Edaravone, an ROS scavenger, ameliorates photoreceptor cell death after experimental retinal detachment

Abstract

Purpose: To investigate whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, would be neuroprotective against photoreceptor cell death in a rat model of retinal detachment (RD).

Methods: RD was induced in adult Brown Norway rats by subretinal injection of sodium hyaluronate. Edaravone (3, 5, or 10 mg/kg) or physiologic saline was administered intraperitoneally once a day until death on day 3 or 5. Oxidative stress in the retina was assessed by 4-hydroxynonenal staining or ELISA for protein carbonyl content. Photoreceptor death was assessed by TUNEL and measurement of the outer nuclear layer thickness. Western blot analysis and caspase activity assays were performed. Inflammatory cytokine secretion and inflammatory cell infiltration were evaluated by ELISA and immunostaining, respectively.

Results: RD resulted in increased generation of ROS. Treatment with 5 mg/kg edaravone significantly reduced the ROS level, along with a decrease in TUNEL-positive cells in the photoreceptor layer. A caspase assay also confirmed decreased activation of caspase-3, -8, and -9 in RD treated with edaravone. The level of the antiapoptotic Bcl-2 was increased in detached retinas after edaravone treatment, whereas the levels of the stress-activated p-ERK1/2 were decreased. In addition, edaravone treatment resulted in a significant decrease in the levels of TNF-α, MCP-1, and macrophage infiltration.

Conclusions: Oxidative stress plays an important role in photoreceptor cell death after RD. Edaravone treatment may aid in preventing photoreceptor cell death after RD by suppressing ROS-induced photoreceptor damage.

Figure 1.

Quantification of oxidative retinal damage in retina with HNE immunostaining and ELISA for PCC. Although there was minimal 4-HNE staining in AR (A), increased 4-HNE staining at the IPL and the ONL was noted 3 days after creation of RD (B). Decreased 4-HNE staining was noted after treatment with both (C) 5 (D) and 10 mg/kg edaravone. (E) There was a significant decrease in PCC 3 days after RD in the edaravone treatment group compared with the saline-treated group (P < 0.01). Data are expressed as the mean ± SE; n = 5–7. **P < 0.01. GCL, ganglion cell layer; Eda, Edaravone. Original magnification: ×200.

Figure 2.

Effect of edaravone on ONL thickness ratio and photoreceptor cell death with TUNEL staining compared with saline treatment 3 and 5 days after RD. Quantification of TUNEL (+) cells showed significantly decreased TUNEL (+) cells/mm² with treatment of (B) 5 and (C) 10 mg/kg edaravone, compared with the saline-treated group (A) at 3 days after RD creation (both, P < 0.01). (D) Five days after RD creation, 10 mg/kg edaravone treatment showed a significant increase in photoreceptor cell death (P = 0.004). (E) Treatment with 5 mg/kg edaravone significantly prevented the reduction of the ONL thickness ratio on days 3 and 5 after RD (P < 0.001 and P = 0.041, respectively). Whereas treatment with 10 mg/kg showed a protective effect on day 3 (P = 0.041), prolonged treatment until day 5 showed no added protective effect (P = 0.25). Data are expressed as the mean ± SE n = 5–7. *P < 0.05. **P < 0.01. GCL, ganglion cell layer; Eda, Edaravone. Original magnification: (A–C) ×10; scale bar, 100 μm.

Figure 3.

Decreases in caspase activation after treatment with edaravone 24 hours after RD creation. Activity of caspase-3 (A), -8 (B), and -9 (C) in AR, RD, and RD treated with 5 mg/kg edaravone. RD induced activation of caspase-8, -9, and -3, 1 day after RD, and treatment with 5 mg/kg edaravone significantly suppressed activation of these caspases. Data are expressed as the mean ± SE, n = 6. *P < 0.05, **P < 0.01.

Figure 4.

ELISA analysis of TNF-α (A) and MCP-1 (B) activity three days after RD creation. While the level of MCP-1 and TNF-α was increased after RD, treatment with edaravone significantly reduced MCP-1 and TNF-α expression. Data are expressed as the mean ± SE, n = 4–6. *P < 0.05, **P < 0.01.

Figure 5.

Evaluation of macrophage infiltration with ED-1 staining in frozen sections. Three days after RD creation, a significantly decreased number of macrophages (arrowhead) was observed in the edaravone-treated group (B) compared with the saline-treated group (A). Quantification of ED-1 positive cells (C). **P < 0.01. Data are expressed as the mean ± SE n = 5–7. GCL, ganglion cell layer; Eda, Edaravone. (A, B) Original magnification: ×10; scale bar, 100 μm.

Figure 6.

Effect of edaravone on levels of Bcl-2, Bax, p-ERK1/2, and total ERK1/2 in AR, RD, and RD treated with 5 mg/kg edaravone (A). Retinas were harvested at 24 hours after RD. Whereas there was no change in the level of Bcl-2 after RD compared to AR, an increased level of Bcl-2 was noted after treatment with edaravone (C). An increased level of Bax was noted after RD, and it did not change with edaravone treatment (D). There was a marked increase in the level of phosph-ERK1/2 after RD. However, a decrease was noted after edaravone treatment (B). There was no change in the total ERK1/2 levels. *P < 0.05, **P < 0.01. Data are expressed as the mean ± SE n = 4–6.