Brimonidine is neuroprotective against glutamate-induced neurotoxicity, oxidative stress, and hypoxia in purified rat retinal ganglion cells

Abstract

Purpose: To investigate the neuroprotective effect of alpha2-adrenergic agonist brimonidine in the presence of glutamate-induced neurotoxicity, oxidative stress, and hypoxia on in vitro cultures of purified rat retinal ganglion cells (RGCs).

Methods: Purified RGC cultures were obtained from retinas of 6-8-day old Wistar rats, following a two-step immunopanning procedure. After 72 h of cultivation, the neuroprotective effect of brimonidine (0.01 microM, 0.1 microM, and 1 microM) was investigated by culturing the RGCs under glutamate, oxidative, and hypoxic stress for a further 72 h, 24 h, and 12 h, respectively. Glutamate neurotoxicity was induced by adding glutamate (25 microM), while oxidative stress was induced by substituting the culture medium with B27 supplement without antioxidants, and hypoxia was induced by cultivation in a controlled-atmosphere incubator with oxygen levels 5% of the normal partial pressure. The RGC viability under each stress condition normalized to that under normal condition was evaluated as live cell percentage based on a total of 7-8 full repeated experiments.

Results: The cell survival percentages of cultures exposed to glutamate, oxidative, and hypoxic stress were 58.2%, 59.3%, and 53.2%, respectively. Brimonidine dose dependently increased RGC survival in the presence of glutamate (80.6% at 1 microM), oxidative (79.8% at 1 microM), and hypoxic (72.3 and 77.4% at 0.1 and 1 microM, respectively) stress. In the presence of alpha2-adrenergic antagonist yohimbine (10 microM), brimonidine (1 microM) showed no protective effects on RGC viability.

Conclusions: At a concentration of 0.1 microM or higher, brimonidine increased survival of purified rat RGCs in the presence of glutamate neurotoxicity, oxidative stress, and hypoxia. The neuroprotective effect of brimonidine is mediated via alpha2-adrenergic receptors at the RGC level.

Figure 1

Retinal ganglion cell viability of seven experiments under (A) glutamate-induced neurotoxicity, (B) oxidative stress, and (C) hypoxic stress, with increasing concentrations of brimonidine. Brimonidine at a concentration of 1 μM significantly increased retinal ganglion cell (RGC) viability in all three stresses. Abbreviations: (-) represents control RGC cultures without glutamate; (+) represents control RGC cultures with glutamate, AO(+)represents medium with anti-oxidant; AO(-) represents medium without anti-oxidant, * represents p<0.01. n=7. Error bar indicates SD.

Figure 2

Retinal ganglion cell viability of eight experiments under (A) glutamate-induced neurotoxicity, (B) oxidative stress, and (C) hypoxic stress. The neuroprotective effect of 1 μM brimonidine was reproduced in all three stresses. The effect of brimonidine on the increase in retinal ganglion cell (RGC) viability was blocked by the α2-adrenergic antagonist yohimbine in all three stresses. Abbreviations: (+) represents control RGC cultures with glutamate; AO(+) represents medium with anti-oxidant; AO(-) represents medium without anti-oxidant; B represents RGC cultures with brimonidine added; B+Y represents RGC cultures with brimonidine and yohimbine added; Y represents RGC cultures with yohimbine added; *, p<0.001; n=7. Error bar indicates SD.