Pregabalin Mediates Retinal Ganglion Cell Survival From Retinal Ischemia/Reperfusion Injury Via the Akt/GSK3β/β-Catenin Signaling Pathway

Abstract

Purpose: Progressive retinal ganglion cell (RGC) loss induced by retinal ischemia/reperfusion (RIR) injury leads to irreversible visual impairment. Pregabalin (PGB) is a promising drug for neurodegenerative diseases. However, with regard to RGC survival, its specific role and exact mechanism after RIR injury remain unclear. In this study, we sought to investigate whether PGB could protect RGCs from mitochondria-related apoptosis induced by RIR and explore the possible mechanisms.

Methods: C57BL/6J mice and primary RGCs were pretreated with PGB prior to ischemia/reperfusion modeling. The retinal structure and cell morphology were assessed by immunochemical assays and optical coherence tomography. CCK8 was used to assay cell viability, and an electroretinogram was performed to detect RGC function. Mitochondrial damage was assessed by a reactive oxygen species (ROS) assay kit and transmission electron microscopy. Western blot and immunofluorescence assays quantified the expression of proteins associated with the Akt/GSK3β/β-catenin pathway.

Results: Treatment with PGB increased the viability of RGCs in vitro. Consistently, PGB preserved the normal thickness of the retina, upregulated Bcl-2, reduced the ratio of cleaved caspase-3/caspase-3 and the expression of Bax in vivo. Meanwhile, PGB improved mitochondrial structure and prevented excessive ROS production. Moreover, PGB restored the amplitudes of oscillatory potentials and photopic negative responses following RIR. The mechanisms underlying its neuroprotective effects were attributed to upregulation of the Akt/GSK3β/β-catenin pathway. However, PGB-mediated neuroprotection was suppressed when using MK2206 (an Akt inhibitor), whereas it was preserved when treated with TWS119 (a GSK3β inhibitor).

Conclusions: PGB exerts a protective effect against RGC apoptosis induced by RIR injury, mediated by the Akt/GSK3β/β-catenin pathway.

Figure 1.

PGB performed neuroprotective effects during RIR injury. (A) A mouse RIR model was successfully established. Microscopic examination showed that the fundus of normal mice had clear optic disc boundaries and well-filled blood vessels; however, in the ischemic group of mice, which showed a sharp increase in IOP, the retinal optic disc boundaries were unclear, blood vessels were cut off, and the retina appeared pale. (B) The mice were pretreated with PGB before modeling, and the neuroprotective effect was evaluated at 24 hours and 7 days after RIR. (C) Representative H&E images of retinal sections (40×). Scale bars: 50 µm. (D) Representative OCT scans in living mice. Scale bars: 200 µm. (E) Representative retinal mounts with fluorescent staining of RGCs (20×). Scale bars: 100 µm. (F–H) Thickness of the whole retina (F) and RNFL and GCC (G) and the number of RGCs (H) decreased under RIR insult, whereas PGB pretreatment rescued the deterioration of the above indicators. The most significant improvement was observed at the dose of 30 mg/kg (n = 6). ^*P  < 0.05 versus control group; ^**P < 0.05 versus NaCl + RIR group; ^***P < 0.05 versus 30-mg/kg PGB + RIR group.

Figure 2.

PGB reduced mitochondria-associated RGC apoptosis induced by RIR injury. (A) PGB pretreatment decreased the expression of pro-apoptotic protein Bax but increased Bcl-2 expression, and the most significant improvement was observed at 30 mg/kg. (B) PGB pretreatment reversed the elevation of cleaved caspase 3/pro-caspase 3 elicited by RIR, and the most significant improvement was observed at 30 mg/kg. (C) PGB (30 mg/kg) pretreatment inhibited ROS generation. (D) RIR injury induced disintegrated cristae and increased matrix density in mitochondria. PGB pretreatment improved the structure of mitochondria following RIR; mitochondria are indicated by the yellow arrow (n = 6; 25,000×). Scale bars: 500 nm. ^*P  < 0.05 versus control group; ^**P  < 0.05 versus NaCl + RIR group; ^***P < 0.05 versus 30-mg/kg PGB + RIR group.

Figure 3.

PGB improved visual function in the retina of RIR mice models. (A) Averaged data of OP amplitudes from recordings obtained 7 days after RIR. (B) Averaged data for PhNR amplitudes from recordings obtained 7 days after RIR. The amplitudes of OPs and PhNRs were decreased after RIR, whereas 30-mg/kg PGB pretreatment restored levels of these indicators (n = 6). ^*P < 0.05 versus control group; ^**P < 0.05 versus NaCl + RIR group.

