Erythropoietin Protects Retina Against Ceramide 2-Induced Damage in Rat

Ceramide plays critical roles in cell proliferation, senescence and apoptosis, and is implicated in neurodegenerative diseases, etc. To clarify if ceramide plays some roles in retinal diseases, we established in vivo and in vitro retinal injury models with ceramide 2 (C2) treatment. In addition, Erythropoietin (EPO), which showed protective effects on retinal cells and blood-retinal barrier (BRB), was also tested for its protection and possible mechanism(s) in these models.

Male Sprague-Dawley rats were divided into four groups, i.e., normal control, vehicle control, C2 treatment, and C2+EPO treatment. After intravitreal injection, the rats were examined for eye fundus, electroretinogram, histological study, and immunostaining, etc. In vitro, retinal neuronal cell line (R28) and the primary human retinal microvascular endothelial cells (HRMECs) were treated with C2, cell viability assay, transendothelial electrical resistance (TEER) and BRB-related molecules were studied to test the protective effect of EPO.

Intravitreal C2-treatment caused significant vision loss in rats, as reflected by reduced b-wave amplitude, increased TUNEL positive cells and GFAP immunostaining in retina. Another major retinal injury observed was BRB breakdown following C2- treatment. Such C2-induced injuries were further confirmed by in vitro study. When HRMECs were treated with C2, the TEER was significantly reduced. The mechanisms for C2 to induce such injuries might be through evidently increased expressions of the related molecules like plasmalemma vesicle-associated protein (PLVAP or PV-1), ecto- 5'-nucleotidase (CD73) and intercellular adhesion molecule-1 (ICAM-1), as observed in C2-treated R28 cells. All these injuries induced by C2 were significantly prevented by EPO both in vivo and in vitro, and its protective mechanisms here might be, in addition to neuroprotective, closely related to its maintenance of BRB integrity, through reducing the expressions of PV-1, CD73 and ICAM-1.

C2 could induce severe retinal injury, and such injuries could be effectively prevented by EPO treatment.