Neuroprotective effects of exogenous erythropoietin in Wistar rats by downregulating apoptotic factors to attenuate N-methyl-D-aspartate-mediated retinal ganglion cells death

The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of μ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: μ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.