Effects of beta-adrenergic receptor antagonists on oxidative stress in purified rat retinal ganglion cells

Abstract

Purpose: To investigate the effect of beta-adrenergic receptor antagonists against oxidative stress on purified rat retinal ganglion cells (RGCs), timolol, betaxolol, carteolol and nipradilol were included in the present study.

Methods: RGCs were purified using a 2 step panning procedure from postnatal days 6-8 using Wistar rats. After 72 h in culture under normal condition, RGCs were exposed to oxidative stress induced by B27 medium without anti-oxidant. To verify whether this stress is apoptotic or necrotic, Annexin V and propidium iodide were used to detect apoptotic and necrotic cells after 2 h stress. The presence of a proinhibitor for intracellular cathepsin B, and an inhibitor for thiol protease (cathepsin B/H/L, calpain), was also assessed to verify necrotic cell death event in oxidative conditions. Next, RGC cultures under oxidative stress were incubated with timolol, betaxolol, carteolol, and nipradilol added, respectively, for 24 h culture. The RGC viability in each condition normalized to that under normal condition was evaluated as live cell percentage based on total experiments of 8-15.

Results: Two h after oxidative stress, Annexin V and propidium iodide positive cells increased. Increased cell death under oxidative stress was significantly reduced by inhibitors for cathepsin or calpain. These data suggest that increased cell death under the current oxidative stress was due to necrosis. Under oxidative stress for 24 h, RGC viability reduced to 52.5-60.2% as compared with normal. With 10 nM and 100 nM timolol, live cell significantly increased to 69.3% and 75.5%, respectively. Both betaxolol and nipradilol enhanced live RGCs significantly in concentration of 100 nM and 1 microM, with viability of 70.5%, 71.6%, and 70.4%, 74.7%, respectively. While with 10 nM, 100 nM and 1 microM addition of carteolol, there was no significant increase in live RGC percentage which ranged from 53.1-55.0%.

Conclusions: Timolol, betaxolol and nipradilol, but not carteolol, showed neuroprotective effects against oxidative stress induced by B27 without antioxidant on purified rat RGCs at concentrations of 10 nM or higher. Although the neuroprotective mechanism of beta-blockers for oxidative stress is still unknown, this additive effect may deserve future studies.

Figure 1

Rat retinal ganglion cell death by oxidative stress. Rat retinal ganglion cells (RGCs) after 72+24 h of culture under AO+ conditions (A, B, C) or AO- conditions (D, E, F). A, D: Phase contrast images of RGCs without labeling. B, E: Live cells with labeling of calcein-AM. C, F: Dead cells (red) with labeling of both calcein-AM and ethidium homodimer-1. The dendrites become shortened and cellular bodies had a deformed appearance (arrows). The scale bar represents 50 μm.

Figure 2

Detection of apoptotic and necrotic retinal ganglion cells under oxidative stress. Apoptotic retinal ganglion cells (RGCs) were significantly increased in the staurosporine-treated RGCs, but not in any other condition. Necrotic RGCs were significantly increased in the AO- and staurosporine conditions. E64-d and Ca-074 Me significantly reduced the necrotic cell percentage compared to the AO- conditions, but the values were not significantly different from the AO+ group. Each value represents mean±SD, (n=8). Asterisk indicates p<0.05, versus all the other groups (Tukey test).

Figure 3

Effect of timolol on oxidative stress-induced retinal ganglion cell damage. Following 24 h under AO- condition in the presence of timolol, the survival rate of RGC was assessed and normalized to that of the control AO+ condition. At 10 and 100 nM, timolol significantly increased retinal ganglion cell viability. Data are expressed as mean±SD, n=8. Asterisk indicates p<0.05; cross symbol indicates p<0.01 by Dunnet test.

Figure 4

Effect of betaxolol on oxidative stress-induced retinal ganglion cell damage. Following 24 h under AO- condition in the presence of betaxolol, the survival rate of RGC was assessed and normalized to that of the control AO+ condition. At 100 nM and 1 μM, betaxolol significantly increased RGC viability. Data are expressed as mean±SD, n=8. Asterisk indicates p<0.05 by Dunnet test.

Figure 5

Effect of carteolol on oxidative stress-induced retinal ganglion cell damage. Following 24 h under AO- condition in the presence of carteolol, the survival rate of RGC was assessed and normalized to that of the control AO+ condition. Carteolol showed no significant effect on oxidative stress-induced damage of RGCs. Data are expressed as mean±SD, n=8.

Figure 6

Effect of nipradilol on oxidative stress-induced retinal ganglion cell damage. Following 24 h under AO- condition in the presence of nipradilol, the survival rate of RGCs was assessed and normalized to that of the control AO+ condition. Nipradilol (100 nM and 1 μM) significantly increased RGC viability. Data are expressed as mean±SD, n=8. Asterisk (*) indicates p<0.05; christ symbol indicates p<0.01 by Dunnet test.