Brimonidine is Neuroprotective in Animal Paradigm of Retinal Ganglion Cell Damage

Abstract

To investigate the neuroprotective effect of brimonidine after retinal ischemia damage on mouse eye. Glaucoma is an optic neuropathy characterized by retinal ganglion cells (RGCs) death, irreversible peripheral and central visual field loss, and high intraocular pressure. Ischemia reperfusion (I/R) injury model was used in C57BL/6J mice to mimic conditions of glaucomatous neurodegeneration. Mouse eyes were treated topically with brimonidine and pattern electroretinogram were used to assess the retinal ganglion cells (RGCs) function. A wide range of inflammatory markers, as well as anti-inflammatory and neurotrophic molecules, were investigated to figure out the potential protective effects of brimonidine in mouse retina. In particular, brain-derived neurotrophic factor (BDNF), IL-6, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor DR-5, TNF-α, GFAP, Iba-1, NOS, IL-1β and IL-10 were assessed in mouse retina that underwent to I/R insult with or without brimonidine treatment. Brimonidine provided remarkable RGCs protection in our paradigm. PERG amplitude values were significantly (p < 0.05) higher in brimonidine-treated eyes in comparison to I/R retinas. Retinal BDNF mRNA levels in the I/R group dropped significantly (p < 0.05) compared to the control group (normal mice); brimonidine treatment counteracted the downregulation of retinal BDNF mRNA in I/R eyes. Retinal inflammatory markers increased significantly (p < 0.05) in the I/R group and brimonidine treatment was able to revert that. The anti-inflammatory IL-10 decreased significantly (p < 0.05) after retinal I/R insult and increased significantly (p < 0.05) in the group treated with brimonidine. In conclusion, brimonidine was effective in preventing loss of function of RGCs and in regulating inflammatory biomarkers elicited by retinal I/R injury.

Keywords: PERG; brimonidine; ischemia-reperfusion; neuroprotection; retinal ganglion cells.

FIGURE 1

RGCs function assessment. (A) Representative PERG waveforms in C57BL6/J mice control, I/R and I/R plus brimonidine. (B) Mouse PERG recording layout. (C) Comparison between PERG amplitude values (µV) and latency values (D) of control, I/R and brimonidine treated mice. Brimonidine significantly counteracted RCGs loss of function induced by I/R injury, after 72 h, in mice retina. In each panel, bars represent the mean values and corresponding standard errors (±SD). One-way ANOVA analysis was performed followed by the Tukey post-hoc test. *p < 0.05 vs. Ctrl; ^† p < 0.05 vs. I/R.

FIGURE 2

BDNF and IL-6 mRNA expression in mice retina. Brimonidine treatment maintained BDNF (A) mRNA levels close to control group, in comparison to I/R injured mice. Furthermore, brimonidine reverted the up-regulation of IL-6 (B) elicited by I/R injury. The mRNA levels were evaluated by RT-PCR; values represent the mRNA fold changes relative to 18 S used as housekeeping gene. Values are reported as a mean ± SD (n = 5). One-way ANOVA analysis was performed followed by the Tukey post-hoc test. *p < 0.05 vs. Ctrl; ^† p < 0.05 vs. I/R.

FIGURE 3

Western Blot. (A) TRAIL and DR5 protein levels in control, I/R and brimonidine-treated mice retina; (B) GFAP, Iba-1, TNF-α and IL-10 proteins in mice retina w or w/o brimonidine; (C) NOS2 and IL-1β proteins in mice retina w or w/o brimonidine. Values represent protein expression relative to β-tubulin, used as housekeeping protein. Values are reported as mean ± SD (n = 5). One-way ANOVA analysis was performed followed by the Tukey post-hoc test. *p < 0.05 vs. Ctrl; ^† p < 0.05 vs. I/R.