nerve and the cerebral spinal fluid, and any change of cerebral fluid components probably affects the MECs function and further impairs the optic nerve.

Objective

This study was designed to investigate the influence of glutamate, a potentially excitotoxic amino acid, to the functional changes of MECs and provide a theoretical evidence for clarifying the mechanism of optic nerve disorders.

Methods

Human MECs strains were cultured in vitro and prepared into cell suspension.The cells were inoculated to 96-well plates with the densities of 1×10⁴/well.The glutamate of 100, 200, 400, 600, 800 and 1 000 μmol/L was added into medium for 12, 24, 36, 48 and 72 hours, respectively, and the cultured cells without glutamate were used as normal control group.MTS assay was employed to measure the proliferative rate (absorbency) of the cells.The regularly cultured MECs were divided into 600 μmol/L glutamate-treated group and normal control group and the cells were treated for 12 and 24 hours respectively, and the expression of superoxide dismutase (SOD) mRNA and heat shock protein 90 (HSP90) mRNA in the cells was detected by real-time PCR; the level of total anti-oxidative capacity (T-AOC) of the cells was processed by enzyme linked immunosorbent assay (ELISA), and the reactive oxygen species (ROS) production was determined by DCFH-DA probe.

Results

Cultured MECs grew well and formed 80% confluence after 72 hours culture.The proliferative rate of the cells were gradually decreased with the increase of glutamate dose and the lapse of affected time, with significant differences among different concentrations of glutamate and various time points (F_{concentration}=52.501, P<0.001; F_time=8.505, P<0.001). The relative expression level of SOD mRNA was significantly reduced in the glutamate-treated group compared with the normal control group in both 24 hours and 48 hours after culture (t=20.278, t=16.724, both at P<0.001), and the expression of HSP90 mRNA in the cells was significantly lower in the glutamate-treated group than that in the normal control group in 24 hours after culture (t=5.065, P=0.002). No significant difference was found in T-AOC activity between glutamate-treated group and normal control group in 24 hours after culture ([30.835±2.094] nmol/(min·L) vs. [32.873±2.317] nmol/(min·L)) (t=1.599, P=1.414). In 48 hours after culture, T-AOC activity was (29.561±1.831) nmol/(min·L) in the glutamate-treated group, which was significantly lower in comparison with normal control group(33.680±2.039) nmol/(min·L) (t=3.682, P=0.004). Fluorescence staining showed that the intensity of green fluorescence of ROS in MECs in the normal control group was weaker than that in the glutamate-treated group under the immunofluorescense microscope.The ROS level was 48.110±1.712 and 40.982±1.853 at 24 hours and 48 hours in the glutamate-treated cells, and which was significantly elevated in comparison with 36.608±1.009 and 37.153±1.424 in the normal control group (t=14.178, P<0.001; t=4.012, P=0.002).

Conclusions

Glutamate inhibits the proliferation of MECs in vitro, and excitatory toxicity of glutamate on MECs probably is associated with oxidative stress response.

Glutamate; Epithelial cells, meninges/pathology; Optic nerve; Oxidative stress; Excitatory amino acid/toxicity; Cell proliferation/drug effects; Humans; Cells, cultured