The Influence of Electrical Stimulation on the Proliferation and Differentiation of Retinal Progenitor Cells

Purpose: This study aimed to evaluate the effects of electrical stimulation on the proliferation and differentiation of retinal progenitor cells (RPCs) and explore the potential mechanism.Methods: RPCs were got from retinas of postnatal days 0-2 GFP+ C57BL/6 mice.Electrical stimulation (ES) was performed with a biphasic current pulse at the pulse width of 2ms and 20Hz pulse frequency.The intensity of the electrical stimulation was 300μA.RPCs were treated with or without ES one hour per day for consecutive 3 days in a state of cell proliferation and differentiation.Cell morphology was observed under a fluorescence microscope to evaluate RPCs viability.And the proliferation ability of RPCs was evaluated by EdU.Immunocytochemistry and western blot analyses were used to observe expression levels of p-Akt, p-Erk, Akt, Erk, rhodopsin, β3-tubulin and PKC-α.Results: RPCs in ES group exhibited higher cell densities and viability than the cells in control group without ES (p<0.05).The relative fluorescence intensity in the ES group was nearly two times that of the control group.Immunocytochemistry and western blot analyses showed that expression levels of p-Akt, p-Erk, Akt and Erk in the ES group was significantly higher than that of the control group (p<0.05).And the relative protein expression levels of rhodopsin (photoreceptor marker),β3-tubulin (panneuron marker) and Pkc-a (bipolar cell marker) in RPCs affected by ES were approximately 1.6-fold, 2.1-fold and 2.4-fold of those in control group, respectively.Conclusion: ES could improve RPCs proliferation via the activation of the Akt and Erk signaling pathways.Moreover, ES markedly induced RPCs differentiation toward retinal neurons.Therefore, ES can be used as a means to modulate the survival, proliferation, and differentiation of RPCs before in vivo implantation.