Coenzyme Q10 protects retinal cells from apoptosis induced by radiation in vitro and in vivo

Abstract

The key pathogenetic event of many retinopathies is apoptosis of retinal cells. Our previous studies have demonstrated that Coenzyme Q10 (CoQ10) prevents apoptosis of corneal keratocytes both in vitro and in vivo, by virtue of its ability to inhibit mitochondrial depolarization, independently of its free radical scavenger role. The aim of this study was to evaluate whether CoQ10 can protect cultured retinal cells and the retinas of rats from radiation-induced apoptosis, if instilled as eye drops in the cornea. In vitro experiments were carried out on cultured ARPE-19 or RGC-5 cells pretreated with CoQ10 before eliciting apoptosis by UV- and γ-radiation, chemical hypoxia (Antimycin A) and serum starvation. Cell viability was evaluated by light microscopy and fluorescence activated cell sorting analysis. Apoptotic events were scored by time-lapse videomicroscopy. Mitochondrial permeability transition was evaluated by JC-1. The anti-apoptotic effectiveness of CoQ10 in retina was also evaluated by an in situ end-labeling assay in Wistar albino rats treated with CoQ10 eye drops prior to UV irradiation of the eye. CoQ10 substantially increased cell viability and lowered retinal cell apoptosis in response both to UV- and γ-radiation and to chemical hypoxia or serum starvation by inhibiting mitochondrion depolarization. In the rat, CoQ10, even when applied as eye drops on the cornea, protected all retina layers from UVR-induced apoptosis. The ability of CoQ10 to protect retinal cells from radiation-induced apoptosis following its instillation on the cornea suggests the possibility for CoQ10 eye drops to become a future therapeutic countermeasure for radiation-induced retinal lesions.

Fig. 1.

Evaluation of the number of living cultured ARPE-19 and RGC-5 cells pretreated (+CoQ10) or not (–CoQ10) with 10 µM CoQ10 for 2 h before being irradiated with UV (15 mJ/cm²) or maintained in a condition of serum starvation (0.5% FBS) or treated with 200 µM Antimycin A (AntA).

Fig. 2.

Evaluation of anti-apoptotic effects of CoQ10 on ARPE-19 cells subjected to (A) UV irradiation (15 mJ/cm²) and (B) γ irradiation (20 µCi/ml). Where specified, the cells were pretreated (+CoQ10) or not (–CoQ10) with CoQ10. Untreated cells served as controls. The cumulative apoptotic events were detected and registered progressively by time-lapse videomicroscopy up to 72 h after stimulation. An apoptotic event was scored the moment the cell, after detachment from the substrate and shrinking, began apoptotic blebbing. Each value is the mean ± SEM of three experiments (*P < 0.005 –CoQ10 vs. control; **P < 0.01 +CoQ10 vs. –CoQ10).

Fig. 3.

(A) Immunoblot analysis of rpe65 or GFP silencing in ARPE-19 cells.

Fig. 4.

Analysis of Δψ in JC-1 labeled mitochondria of ARPE-19 cells.

Fig. 5.

Topical application of CoQ10 to rat cornea prevents retinal apoptosis in response to UV irradiation. (A) Representative microscopy image of the retina from UV-irradiated mice pretreated with CoQ10 (+CoQ10) or with the vehicle alone (–CoQ10) analyzed using the In Situ End Labeling (ISEL) technique of nicked DNA to detect DNA fragmentation. GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer) and ONL (outer nuclear layer) are indicated. (B) The numbers of ISEL-positive (brown) and -negative (light blue) nuclei within at least three fields of retina specimens were scored by two different observers. The number of apoptotic cells was expressed as a percentage of total number of counted cells. Data are means ± SEM of three experiments; *P < 0.01.