Figure 4.

PGB protected against RIR injury via the Akt/GSK3β/β-catenin pathway. (A) The results of WB showed that the expression of p-Akt, p-GSK3β, and β-catenin was downregulated after RIR, whereas PGB (30 mg/kg) pretreatment reversed these findings. (B, D) Representative IF images of retinal sections (40×). Scale bars: 50 µm. The ratios of RGCs in the GCL expressing p-Akt, p-GSK3β, and β-catenin were downregulated after RIR, but PGB pretreatment upregulated the proportion of positive cells. (C) PGB pretreatment inhibited the activity of GSK3β (n = 6). ^*P < 0.05 versus control group; ^**P < 0.05 versus NaCl + RIR group.

Figure 5.

The neuroprotective effect of PGB required the phosphorylation of Akt and inactivation of GSK3β. (A, B) The results of WB showed that MK2206 (10 nM) inhibited the phosphorylation of Akt and GSK3β stimulated by PGB. (C, D) Representative IF images of retinal sections (40×). Scale bars: 50 µm. MK2206 inhibited the increase in the ratio of RGCs, with p-Akt⁺ and p-GSK3β⁺ stimulated by PGB (30 mg/kg). (E) Consistent with the effect of PGB pretreatment, TWS119 (10 nM) inhibited the activity of GSK3β. (F) WB showed that TWS119 enhanced the expression of β-catenin (n = 6). ^*P < 0.05 versus control group; ^**P < 0.05 versus NaCl + RIR group; ^***P < 0.05 versus 30-mg/kg PGB + dimethyl sulfoxide (DMSO) + RIR group.

Figure 6.

PGB pretreatment prevented mitochondrial impairment and retinal dysfunction by the phosphorylation of Akt and inactivation of GSK3β. (A) PGB (30 mg/kg) and TWS119 (10 nM) pretreatment improved the structure of mitochondria in the retina of RIR mice models; mitochondria are indicated by the yellow arrow (25,000×). Scale bars: 500 nm. (B, D) MK2206 (10 nM) reversed the effects of PGB and decreased the thickness of the RNFL and GCC, and TWS119 maintained the effects of PGB and reserved the increased thickness of RNFL and GCC. Scale bars: 200 µm. (C, E) Representative ERG waveforms in living mice. MK2206 disrupted the visual function balance of RGCs in the presence of PGB, with a decrease in the amplitudes of OPs and PhNRs. However, TWS119 maintained the normal level of the amplitudes of OPs and PhNRs (n = 6). ^*P < 0.05 versus control group; ^**P < 0.05 versus NaCl + RIR group; ^***P < 0.05 versus 30-mg/kg PGB + DMSO + RIR group.

Figure 7.

PGB protected primary RGCs from OGD/R injury via the Akt/GSK3β/β-catenin pathway. (A) IF identification of primary mice RGCs (40×). Scale bars: 50 µm. (B) Primary RGCs pretreated with PGB (50 µM, 100 µM, or 200 µM) for 24 hours were subjected to OGD/R. OGD/R induced a considerable decrease in cell viability, whereas PGB improved cell viability. The most significant improvement was observed at 100 µM. (C, D) Primary RGCs pretreated with MK2206 (5 µM) were treated with PGB (100 µM) for 24 hours prior to the onset of OGD/R. The ROS level was measured through the fluorescent probe, DCFH-DA, and the ratio of ROS⁺ cells was measured. PGB pretreatment inhibited ROS generation, but MK2206 counteracted the effect. (40×). Scale bars: 50 µm. (E) Representative IF images of primary RGCs (40×). Scale bars: 50 µm. The ratios of RGCs expressing p-Akt, p-GSK3β, and β-catenin were downregulated after OGD/R, which was countered by PGB pretreatment. However, MK2206 decreased the ratios of positive cells (n = 6). ^*P < 0.05 versus control group; ^**P < 0.05 versus NaCl + OGD/R group; ^***P < 0.05 versus PGB 100 µM + OGD/R group; ^****P < 0.05 versus PGB + DMSO + OGD/R group.

Figure 8.

PGB enhanced RGC survival following RIR injury via the Akt/GSK3β/β-catenin signaling pathway.