Efficacy of intravitreal injection of recombinant erythropoietin for protection of retinal ganglion cells in a rat model of retinal ischemia

Abstract

Purpose: Erythropoietin (EPO) is a hematopoietic cytokine capable of inhibitting the apoptosis of erythrocyte progenitors, and of stimulating their proliferation and differentiation in response to hypoxia. The use of recombinant human EPO-alpha (EPOrh) is largely recommended to treat anemia. In addition, the non-haematopoietic, neuroprotective and neurotrophic functions of EPOrh described by several reports have led to propose therapies for several central nervous system and retinal diseases. The aim of our study was to investigate the effects of intravitreal administration of EPOrh in a model of retinal ischemia by irreversible bilateral occlusion of the carotid artery (IBCAO) in rats.

Methods: All animals had their retinal function evaluated by ERG/EVPs before the IBCAO induction. Two days after IBCAO induction, we performed the unilateral intravitreous injection of EPOrh (400 ng) or saline. Seven days after the injection, the ERG/EVPs were repeated and the animals were euthanized. The eyes were dissected, fixed in 4% paraformaldehyde with 30% sucrose overnight and processed for immunofluorescence or western blotting using antibodies anti-Brn3a, anti-GFAP and anti-macrosialin, or stained by hematoxylin of Harris. All experimental procedures were approved by Animal Ethics Committee (CEUA/UFRJ).

Results: After IBCAO, there was a 95% reduction of retinal ganglion cells (RGCs) in ischemic retinas compared to the control animals (Sham). Retinas that received a single intravitreal injection of EPOrh preserved 57 % of RGCs Brn-3a positive cells as compared to those that received or not only a saline intravitreal injection (vehicle). In addition, a down-regulation of inflammatory response was observed in ischemic retinas treated with EPOrh, indicated by a decrease of GFAP (astrocytes/Müller glia, glial reactivity) and macrosialin (activated microglia) protein expression. Finally, the EVPs (evoked potentials) in retinas treated wih EPOrh showed a trend to preserve the amplitude of waves in response to flash stimuli.

Conclusions: Our results indicate the neuroprotective potential of intravitreal administration of EPOrh in IBCAO, which could be assumed as a therapeutical approach for patients affected by ocular ischemic syndromes.

Keywords: 615 neuroprotection • 531 ganglion cells • 572 ischemia