Mechanism of microglia promoting retinal ganglion cell death in vitro

Abstract

Objective: To investigate the role and mechanism of microglial activation in the process of retinal ganglion cell (RGC) death in the oxygen-glucose deprivation/reperfusion (OGD/R) model which mimicked retinal ischemia/reperfusion injury in vitro. Methods: Experimental study. Primary RGCs from C57BL/6 mice and BV2 microglia were co-cultured or cultured alone. The OGD/R model was established in vitro (reoxygenation time was set to 6 h, 24 h, 36 h, 48 h). BV2 microglial activation was assessed by immunofluorescence staining of ionized calcium binding adapter molecule 1 (iba1), and the survival rate of RGCs was detected by the Cell Counting Kit-8. The apoptosis rate of RGC was detected by using apoptosis detection kit. The levels of Toll-like receptor-4 (TLR4) and Nod-like receptor family pyrin domain containing protein 3 (NLRP3) in BV2 cells were detected by PCR, Western-blot and immunofluorescence staining. The activity of caspase-8 in BV2 cells was detected by the CaspGLOW Kit, and the content of interleukine-1β (IL-1β) in the supernatant was detected by enzyme linked immunosorbent assay. After the corresponding pathways were blocked by TLR4 small interfering RNA (siRNA) transfection or caspase-8 inhibitor, the expression changes of TLR4 and NLRP3, the activity of caspase-8, and the difference of IL-1β content could be observed as well as the activity of RGCs co-cultured with BV2. Statistical analysis was performed using analysis of variance. Results: Under co-culture of RGC and BV2 cells, cellular immunofluorescence detection showed that the expression of iba1 in BV2 cells increased, which indicated BV2 cells were activated significantly in the OGD/R model. In the OGD/R model, the apoptosis rate of RGC co-cultured with BV2 cells (71.1%±3.2%) was significantly higher than that of RGC cultured alone (35.1%±1.8%) (t=10.10, P<0.01). Cellular immunofluorescence detection showed that the expression of TLR4 and NLRP3 in BV2 cells in the OGD/R model increased significantly when BV2 cells were cultured alone, and their mRNA levels increased significantly with prolongation of reoxygenation time (F=64.45, 72.74; P<0.01), and peaked at OGD/R 24 h (TLR4 mRNA, relative ratio to control was 2.83±0.23; NLRP3 mRNA, relative ratio to control was 3.12±0.27). Caspase-8 activity also increased with prolonged reoxygenation time, the difference was statistically significant (F=93.57, P<0.01), and peaked at OGD/R 24 h (relative ratio to control was 2.92±0.31). After transfection of BV2 cells with TLR4 siRNA, its caspase-8 activity was significantly inhibited, but using caspase-8 inhibitor did not affect the up-regulation of TLR4 expression in BV2 cells. However, the mature IL-1β secreted by BV2 cells exposed to OGD/R was significantly reduced by using caspase-8 inhibitor (from 3.52±0.55 to 1.39±0.37, t=7.19, P<0.01), meanwhile, the expression of NLRP3 was also significantly decreased after caspase-8 inhibitor pretreatment (from 2.79±0.23 to 1.37±0.19, t=9.37, P<0.01). In the OGD/R model, the activity of RGC cells co-cultured with TLR4 siRNA-transfected BV2 cells was 74.5%±1.2%, and the activity of RGC cells co-cultured with BV2 cells treated with caspase-8 inhibitor was 62.8%±1.5%, those were both higher than that of RGC cells co-cultured with untreated BV2 cells (36.7%±0.3%), and the difference was statistically significant (t=11.60, 6.83; both P<0.01). Conclusion: TLR4-caspase-8-NLRP3 inflammasome pathway is activated in microglia exposed to OGD/R, resulting in the production of IL-1β, thereby contributing to the death of RGCs. (Chin J Ophthalmol, 2020, 56: 32-40).